Title:
IMMUNOGLOBULIN FUSION PROTEINS AND COMPOSITIONS THEREOF
Kind Code:
A1


Abstract:
Disclosed herein are immunoglobulin fusion proteins that have a first antibody region attached to an extender fusion region. The extender fusion region contains a therapeutic agent and a beta strand secondary structure. The extender fusion region may contain 7 or fewer consecutive amino acids based on or derived from an ultralong CDR3. Alternatively, the extender fusion region contains a rigid stalk protein structure, but does not contain an amino acid sequence based on or derived from an ultralong CDR3. The extender fusion region may also have one or more linkers or proteolytic cleavage sites. The immunoglobulin fusion proteins may have additional therapeutic agents and extender fusion regions. Also disclosed herein are pharmaceutical compositions of immunoglobulin fusion proteins and methods for using the immunoglobulin fusion proteins for the treatment or prevention of a disease or condition in a subject.



Inventors:
Wang, Feng (Carlsbad, CA, US)
Zhang, Yong (Temple City, CA, US)
Liu, Tao (San Diego, CA, US)
DU, Juanjuan (San Diego, CA, US)
Wang, Ying (San Diego, CA, US)
Liu, Yan (San Diego, CA, US)
Schultz, Peter G. (La Jolla, CA, US)
Application Number:
14/903489
Publication Date:
08/18/2016
Filing Date:
07/11/2014
Assignee:
THE CALIFORNIA INSTITUTE FOR BIOMEDICAL RESEARCH (La Jolla, CA, US)
THE SCRIPPS RESEARCH INSTITUTE (La Jolla, CA, US)
Primary Class:
International Classes:
C07K16/28; C07K14/505; C07K14/535; C07K14/61; C07K16/32
View Patent Images:



Primary Examiner:
HISSONG, BRUCE D
Attorney, Agent or Firm:
WILSON SONSINI GOODRICH & ROSATI (PALO ALTO, CA, US)
Claims:
1. 1.-61. (canceled)

62. An immunoglobulin fusion protein comprising: a) a first antibody region; and b) a first extender fusion region comprising a first therapeutic agent attached to a first extender peptide, wherein the first extender peptide comprises a first beta strand secondary structure region having a first beta strand secondary structure; wherein the first extender fusion region does not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain of SEQ ID NO: 248.

63. The immunoglobulin fusion protein of claim 62, wherein the first extender fusion region comprises a second extender peptide comprising a second beta strand secondary structure region having a second beta strand secondary structure, and wherein the first beta strand secondary structure and second beta strand secondary structure form a beta sheet.

64. The immunoglobulin fusion protein of claim 62, wherein the first extender fusion region replaces a portion of a light chain of the first antibody region or a portion of the heavy chain of the first antibody region.

65. The immunoglobulin fusion protein of claim 62, wherein the first antibody region comprises an antibody selected from an anti-Her2 antibody, trastuzumab, an anti-CD47 antibody, palivizumab, and antigen binding fragments thereof.

66. The immunoglobulin fusion protein of claim 62, wherein the first antibody region comprises a heavy chain, wherein the heavy chain is represented by an amino acid sequence that is at least 90% homologous to a sequence selected from SEQ ID NOS: 24-27, 29-33, 36-39, and 251-253, and a light chain, wherein the light chain is represented by an amino acid sequence that is at least 90% homologous to a sequence selected from SEQ ID NOS: 21-23, 28, 34, 35, 40, 248-250 and 278.

67. The immunoglobulin fusion protein of claim 62, wherein the first beta strand secondary structure region comprises an amino acid sequence that is at least 90% homologous to a sequence selected from SEQ ID NOs: 109-128, 305-308.

68. The immunoglobulin fusion protein of claim 62, wherein the first beta strand secondary structure region comprises an amino acid sequence of ETKKYQXnS (SEQ ID NO:110), wherein n=1-8 and X is selected from a basic amino acid, an acidic amino acid, a polar amino acid, and a charged amino acid.

69. The immunoglobulin fusion protein of claim 63, wherein the first beta strand secondary structure region comprises an amino acid sequence of ETKKYQXnS (SEQ ID NO: 306), wherein n=1-8 and X is selected from a basic amino acid, an acidic amino acid, a polar amino acid, and a charged amino acid, and wherein the second beta strand secondary structure region comprises an amino acid sequence of SX1TX2NX3 (SEQ ID NO: 128), wherein X1, X2, and X3 are independently selected from polar amino acids.

70. The immunoglobulin fusion protein of claim 63, wherein the second beta strand secondary structure region comprises an amino acid sequence of YX1YX2Y, and wherein X1 and X2 are independently selected from polar amino acids.

71. The immunoglobulin fusion protein of claim 62, wherein the immunoglobulin fusion protein comprises an amino acid sequence that is at least about 90% identical to an amino acid sequence of any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304.

72. The immunoglobulin fusion protein of claim 62, wherein the immunoglobulin fusion protein further comprises a second therapeutic agent.

73. The immunoglobulin fusion protein of claim 72, wherein the immunoglobulin fusion protein further comprises a second antibody region, wherein the second antibody region comprises a second extender fusion region attached to the second antibody region, and wherein the second extender fusion region comprises the second therapeutic agent and an extender peptide comprising a) an amino acid sequence having an alpha helical secondary structure, or b) an amino acid sequence having a beta strand secondary structure, or c) an amino acid sequence having no regular secondary structure.

74. The immunoglobulin fusion protein of claim 72, further comprising a second antibody region, wherein the second therapeutic agent is directly attached to the second antibody region.

75. The immunoglobulin fusion protein of claim 62, wherein first therapeutic agent is selected from bGCSF, hGCSF, bGMCSF, hGMCSF, GDF11, interferon-beta, interferon-alpha, interleukin 11 (IL-11), exendin-4, GLP-1, relaxin, oxyntomodulin, leptin, betatrophin, bovine growth hormone (bGH), human growth hormone (hGH), parathyroid hormone, erythropoietin, Moka1, VM-24, Mamba1, angiopoeitin-like 3 (ANGPTL3), CVX15, a CXCR4 ligand, a neutrophil elastase inhibitor, and homologs thereof.

76. The immunoglobulin fusion protein of claim 62, wherein the first therapeutic agent is represented by an amino acid sequence that is at least 90% homologous to a sequence selected from any one of SEQ ID NOs: 200-235 and/or encoded by a nucleic acid sequence that is at least 90% homologous to a sequences selected from SEQ ID NOs: 167-199.

77. The immunoglobulin fusion protein of claim 62, wherein the first antibody region comprises a BVK antibody or antigen binding fragment thereof and the first therapeutic agent comprises a Moka1 peptide, and wherein the first extender fusion region is grafted into a heavy chain of the BVK antibody.

78. The immunoglobulin fusion protein of claim 77, wherein the Moka1 peptide is represented by an amino acid sequence that is at least about 90% homologous to SEQ ID NO. 202.

79. The immunoglobulin fusion protein of claim 77, wherein the heavy chain of the BVK antibody is encoded by a nucleotide sequence that is at least 90% homologous to SEQ ID NO. 1.

80. The immunoglobulin fusion protein of claim 77, wherein the first beta strand secondary structure is represented by an amino acid sequence selected from SEQ ID NOS: 111, 119, and combinations thereof.

81. The immunoglobulin fusion protein of claim 77, wherein: a) the BVK antibody comprises a heavy chain encoded by a nucleotide sequence of SEQ ID NO:1, and a light chain encoded by a nucleotide sequence of SEQ ID NO: 14; b) the Moka1 peptide is represented by an amino acid sequence of SEQ ID NO: 202; c) the first beta strand secondary structure is represented by an amino acid sequence of SEQ ID NO: 111; and d) a second beta strand secondary structure is represented by an amino acid sequence of SEQ ID NO: 119, wherein the Moka1 peptide is grafted into the heavy chain of the BVK antibody.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage entry of International Application No. PCT/US2014/046429, filed Jul. 11, 2014; which claims the benefit of U.S. Provisional Application No. 61/845,287 filed Jul. 11, 2013; U.S. Provisional Application No. 61/845,280 filed Jul. 11, 2013; U.S. Provisional Application No. 61/925,904 filed Jan. 10, 2014; and U.S. Provisional Application No. 62/017,713 filed Jun. 26, 2014, all of which are incorporated by reference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 6, 2016, is named 41135-707-831-SEQUENCE.txt and is 587,784 bytes in size.

BACKGROUND OF THE INVENTION

Antibodies are natural proteins that the vertebrate immune system forms in response to foreign substances (antigens), primarily for defense against infection. For over a century, antibodies have been induced in animals under artificial conditions and harvested for use in therapy or diagnosis of disease conditions, or for biological research. Each individual antibody producing cell produces a single type of antibody with a chemically defined composition. However, antibodies obtained directly from animal serum in response to antigen inoculation actually comprise an ensemble of non-identical molecules (e.g., polyclonal antibodies) made from an ensemble of individual antibody producing cells.

Antibody fusion constructs can be used to improve the delivery of drugs or other agents to target cells, tissues and tumors. Antibody fusion constructs may comprise a chemical linker to attach a drug or other agent to an antibody. Exemplary antibody fusion constructs and methods of producing antibody fusion constructs are disclosed in US patent application numbers 20060182751, 20070160617 and U.S. Pat. No. 7,736,652.

Disclosed herein are novel immunoglobulin fusion proteins and methods of producing such immunoglobulin fusion proteins. Further disclosed herein are uses of the immunoglobulin fusion proteins for the treatment of various diseases and conditions. Methods of extending the half-life of a therapeutic agent are also disclosed herein.

SUMMARY OF THE INVENTION

Disclosed herein are immunoglobulin fusion proteins comprising a first antibody region; and a first extender fusion region comprising a first therapeutic agent attached to a first extender peptide, wherein the first extender peptide comprises a first beta strand secondary structure region containing a first beta strand secondary structure; and wherein the first extender fusion region does not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain of SEQ ID NO: 248. The first extender fusion region may not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain selected from a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, and an F18 ultralong complementary determining region 3 heavy chain. The first extender fusion region may not contain more than 7 consecutive amino acids that are based on or derived from a bovine ultralong CDR3. The first extender fusion region may not comprise an amino acid sequence that is based on or derived from a bovine ultralong CDR3. The first antibody region may be a human antibody or human antibody fragment and wherein the first beta strand secondary structure is based on or derived from a human beta strand secondary structure. The first antibody region may be based on or derived from a monoclonal antibody. The first antibody region may be based on or derived from an antibody designed to selectively interact with a target selected from a cancer cell and a virus. The first antibody region may be based on or derived from an anti-Her2 antibody, trastuzumab, an anti-CD47 antibody and palivizumab. The first extender peptide may comprise an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 109-128, 305 and 308. The first extender fusion region may further comprise a second extender peptide, wherein the second extender peptide comprises a second beta strand secondary structure and wherein the first beta strand secondary structure and the second beta strand secondary structure form a beta sheet. The first extender peptide may comprise an amino acid sequence that is at least about 50% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 109-114 and 305. The second extender peptide may comprise an amino acid sequence that is at least about 50% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 115-128 and 308. The first extender peptide may comprise an amino acid sequence of ETKKYQXnS (SEQ ID NO: 110). The first extender peptide may comprise an amino acid sequence of ETKKYQXnS (SEQ ID NO: 305), wherein n is equal to a number selected from 1-8. The immunoglobulin fusion proteins disclosed herein may further comprise a second extender peptide, wherein the second extender peptide comprises an amino acid sequence of SX1TX2NX3 (SEQ ID NO: 306). X1, X2 and X3 may be independently selected from a polar amino acid. The polar amino acid may be Y. The immunoglobulin fusion proteins may further comprise a second extender peptide, wherein the second extender peptide comprises an amino acid sequence of YX1YX2Y (SEQ ID NO: 128). The first extender fusion region may comprise one or more linkers selected from SEQ ID NOs:161-166 and 309. The immunoglobulin fusion proteins may have the Formula II: A1-E1-T1-E2 or Formula IIA:

embedded image

wherein A1 is the first antibody region; E1 is the first extender peptide, E2 is a second extender peptide; and T1 is the first therapeutic agent. The fusion protein may comprise an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The immunoglobulin fusion proteins may comprise an amino acid sequence that is at least about 50% identical to an amino acid sequence of any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The immunoglobulin fusion proteins may further comprise a second antibody region based on or derived from an antibody or fragment thereof. The immunoglobulin fusion proteins may further comprise a second extender fusion region. The immunoglobulin fusion protein may further comprise a second therapeutic agent. The immunoglobulin fusion proteins may have the Formula III: (A1-E1-T1-E2)-(A2-E3-T2-E4) or Formula IIIA

embedded image

wherein: A1 is the first antibody region; E1 is the first extender peptide; T1 is the first therapeutic agent; A2 is a second antibody region; E2, E3, E4 are extender peptides; and T2 is a second therapeutic agent. The first therapeutic agent may be based on or derived from a biomolecule selected from a peptide and a protein. The protein or peptide may be selected from a growth factor, a cytokine, a chemokine, a hormone and a toxin. The peptide or protein may not be naturally occurring. The first therapeutic agent may be based on or derived from a cyclic peptide. The first therapeutic agent may be a conformationally constrained peptide. The first therapeutic agent may be selected from an agonist, an antagonist, a ligand and a substrate. The first therapeutic agent may be selected from bGCSF, hGCSF, bGMCSF, hGMCSF, GDF11, interferon-beta or interferon-alpha, interleukin 11 (IL-11), exendin-4, GLP-1, relaxin, oxyntomodulin, leptin, betatrophin, bovine growth hormone (bGH), human growth hormone (hGH), parathyroid hormone, erythropoietin, Moka1, VM-24, Mamba1, angiopoeitin-like 3 (ANGPTL3), a homolog thereof and a derivative thereof. The first therapeutic agent may interact with a target selected from CXCR4 and a neutrophil elastase inhibitor. The first therapeutic agent may be based on or derived from an amino acid sequence selected from any one of SEQ ID NOs: 263-298 or encoded by a nucleic acid sequence based on or derived from any one of SEQ ID NOs: 227-262. The first therapeutic agent may be based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 263-298 or encoded by a nucleic acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 227-262. The extender fusion region may further comprise one or more proteolytic cleavage sites. The one or more proteolytic cleavage sites may be at the N-terminus, C-terminus, or N- and C-termini of a therapeutic agent. The one or more proteolytic cleavage sites may be within a therapeutic agent. The one or more proteolytic cleavage sites may comprise a Factor Xa cleavage site. The one or more proteolytic cleavage sites may be in one or more extender peptides. The one or more proteolytic cleavage sites may be in the antibody region.

Further disclosed herein are polynucleotides comprising a nucleic acid sequence encoding an immunoglobulin fusion protein comprising a first antibody region; and a first extender fusion region comprising a first therapeutic agent attached to a first extender peptide, wherein the first extender peptide comprises a region containing a first beta strand secondary structure; and wherein the first extender fusion region does not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain of SEQ ID NO: 248.

Disclosed herein are vectors comprising a polynucleotide comprising a nucleic acid sequence encoding an immunoglobulin fusion protein comprising a first antibody region; and a first extender fusion region comprising a first therapeutic agent attached to a first extender peptide, wherein the first extender peptide comprises a region containing a first beta strand secondary structure; and wherein the first extender fusion region does not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain of SEQ ID NO: 248.

Further disclosed herein are host cells comprising a polynucleotide comprising a nucleic acid sequence encoding an immunoglobulin fusion protein comprising a first antibody region; and a first extender fusion region comprising a first therapeutic agent attached to a first extender peptide, wherein the first extender peptide comprises a region containing a first beta strand secondary structure; and wherein the first extender fusion region does not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain of SEQ ID NO: 248.

Disclosed herein are methods of producing an immunoglobulin fusion protein, the method comprising culturing a host cell comprising a polynucleotide comprising a nucleic acid sequence encoding an immunoglobulin fusion protein comprising a first antibody region; and a first extender fusion region comprising a first therapeutic agent attached to a first extender peptide, wherein the first extender peptide comprises a region containing a first beta strand secondary structure; and wherein the first extender fusion region does not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain of SEQ ID NO: 248 under conditions wherein the polynucleotide sequence is expressed, thereby producing an immunoglobulin fusion protein.

Further disclosed herein are pharmaceutical compositions comprising an immunoglobulin fusion protein comprising a first antibody region; and a first extender fusion region comprising a first therapeutic agent attached to a first extender peptide, wherein the first extender peptide comprises a region containing a first beta strand secondary structure; and wherein the first extender fusion region does not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain of SEQ ID NO: 248. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient.

Disclosed herein are methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an immunoglobulin fusion protein comprising a first antibody region; and a first extender fusion region comprising a first therapeutic agent attached to a first extender peptide, wherein the first extender peptide comprises a region containing a first beta strand secondary structure; and wherein the first extender fusion region does not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain of SEQ ID NO: 248.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of the disclosure, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the disclosure, shown in the figures are embodiments which are presently preferred. It should be understood, however, that the disclosure is not limited to the precise arrangements, examples and instrumentalities shown. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

In some figures, trastuzumab is referred to as Herceptin. It is to be understood that trastuzumab and Herceptin may be used interchangeably throughout this disclosure. In some figures, an immunoglobulin fusion protein is described in the following order: antibody, secondary structure (e.g. beta strand), therapeutic agent, and antibody region to which the therapeutic agent is attached; for example, trastuzumab-beta hEPO (CDRH3). Wherein the secondary structure is a beta sheet or a coiled coil, the secondary structure may simply be referred to as beta or coil, respectively. The immunoglobulin fusion protein comprising a secondary structure may additionally comprise a linker, although the presence of the linker may not be indicated in the description or name of the immunoglobulin fusion protein. The immunoglobulin fusion protein may be described in any other manner, for example, trastuzumab-CDRH3-direct-hEPO is the same fusion as trastuzumab-direct hEPO (CDRH3), wherein direct may be indicative of the absence of a secondary structure and/or the presence of a linker. In some instances, an antibody is abbreviated in the figures, for example, bAb or BLVH12 are abbreviations for bovine antibody. PBS is an abbreviation of phosphate buffered saline. In some instances, hAb is an abbreviation for Herceptin or trastuzumab antibody. In some instances, H2 is an abbreviation of CDRH2, H3 is an abbreviation of CDRH3, and L3 is an abbreviation of CDRL3. In some instances, CDRH3 and CDR3H indicate a complementary determining region 3 of a heavy chain, CDRH2 and CDR2H indicate a complementary determining region 3 of a heavy chain, and CDRL3 and CDR3L indicate a complementary determining region 3 of a light chain.

FIG. 1 depicts an exemplary schematic of various immunoglobulin fusion proteins with an extender peptide comprising a beta strand structure.

FIG. 2 depicts an exemplary schematic of various immunoglobulin fusion proteins with extender peptides.

FIGS. 3A-3G depict exemplary schematics of various non-antibody regions.

FIG. 4 depicts an exemplary schematic of various extender peptides. FIG. 4 discloses SEQ ID NOS 119, 111, 120, 111, 121, 111, 122, 111, 123, 111, 124, 111, 119, 111, 125, 112, 126, 113, 127 and 114, respectively, in order of appearance.

FIG. 5 depicts an SDS-PAGE of an immunoglobulin-beta-strand bovine granulocyte colony-stimulating factor (bGCSF) based fusion protein.

FIG. 6 depicts a graph of the in vitro proliferative activity of immunoglobulin-beta-strand bovine granulocyte colony-stimulating factor (bGCSF) based fusion proteins in mouse NFS-60 cells.

FIG. 7 depicts a graph of the binding affinity of a trastuzumab-beta-strand bovine granulocyte colony-stimulating factor (bGCSF) based fusion protein to a Her2 receptor.

FIG. 8 depicts an SDS-PAGE of an trastuzumab-beta-strand-Exendin-4 based fusion protein.

FIG. 9 depicts in vitro activities of trastuzumab-beta strand-Exendin-4 L1 IgGs.

FIG. 10 depicts an SDS-PAGE gel of trastuzumab-beta strand-Mokatoxin-1 (Moka1) L1 IgG (1) and trastuzumab-beta strand-Vm24 L1 IgG (2).

FIG. 11 depicts in vitro activity of trastuzumab-beta-Moka1 and trastuzumab-beta-Vm24 IgGs, specifically in vitro inhibition of T-cell activation.

FIG. 12 depicts an SDS-PAGE gel of trastuzumab-CDR3H-beta strand-human erythropoietin (hEPO) IgG.

FIG. 13 depicts ESI-MS of the heavy chain of trastuzumab-CDR3H-beta-hEPO fusion protein treated with Peptide-N-Glycosidase and DTT (Exp: 68789 Da; Obs: 68669 Da (matching the mass of the heavy chain of trastuzumab-CDR3H-beta-hEPO without Glu1) and 69614 Da (due to O-glycosylation on hEPO)).

FIG. 14 depicts in vitro activity of trastuzumab-CDR3H-beta-hEPO IgG on proliferation of TF-1 cells.

FIG. 15 depicts an SDS-PAGE gel of trastuzumab (CDRH3)-beta human growth hormone fusion protein.

FIG. 16 depicts an SDS-PAGE gel of trastuzumab (CDRH2)-beta human growth hormone fusion protein.

FIG. 17 depicts an exemplary schematic representation of engineering Moka1 toxin or Vm24 toxin into an ultralong CDR3H.

FIG. 18 depicts an SDS-PAGE gel of a BLV1H12-CDR3H-beta Moka1 IgG with (L1) or without (L0) a GGGGS linker (SEQ ID NO: 164).

FIG. 19 depicts BLV1H12-CDR3H-beta Moka1 IgG inhibits human peripheral blood mononuclear cells (PBMCs) activation.

FIG. 20 depicts BLV1H12-CDR3H-beta Moka1 IgG inhibits human T-cell activation.

FIG. 21 depicts an SDS-PAGE gel of a BLV1H12-CDR3H-beta VM24 IgG with one linker (L1) or two (L2) GGGGS linkers (SEQ ID NO: 164). FIG. 21 discloses ‘GGGSGGGGS’ as SEQ ID NO: 165.

FIG. 22 depicts BLV1H12-CDR3H-beta VM24 IgG inhibits human T-cell activation.

FIG. 23 depicts an exemplary schematic representation of engineering human erythropoietin (hEPO) into an ultralong CDR3H.

FIG. 24 depicts an SDS-PAGE gel of BLV1H12-CDRH3-beta hEPO fusion protein.

FIG. 25 depicts in vitro activities of BLV1H12-CDRH3-beta hEPO fusion protein.

FIG. 26 depicts pharmacokinetics of BLV1H12-CDRH3-beta hEPO and trastuzumab-CDRH3-beta hEPO fusion proteins in mice.

FIG. 27 depicts pharmacodynamics of BLV1H12-CDRH3-beta hEPO and trastuzumab-CDRH3-beta hEPO fusion proteins in mice.

FIG. 28 depicts an exemplary schematic representation of engineering glucagon-like peptide 1 (GLP-1) or Exendin-4 (Ex-4) into an ultralong CDR3H.

FIG. 29 depicts an SDS-PAGE gel of BLV1H12-CDRH3-beta GLP-1 and BLV1H12-CDRH3-beta Ex-4 with and without protease treatment.

FIG. 30 depicts in vitro activities of BLV1H12-CDRH3-beta GLP1/Ex-4 Clip fusion proteins on activating GLP1 receptor (GLP1R).

FIGS. 31 A-B depict plasma stabilities of BLV1H12-CDRH3-beta Ex-4 RN fusion proteins. Percentages of Ab-Ex-4 RN were determined on the basis of activities measured at time 0 through in vitro assay using HEK293 cells with GLP-1 receptor-CRE (cAMP response element)-Luc.

FIG. 32 depicts pharmacokinetics of BLV1H12-CDRH3-beta Ex-4 RN fusion protein in mice. Amount of Ex-4 and Ab-Ex-4 RN in mice plasma were determined through in vitro activity assay using HEK293 cells with GLP-1 receptor-CRE (cAMP response element)-Luc. Plasma concentrations at first time point (30 min) were taken as the maxima.

FIGS. 33A-B depict BLV1H12-CDRH3-beta Ex-4 RN fusion protein reduces blood glucose levels (30 min) in mice by oral glucose tolerance test in CD1 mice (3 g/kg), I.V. injection (200 ul/injection); N=5; Ex-4: 0.5 ug; Ab-Ex-4 RN: 100 ug.

FIGS. 34 A-B depict BLV1H12-CDRH3-beta Ex-4 RN fusion protein provides extended control of blood glucose levels in mice by oral glucose tolerance test (OGTT) in CD1 mice 24 h post treatment of fusion protein. OGTT in CD1 mice (3 g/kg). S.C. injection (200 ul/injection); N=5; Ab: 100 & 200 ug; Ex-4: 0.5 ug; Ab-Ex-4 RN: 100 & 200 ug. ** p<0.01.

FIGS. 35 A-B depict BLV1H12-CDRH3-beta Ex-4 RN fusion protein provides extended control of blood glucose levels in mice by oral glucose tolerance test (OGTT) in CD1 mice 48 h post treatment of fusion protein. OGTT in CD1 mice (3 g/kg). S.C. injection (200 ul/injection); N=5; Ab: 100 & 200 ug; Ex-4: 0.5 ug; Ab-Ex-4 RN: 100 & 200 ug.* p<0.05, ** p<0.01.

FIG. 36 depicts an exemplary scheme for grafting bovine granulocyte colony-stimulating factor (GCSF) onto the ‘knob’ domain of bovine BLV1H12 antibody with ultralong CDR3H region. FIG. 36 discloses ‘(GGGGS)n’ as SEQ ID NO: 164.

FIGS. 37 A-E depicts proliferative activities of BLV1H12 (Ab)-bovine granulocyte colony-stimulating factor (GCSF) fusion proteins on mouse NFS-60 cells. Ln=(GGGGS)n, n=0 or 1 (SEQ ID NO: 164).

FIGS. 38 A-E depicts proliferative activities of BLV1H12 (Ab)-bovine granulocyte colony-stimulating factor (GCSF) fusion proteins on human granulocyte progenitors. Ln=(GGGGS)n, n=0 or 1 (SEQ ID NO: 164).

FIGS. 39 A-B depict pharmacokinetics of BLV1H12-beta-bovine granulocyte colony-stimulating factor (GCSF) fusion proteins in mice. Ln=(GGGGS)n, n=0 or 1 (SEQ ID NO: 164).

FIGS. 40 A-B depict proliferative activities of BLV1H12 (Ab)-bovine granulocyte colony-stimulating factor (GCSF) fusion proteins on mice neutrophils, blood stained and counted at the 10th day post-injection. N.C.: negative control. Ln=(GGGGS)n, n=0 or 1 (SEQ ID NO: 164).

FIG. 41A depicts an SDS-PAGE gel of BLV1H12 Fab-CDR3H-beta human growth hormone fusion protein.

FIG. 41B depicts an SDS-PAGE gel of BLV1H12 hFc(IgG)-CDR3H-beta human growth hormone fusion protein.

FIGS. 42 A-C depict proliferation of NB2 cells (A), Ba/F3 cells (B) and IM9 STATS (C) by BLV1H12 Fab-CDR3H-beta human growth hormone fusion protein and BLV1H12 hFc(IgG)-CDR3H-beta human growth hormone fusion protein.

FIG. 43A depicts an SDS-PAGE gel of BLV1H12-CDR3H-beta human leptin fusion protein.

FIG. 43B depicts leptin receptor activity with the addition of human Leptin or BLV1H12-CDR3H-beta human leptin fusion protein.

FIGS. 44 A-C depict SDS-PAGE gels of expressed and purified (A) BLV1H12-CDR3H-beta human relaxin clip fusion protein (SEQ ID NOs: 274 and 40), (B) BLV1H12-CDR3H-beta human relaxin clip fusion protein with engineered connector peptide (SEQ ID NOs: 276 and 40), (C) BLV1H12-CDR3H-beta human relaxin clip fusion protein with GGSIEGR linker (SEQ ID NO: 307) (SEQ ID NOs: 275 and 40).

FIG. 45 depicts (A) crystal structure of CXCR4 (green) in complex with a β-hairpin peptide antagonist CVX15 (yellow) (PDB code 3OE0), (B) crystal structure of bovine antibody BLV1H12 (PDB code 4K3D) depicts a disulfide cross-linked “knob” domain (red) grafted onto a solvent-exposed β-strand “stalk” (yellow), (C) a cartoon representation of the anti-CXCR4 antibody design. The loop region of the β-hairpin that resides outside the binding pocket of CXCR4 (blue) is removed and the anti-parallel β-strand region (green) is reconnected by selected β-turns to generate an inverted β-hairpin that is fused to the knob domain truncated bovine antibody scaffold. (D) A schematic representation of CVX15 and the engineered CDRs with β-turn promoting residues highlighted in bold. Potential interactions of bAb-AC1 with the CXCR4 ligand-binding pocket (blue box) are depicted on the basis of an analysis of the CXCR4-CVX15 complex. FIG. 45 discloses ‘Tyr-Arg-Lys-Cys-Arg-Gly-Gly-Arg-Arg-Trp-Cys-Tyr-Gln-Lys’ as SEQ ID NO: 231.

FIG. 46 depicts SDS-PAGE gel of purified BLV1H12-beta BCCX2 HC 1 (bAb-AC1) (SEQ ID NOs: 92 and 40) and BLV1H12-beta BCCX2 HC 4 (bAb-AC4) (SEQ ID NOs: 95 and 40) with or without the addition of reducing reagent DTT.

FIG. 47 depicts SDS-PAGE gel of purified trastuzumab-beta BCCX2 HC long (HLCX) (SEQ ID NOs: 96 and 40), trastuzumab-beta BCCX2 HC medium HMCX (SEQ ID NOs: 97 and 40), trastuzumab-beta BCCX2 HC short (HSCX) (SEQ ID NOs: 98 and 40), with or without reducing reagent DTT.

FIG. 48 depicts flow cytometry analysis of interactions between CXCR4 and engineered antibodies.

FIG. 49 depicts SDF-1 binds to CXCR4 with a Kd value of 14.2±1.2 nM determined by Tag-lite HTRF binding assay. Tag-lite labeled CXCR4 cells were incubated with increasing concentrations of the fluorescent SDF-1 for 3 h at room temperature. The signal was recorded by an EnVision multi-label plate reader (PerkinElmer) at 620 nm and 665 nm with 340 nm excitation.

FIG. 50A depicts specific binding between BLV1H12-beta BCCX2 HC 1, BLV1H12-beta BCCX2 HC 2, BLV1H12-beta BCCX2 HC3 and CXCR4 determined by a Tag-lite HTRF binding assay.

FIG. 51 depicts competition between BLV1H12-beta BCCX2 IgG fusions and 12G5 (66.6 nM) for binding to Jurkat cells in a dose dependent manner with an IC50 value of 39.3 nM.

FIG. 52 depicts flow cytometry analysis of bAb-AC4 (1 μg/mL) binding to (A) Jurkat cells and (B) CHO cells. Cells were first blocked with blocking buffer (PBS supplemented with 3% BSA) at 4° C. for 10 min and then incubated with various concentrations of antibodies in blocking buffer for 1 h. Cells were then washed with PBS and incubated with Alexa Fluor 647 conjugated goat anti-human IgG (0.5 μg/ml) in blocking buffer. After incubation, cells were washed and analyzed by flow cytometry. (C) bAb-AC4 (200 nM) completely blocks the binding between 12G5 (66 nM) and Jurkat cells. Jurkat cells were pre-incubated with 200 nM bAb-AC4 in blocking buffer at 4° C. for 30 min. Fluorescein conjugated 12G5 was added in blocking buffer to a final concentration of 10 μg/mL for an additional 30 min Cells were then washed with PBS and analyzed by a flow cytometer.

FIG. 53 depicts specific bindings between bAb-AC4 and CXCR4 receptor determined by Tag-lite HTRF binding assay with a Ki value of 0.9 nM. Tag-lite labeled CXCR4 cells were incubated with increasing concentrations of bAb-AC4 in the presence of 50 nM fluorescent ligand for 3 h at room temperature. The signal was recorded by an EnVision multi-label plate reader (PerkinElmer) at 620 nm and 665 nm with 340 nm excitation.

FIG. 54 depicts a cartoon representation of the migration assay. CXCR4 expressing cells migrate from top well of the transwell plate through a fibronectin layer to the bottom chamber filled with 10 ng/ml SDF-1.

FIGS. 55 A-C depict (A) 300 nM of bAb-AC4 efficiently blocks SDF-1 induced CXCR4 activation measured by intracellular calcium flux. (B) The antibodies bAb-AC1 and bAb-AC4 potently inhibit SDF-1 induced migration of Ramos cells in a dose dependent manner with EC50 values of 8.5 nM and 3.2 nM, respectively. At saturating concentration, they are able to completely inhibit SDF-1 induced chemotaxis. (C) Bottom chamber images of SDF-1 induced Ramos cell migration after treatment with no antibody, 12G5, or bAb-AC4 at 300 nM, respectively.

FIG. 56 depicts pretreatment with 300 nM antibodies significantly reduced (bAb-AC1) or completely blocked (bAb-AC4) SDF-1 induced calcium flux in Ramos cells. Fluo-4 loaded Ramos cells were washed with HBSS/HEPES twice and re-suspended in assay buffer (HBSS with 30 mM HEPES and 2.5 mM probenecid) at a density of 106 cells/ml. Antibodies were added and incubated with loaded cells for 1 h and calcium flux signals were recorded on a fluorescence laser-imaging plate reader immediately upon addition of SDF-1 at a final concentration of 50 nM.

FIG. 57 depicts flow cytometry analysis of Jurkat cell binding by trastuzumab-beta BCCX2 HC long (HLCX) (SEQ ID NOs: 96 and 40), trastuzumab-beta BCCX2 HC medium HMCX (SEQ ID NOs: 97 and 40), trastuzumab-beta BCCX2 HC short (HSCX) (SEQ ID NOs: 98 and 40), at 0.1 ug/ml.

FIG. 58 depicts inhibition of SDF-1 induced Ramos cell migration by HLCX, HMCX, HSCX.

FIG. 59 A depicts CXCR4 receptor binding with anti-CXCR4 bAb fusion proteins.

FIG. 59 B depicts CXCR4 receptor binding with anti-CXCR4 hAb fusion proteins.

FIG. 60 A depicts an SDS-PAGE gel of BLV1H12 Fab-CDRH3-beta-bovine trypsin inhibitor (BTI) fusion protein after purification.

FIG. 60 B depicts BLV1H12 Fab-CDRH3-beta-bovine trypsin inhibitor (BTI) fusion protein potently inhibits trypsin enzymatic activity.

FIG. 60 C depicts kinetic characterization of BTI fusion protein-trypsin interaction by biolayer interferometry experiment, blue curves represent experimental data and red curves represent the statistical fitting of curves.

FIG. 61 depicts an SDS-PAGE gel of human BVK antibody Fab-CDRH3-beta-elastase (BEI) fusion protein.

FIG. 62 depicts rationally designed anti-Neutrophil Elastase (NE) antibody fusion proteins BEI1 and BEI2 potently inhibit human neutrophil elastase activity with low nanomolar inhibition constants (18.7 nM and 18.2 nM respectively)

FIG. 63 depicts SDS-PAGE gels of human BVK CDRH3 beta neutrophil elastase inhibitor (HEI) fusion proteins.

FIG. 64 depicts HEI fusion proteins inhibit elastase proteolytic activity in vitro. HEI fusion proteins include, Human BVK-beta HEI HC 4 (SEQ ID NOs: 103 and 102), Human BVK-beta HEI HC 5 (SEQ ID NOs: 104 and 102), Human BVK-beta HEI HC 6 (SEQ ID NOs: 105 and 102), Human BVK-beta HEI HC 7 (SEQ ID NOs: 106 and 102), Human BVK-beta HEI HC 8 (SEQ ID NOs: 107 and 102), and Human BVK-beta HEI HC 9 (SEQ ID NOs: 108 and 102).

FIGS. 65 A-B depict selectivity of HEI fusion proteins. None of the HEI fusion proteins have any effect on the activity of trypsin (A) or chymotryspin (B).

FIGS. 66 A-B depict in vitro proliferative activities of trastuzumab fusion antibody containing a CDR3H beta sheet human erythropoietin (H3-beta/hEPO) extender fusion region and a CDR3L coiled coil human granulocyte colony stimulating factor (L3-coil/hGCSF) extender fusion region.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are immunoglobulin fusion proteins comprising a first antibody region; and a first extender fusion region comprising a first therapeutic agent attached to a first extender peptide, wherein the first extender peptide comprises a first beta strand secondary structure region containing a first beta strand secondary structure; and wherein the first extender fusion region does not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain of SEQ ID NO: 248. The first extender fusion region may not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain selected from a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, and an F18 ultralong complementary determining region 3 heavy chain. The first extender fusion region may not contain more than 7 consecutive amino acids that are based on or derived from a bovine ultralong CDR3.

Disclosed herein are immunoglobulin fusion proteins and methods of producing such immunoglobulin fusion proteins. The immunoglobulin fusion protein may comprise (a) a non-antibody region; and (b) a first antibody region comprising 6 or fewer consecutive amino acids of a complementarity determining region 3 (CDR3).

Further disclosed herein are immunoglobulin fusion proteins comprising (a) a non-antibody region; and (b) an antibody region, wherein the antibody region comprises an antibody sequence comprising 6 or fewer consecutive amino acids of a complementarity determining region (CDR). The CDR may be a CDR1. The CDR may be a CDR2. The CDR may be a CDR3.

Further disclosed herein are immunoglobulin fusion proteins comprising (a) a non-antibody region; and (b) an antibody region, wherein the non-antibody region replaces at least a portion of an antibody from which the antibody region is based on or derived from. The non-antibody region may replace at least a portion of a complementarity determining region. The non-antibody region may replace at least a portion of a variable domain. The non-antibody region may replace at least a portion of a constant domain. The non-antibody region may replace at least a portion of a heavy chain. The non-antibody region may replace at least a portion of a light chain.

Further disclosed herein are immunoglobulin fusion proteins comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises: (a) a first extender peptide, wherein the first extender peptide comprises an amino acid sequence comprising a beta strand secondary structure, and wherein the first extender peptide comprises (i) 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that is not based on or derived from an ultralong CDR3; and (b) a therapeutic agent.

Further disclosed herein are immunoglobulin fusion proteins comprising an antibody region attached to a non-antibody region, wherein the non-antibody region comprises: (a) a first extender peptide comprising at least one secondary structure; and (b) a therapeutic agent. The secondary structure may be a beta strand.

Further disclosed herein are dual fusion proteins comprising two or more therapeutic agents attached to an antibody or fragment thereof. At least one therapeutic agent may be inserted into the antibody or fragment thereof. Two or more therapeutic agents may be inserted into the antibody or fragment thereof. The therapeutic agents may replace at least a portion of the antibody or fragment thereof.

Exemplary immunoglobulin fusion proteins with an extender peptide comprising a beta strand are depicted in FIG. 1. As shown in FIG. 1, an antibody region (1110) comprising two immunoglobulin heavy chains (1115, 1120) and two immunoglobulin light chains (1125, 1130) is attached to an extender fusion region (1135) comprising two extender peptides (1140, 1145) and a therapeutic agent (1150) to produce immunoglobulin fusion proteins (1160, 1170, 1180). As shown in FIG. 1, the immunoglobulin fusion protein (1160) comprises an extender fusion region attached to one of the immunoglobulin heavy chains of the antibody region. As shown in FIG. 1, the immunoglobulin fusion protein (1170) comprises an extender fusion region attached to one of the immunoglobulin light chains of the antibody region. Also shown in FIG. 1, the immunoglobulin fusion protein (1180) comprises two extender fusion regions attached two immunoglobulin chains of the antibody region. The two extender peptides may form a beta sheet. The two extender peptides may form anti-parallel beta strands.

Additional exemplary immunoglobulin fusion proteins are depicted in FIG. 2. Formula IA of FIG. 2 depicts an immunoglobulin fusion protein comprising an antibody region (A1) attached to an extender fusion region comprising an extender peptide (E1) attached to a therapeutic agent (T1).

Formula IIA of FIG. 2 depicts an immunoglobulin fusion protein comprising an antibody region (A1) attached to an extender fusion region comprising two extender peptides (E1 and E2) attached to a therapeutic agent (T1). Formula IIIA of FIG. 2 depicts an immunoglobulin dual fusion protein comprising two antibody regions (A1 and A2) attached to each other. The immunoglobulin dual fusion protein may comprise (a) a first antibody region (A1) attached to a first extender fusion region comprising two extender peptides (E1 and E2) attached to a first therapeutic agent (T1); and (b) a second antibody region (A2) attached to a second extender fusion region comprising two extender peptides (E3 and E4) attached to a second therapeutic agent (T2).

Formula IVA of FIG. 2 depicts an immunoglobulin fusion protein comprising an antibody region (A1) attached to an extender fusion region comprising a linker (L1) attached to a therapeutic agent (T1), with the linker and therapeutic agent located between two extender peptides (E1 and E2).

Formula VA of FIG. 2 depicts an immunoglobulin fusion protein comprising an antibody region (A1) attached to an extender fusion region comprising a proteolytic cleavage site (P1) attached to a therapeutic agent (T1), with the proteolytic cleavage site and therapeutic agent located between two extender peptides (E1 and E2). Formula VB of FIG. 2 shows the clipped version of Formula VA, wherein the proteolytic cleavage site is cleaved by a protease, which results in release of one end of the therapeutic agent.

Formula VIA of FIG. 2 depicts an immunoglobulin fusion protein comprising an antibody region (A1) attached to an extender fusion region comprising a therapeutic agent (T1) attached to a linker (L1) and a proteolytic cleavage site (P1), which the therapeutic agent, linker and proteolytic cleavage site located between two extender peptides (E1 and E2). Formula VIB of FIG. 2 shows the clipped version of Formula VIA, wherein the proteolytic cleavage site is cleaved by a protease, which results in release of one end of the therapeutic agent.

Formula VIIA of FIG. 2 depicts an immunoglobulin dual fusion protein comprising two antibody regions (A1 and A2). The first antibody region (A1) is attached to a first extender fusion region comprising a therapeutic agent (T1) with two linkers (L1 and L2) on each end, with the therapeutic agent and linkers located between two extender peptides (E1 and E2). The second antibody region (A2) is attached to a second extender fusion region comprising a therapeutic agent (T2) attached to a proteolytic cleavage site (P1). The therapeutic agent and proteolytic cleavage site in the second extender fusion region are flanked by two linkers (L3 and L4). The therapeutic agent, proteolytic cleavage site and the two linkers of the second extender region are flanked by two extender peptides (E1 and E2).

Formula VIIIA of FIG. 2 depicts an immunoglobulin fusion protein comprising an antibody region (A1) attached to an extender fusion region comprising two extender peptides (E1 and E2), two linkers (L1 and L2), two proteolytic cleavage sites (P1 and P2) and a therapeutic agent (T1). Formula VIIIB of FIG. 2 shows the clipped version of Formula VIIIA, wherein the proteolytic cleavage sites located on the N- and C-termini of the therapeutic agent are cleaved by a protease, which results in release of the therapeutic agent from the immunoglobulin fusion protein.

Further disclosed herein are methods of treating a disease or condition in a subject in need thereof. The method may comprise administering to the subject an immunoglobulin fusion protein comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide; and (b) a therapeutic agent. The extender peptide may be based on or derived from an ultralong CDR3. The extender peptide may comprise 7 or fewer amino acids from an ultralong CDR3 sequence. Alternatively, or additionally, the extender peptide does not comprise an amino acid sequence based on or derived from an ultralong CDR3. The extender peptide may comprise one or more secondary structures. The one or more secondary structures may be a beta strand. The method may comprise administering to the subject an immunoglobulin fusion protein comprising a first antibody region; and a first extender fusion region comprising a first therapeutic agent attached to a first extender peptide, wherein the first extender peptide comprises a region containing a first beta strand secondary structure; and wherein the first extender fusion region does not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain of SEQ ID NO: 248. The first extender fusion region may not contain more than 7 consecutive amino acids from an ultralong complementary determining region 3 heavy chain selected from a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, and an F18 ultralong complementary determining region 3 heavy chain. The first extender fusion region may not contain more than 7 consecutive amino acids that are based on or derived from a bovine ultralong CDR3. The first extender fusion region may not comprise an amino acid sequence that is based on or derived from a bovine ultralong CDR3.

Further disclosed herein are methods of extending the half-life of a therapeutic agent. The method may comprise attaching an extender peptide to a therapeutic agent to produce an extender fusion peptide. The method may further comprise attaching an antibody region to the extender peptide, therapeutic agent, or extender fusion peptide. The method may comprise incorporating a therapeutic agent into an antibody region. The method may comprise incorporating an antibody region into a therapeutic agent. The method may comprise grafting a therapeutic agent into an antibody region. The method may comprise grafting an antibody region into a therapeutic agent.

Further disclosed herein are methods of extending the half-life of a therapeutic agent. The method may comprise attaching an antibody region to the therapeutic agent to produce an immunoglobulin fusion protein. The method may further comprise attaching one or more linkers or proteolytic cleavage sites to the immunoglobulin fusion protein. The one or more linkers may be attached to an N- and/or C-terminus of the therapeutic agent. The one or more proteolytic cleavage sites may be attached to an N- and/or C-terminus of the therapeutic agent. The one or more proteolytic cleavage sites may be inserted into the therapeutic agent.

Further disclosed herein are methods of improving the delivery of a therapeutic agent. The method may comprise attaching an extender peptide to a therapeutic agent. The method may further comprise attaching an antibody region to the extender peptide, therapeutic agent, or extender fusion peptide.

Further disclosed herein are methods of improving the delivery of a therapeutic agent. The method may comprise attaching an antibody region to a therapeutic agent to produce an immunoglobulin fusion protein. The method may further comprise attaching one or more linkers or proteolytic cleavage sites to the immunoglobulin fusion protein. The one or more linkers may be incorporated at an N- and/or C-terminus of the therapeutic agent. The one or more proteolytic cleavage sites may be incorporated at an N- and/or C-terminus of the therapeutic agent. The one or more proteolytic cleavage sites may be incorporated within the therapeutic agent. The one or more proteolytic cleavage sites may be incorporated within antibody region. The one or more proteolytic cleavage sites may be incorporated within the extender fusion region. The one or more proteolytic cleavage sites may be incorporated within the extender peptide. The one or more linkers may be attached to an N- and/or C-terminus of the therapeutic agent. The one or more proteolytic cleavage sites may be attached to an N- and/or C-terminus of the therapeutic agent. The one or more proteolytic cleavage sites may be inserted into the therapeutic agent.

Before the present methods and compositions are described, it is to be understood that this invention is not limited to a particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

The terms “homologous,” “homology,” or “percent homology” when used herein to describe to an amino acid sequence or a nucleic acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the peptide” includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Immunoglobulin Fusion Proteins

The immunoglobulin fusion proteins disclosed herein may comprise one or more antibody regions. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. The immunoglobulin domain may be from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody. The immunoglobulin domain may be from a recombinant antibody. The immunoglobulin domain may be from a mammalian antibody. The mammalian antibody may be a human antibody. The human antibody may be a human engineered or fully human antibody. The mammalian antibody may be a murine antibody. The mammalian antibody may be a non-human primate antibody. The mammalian antibody may be a bovine antibody.

The immunoglobulin domain may be a human antibody, wherein a portion of the human antibody is replaced with a non-human peptide. The immunoglobulin domain may be a human antibody, wherein a non-human peptide is added to the human antibody. The non-human peptide may be a portion of a non-human antibody or non-human antibody fragment. The portion of the non-human antibody or non-human antibody fragment may be a portion of a bovine antibody or a fragment thereof. The portion of the non-human antibody or non-human antibody fragment may be a CDR. The CDR may be a CDR3. The CDR may be a CDR2. The CDR may be a CDR1. The CDR may be an ultralong CDR. The CDR may be an ultralong CDR3. The CDR may be a bovine ultralong CDR. The CDR may be a bovine ultralong CDR3. The human antibody may have a sequence that is about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous to a fully human antibody. The human antibody may have a sequence that is less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14% or less than about 15% homologous to a non-human peptide, a non-human protein or a non-human antibody. The portion of the human antibody that may be replaced may be a CDR or a portion thereof. The portion of the human antibody that may be replaced may be at least a portion of a variable fragment of the human antibody. The portion of the human antibody replaced may be at least a portion of a Fab of the human antibody. The portion of the human antibody replaced may be a portion a light chain or heavy chain of the human antibody. The non-human peptide may be less than about 4, less than about 5, less than about 6, less than about 7, less than about 8, less than about 9, less than about 10, less than about 11, less than about 12, less than about 13, less than about 14, less than about 15, less than about 16, less than about 17, less than about 18, less than about 19, less than about 20, less than about 22, less than about 23, less than about 24, less than about 25, less than about 26, less than about 27, less than about 28, less than about 29 or less than about 30 amino acids. The non-human peptide may be less than about 35 amino acids. The non-human peptide may be less than about 8 amino acids. The non-human peptide may be less than about 7 amino acids. The non-human peptide may be less than about 6 amino acids.

The immunoglobulin domain may be from a humanized antibody. The humanized antibody may comprise a portion that is less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9% or less than about 10% homologous to a non-human antibody. The humanized antibody may be at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98%, or at least about 99% homologous to a human antibody. By non-limiting example, the non-human antibody may be a non-human primate antibody. By non-limiting example, the non-human antibody may be a bovine antibody. The non-human antibody may be a murine antibody. The humanized antibody may be a monoclonal antibody.

The immunoglobulin fusion protein may comprise an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The immunoglobulin fusion protein may comprise an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The immunoglobulin fusion protein may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The immunoglobulin fusion protein may comprise an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The immunoglobulin fusion protein may comprise an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304.

The immunoglobulin fusion protein may comprise an amino acid sequence comprising 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids based on or derived from any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The immunoglobulin fusion protein may comprise an amino acid sequence comprising 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 450, 500 or more amino acids based on or derived from any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The immunoglobulin fusion protein may comprise an amino acid sequence comprising 10 or more amino acids based on or derived from any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The immunoglobulin fusion protein may comprise an amino acid sequence comprising 50 or more amino acids based on or derived from any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The immunoglobulin fusion protein may comprise an amino acid sequence comprising 100 or more amino acids based on or derived from any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The immunoglobulin fusion protein may comprise an amino acid sequence comprising 200 or more amino acids based on or derived from any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The amino acids may be consecutive. Alternatively, or additionally, the amino acids are nonconsecutive.

The immunoglobulin fusion protein may be encoded by a nucleotide sequence that is based on or derived from any one of SEQ ID NOs: 41-75, 279-296, 299, 301, and 303. The immunoglobulin fusion protein may be encoded by a nucleotide sequence that is at least about 50% homologous to any one of SEQ ID NOs: 41-75, 279-296, 299, 301, and 303. The immunoglobulin fusion protein may be encoded by a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to any one of SEQ ID NOs: 41-75 279-296, 299, 301, and 303. The immunoglobulin fusion protein may be encoded by a nucleotide sequence that is at least about 70% homologous to any one of SEQ ID NOs: 41-75 279-296, 299, 301, and 303. The immunoglobulin fusion protein may be encoded by a nucleotide sequence that is at least about 80% homologous to any one of SEQ ID NOs: 41-75 279-296, 299, 301, and 303.

The immunoglobulin fusion protein may be encoded by a nucleotide sequence comprising 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides based on or derived from any one of SEQ ID NOs: 41-75 279-296, 299, 301, and 303. The immunoglobulin fusion protein may be encoded by a nucleotide sequence comprising 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 450, 500 or more nucleotides based on or derived from any one of SEQ ID NOs: 41-75 279-296, 299, 301, and 303. The immunoglobulin fusion protein may be encoded by a nucleotide sequence comprising 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more nucleotides based on or derived from any one of SEQ ID NOs: 41-75 279-296, 299, 301, and 303. The immunoglobulin fusion protein may be encoded by a nucleotide sequence comprising 1100, 1200, 1300, 1400, 1500 or more nucleotides based on or derived from any one of SEQ ID NOs: 41-75 279-296, 299, 301, and 303. The immunoglobulin fusion protein may be encoded by a nucleotide sequence comprising 100 or more nucleotides based on or derived from any one of SEQ ID NOs: 41-75 279-296, 299, 301, and 303. The immunoglobulin fusion protein may be encoded by a nucleotide sequence comprising 500 or more nucleotides based on or derived from any one of SEQ ID NOs: 41-75 279-296, 299, 301, and 303. The immunoglobulin fusion protein may be encoded by a nucleotide sequence comprising 1000 or more nucleotides based on or derived from any one of SEQ ID NOs: 41-75 279-296, 299, 301, and 303. The immunoglobulin fusion protein may be encoded by a nucleotide sequence comprising 1300 or more nucleotides based on or derived from any one of SEQ ID NOs: 41-75 279-296, 299, 301, and 303. The nucleotides may be consecutive. Alternatively, or additionally, the nucleotides are nonconsecutive.

The immunoglobulin fusion protein may further comprise one or more immunoglobulin light chains. The immunoglobulin fusion protein may comprise at least two immunoglobulin light chains. The immunoglobulin light chain may comprise an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The immunoglobulin light chain may be encoded by a nucleotide sequence based on or derived from any one of SEQ ID NOs: 257-259. The immunoglobulin light chain may comprise an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The immunoglobulin light chain may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The immunoglobulin light chain may comprise an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The immunoglobulin light chain may comprise an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250.

The immunoglobulin light chain may comprise an amino acid sequence comprising 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids based on or derived from any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The immunoglobulin light chain may comprise an amino acid sequence comprising 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 450, 500 or more amino acids based on or derived from any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The immunoglobulin light chain may comprise an amino acid sequence comprising 10 or more amino acids based on or derived from any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The immunoglobulin light chain may comprise an amino acid sequence comprising 50 or more amino acids based on or derived from any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The immunoglobulin light chain may comprise an amino acid sequence comprising 100 or more amino acids based on or derived from any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The immunoglobulin light chain may comprise an amino acid sequence comprising 200 or more amino acids based on or derived from any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The amino acids may be consecutive. Alternatively, or additionally, the amino acids are nonconsecutive.

The immunoglobulin light chain may be encoded by a nucleotide sequence that is based on or derived from any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence that is at least about 50% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence that is at least about 70% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence that is at least about 80% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259.

The immunoglobulin light chain may be encoded by a nucleotide sequence comprising 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides based on or derived from any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence comprising 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 450, 500 or more nucleotides based on or derived from any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence comprising 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more nucleotides based on or derived from any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence comprising 1100, 1200, 1300, 1400, 1500 or more nucleotides based on or derived from any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence comprising 100 or more nucleotides based on or derived from any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence comprising 500 or more nucleotides based on or derived from any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence comprising 1000 or more nucleotides based on or derived from any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence comprising 1300 or more nucleotides based on or derived from any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The nucleotides may be consecutive. Alternatively, or additionally, the nucleotides are nonconsecutive.

The immunoglobulin fusion protein may further comprise one or more immunoglobulin heavy chains. The immunoglobulin fusion protein may comprise at least two immunoglobulin heavy chains. The immunoglobulin heavy chain may comprise an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 24-27, 29-33, 36-39 and 251-253. The immunoglobulin heavy chain may comprise an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 24-27, 29-33, 36-39 and 251-253. The immunoglobulin heavy chain may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to any one of SEQ ID NOs: 24-27, 29-33, 36-39 and 251-253. The immunoglobulin heavy chain may comprise an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 24-27, 29-33, 36-39 and 251-253. The immunoglobulin heavy chain may comprise an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 24-27, 29-33, 36-39 and 251-253. The immunoglobulin heavy chain may not comprise an amino acid sequence selected from SEQ ID NOs: 248-250. The immunoglobulin heavy chain may not comprise an amino acid sequence based on or derived from SEQ ID NOs: 248-250. The immunoglobulin heavy chain may not comprise more than about 6, about 7, about 8, about 15, about 20 or about 35 consecutive amino acids from SEQ ID NOs: 248-250.

The immunoglobulin heavy chain may comprise an amino acid sequence comprising 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids based on or derived from any one of SEQ ID NOs: 24-27, 29-33, 36-39 and 251-253. The immunoglobulin heavy chain may comprise an amino acid sequence comprising 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 450, 500 or more amino acids based on or derived from any one of SEQ ID NOs: 24-27, 29-33, 36-39 and 251-253. The immunoglobulin heavy chain may comprise an amino acid sequence comprising 10 or more amino acids based on or derived from any one of SEQ ID NOs: 24-27, 29-33, 36-39 and 251-253. The immunoglobulin heavy chain may comprise an amino acid sequence comprising 50 or more amino acids based on or derived from any one of SEQ ID NOs: 24-27, 29-33, 36-39 and 251-253. The immunoglobulin heavy chain may comprise an amino acid sequence comprising 100 or more amino acids based on or derived from any one of SEQ ID NOs: 24-27, 29-33, 36-39 and 251-253. The immunoglobulin heavy chain may comprise an amino acid sequence comprising 200 or more amino acids based on or derived from any one of SEQ ID NOs: 24-27, 29-33, 36-39 and 251-253. The amino acids may be consecutive. Alternatively, or additionally, the amino acids are nonconsecutive.

The immunoglobulin heavy chain may be encoded by a nucleotide sequence that is based on or derived from any one of SEQ ID NOs: 5-13, 16-19 and 254-256. The immunoglobulin heavy chain may be encoded by a nucleotide sequence that is at least about 50% homologous to any one of SEQ ID NOs: 5-13, 16-19 and 254-256. The immunoglobulin heavy chain may be encoded by a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to any one of SEQ ID NOs: 5-13, 16-19 and 254-256. The immunoglobulin heavy chain may be encoded by a nucleotide sequence that is at least about 70% homologous to any one of SEQ ID NOs: 5-13, 16-19 and 254-256. The immunoglobulin heavy chain may be encoded by a nucleotide sequence that is at least about 80% homologous to any one of SEQ ID NOs: 5-13, 16-19 and 254-256. The immunoglobulin heavy chain may not be encoded by a nucleotide sequence selected from SEQ ID NOs: 254-256. The immunoglobulin heavy chain may not be encoded by a nucleotide sequence based on or derived from SEQ ID NOs: 254-256.

The immunoglobulin heavy chain may be encoded by a nucleotide sequence comprising 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides based on or derived from any one of SEQ ID NOs: 5-13, 16-19 and 254-256. The immunoglobulin heavy chain may be encoded by a nucleotide sequence comprising 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 450, 500 or more nucleotides based on or derived from any one of SEQ ID NOs: 5-13, 16-19 and 254-256. The immunoglobulin heavy chain may be encoded by a nucleotide sequence comprising 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more nucleotides based on or derived from any one of SEQ ID NOs: 5-13, 16-19 and 254-256. The immunoglobulin heavy chain may be encoded by a nucleotide sequence comprising 1100, 1200, 1300, 1400, 1500 or more nucleotides based on or derived from any one of SEQ ID NOs: 5-13, 16-19 and 254-256. The immunoglobulin heavy chain may be encoded by a nucleotide sequence comprising 100 or more nucleotides based on or derived from any one of SEQ ID NOs: 5-13, 16-19 and 254-256. The immunoglobulin heavy chain may be encoded by a nucleotide sequence comprising 500 or more nucleotides based on or derived from any one of SEQ ID NOs: 5-13, 16-19 and 254-256. The immunoglobulin heavy chain may be encoded by a nucleotide sequence comprising 1000 or more nucleotides based on or derived from any one of SEQ ID NOs: 5-13, 16-19 and 254-256. The immunoglobulin heavy chain may be encoded by a nucleotide sequence comprising 1300 or more nucleotides based on or derived from any one of SEQ ID NOs: 4-6 and 8-11. The nucleotides may be consecutive. Alternatively, or additionally, the nucleotides are nonconsecutive.

The immunoglobulin fusion protein may comprise (a) a first immunoglobulin fusion heavy chain comprising an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 24-27, 29-33 and 36-39; and (b) a first immunoglobulin light chain comprising an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 278. The immunoglobulin fusion protein may comprise (a) a first immunoglobulin fusion heavy chain comprising an amino acid sequence that is at least about 50% identical to SEQ ID NOs: 24-27, 29-33 and 36-39; and (b) a first immunoglobulin light chain comprising an amino acid sequence that is at least about 50% identical to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 278. The first immunoglobulin fusion heavy chain may comprise an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, or 97% identical to any one of SEQ ID NOs24-27, 29-33 and 36-39. The first immunoglobulin light chain comprising an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, or 97% identical to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 278.

The immunoglobulin fusion protein may comprise (a) a first immunoglobulin fusion heavy chain encoded by a nucleotide sequence of any one of SEQ ID NOs: 5-13 and 16-19; and (b) a first immunoglobulin light chain encoded by a nucleotide sequence of any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297. The immunoglobulin fusion protein may comprise (a) a first immunoglobulin fusion heavy chain encoded by a nucleotide sequence that is at least 50% or more homologous to a nucleotide sequence of any one of SEQ ID NOs: 5-13 and 16-19; and (b) a first immunoglobulin light chain encoded by a nucleotide sequence that is at least 50% or more homologous to a nucleotide sequence of any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297. The first immunoglobulin fusion heavy chain encoded by a nucleotide sequence that is at least 60%, 70%, 75%, 80%, 90%, 95%, or 97% or more homologous to a nucleotide sequence of any one of SEQ ID NOs: 5-13 and 16-19. The first immunoglobulin light chain encoded by a nucleotide sequence that is at least 60%, 70%, 75%, 80%, 90%, 95%, or 97% or more homologous to a nucleotide sequence of any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297.

The immunoglobulin fusion protein may comprise (a) a first immunoglobulin heavy chain comprising an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 24-27, 29-33, and 36-39; and (b) a first immunoglobulin fusion light chain comprising an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 278. The immunoglobulin fusion protein may comprise (a) a first immunoglobulin heavy chain comprising an amino acid sequence that is at least about 50% identical to any one of SEQ ID NOs: 24-27, 29-33, and 36-39; and (b) a first immunoglobulin fusion light chain comprising an amino acid sequence that is at least about 50% identical to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 278. The first immunoglobulin heavy chain may comprise an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, or 97% identical to any one of SEQ ID NOs: 24-27, 29-33, and 36-39. The first immunoglobulin fusion light chain comprising an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, or 97% identical to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 278.

The immunoglobulin fusion protein may comprise (a) a first immunoglobulin heavy chain encoded by a nucleotide sequence of any one of SEQ ID NOs: 5-13 and 16-19; and (b) a first immunoglobulin fusion light chain encoded by a nucleotide sequence of any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin fusion protein may comprise (a) a first immunoglobulin heavy chain encoded by a nucleotide sequence that is at least 50% or more homologous to a nucleotide sequence of any one of SEQ ID NOs: 5-13 and 16-19; and (b) a first immunoglobulin fusion light chain encoded by a nucleotide sequence that is at least 50% or more homologous to a nucleotide sequence of any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The first immunoglobulin heavy chain encoded by a nucleotide sequence that is at least 60%, 70%, 75%, 80%, 90%, 95%, or 97% or more homologous to a nucleotide sequence of any one of SEQ ID NOs: 5-13 and 16-19. The first immunoglobulin fusion light chain encoded by a nucleotide sequence that is at least 60%, 70%, 75%, 80%, 90%, 95%, or 97% or more homologous to a nucleotide sequence of any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259.

The immunoglobulin fusion protein may comprise (a) a first immunoglobulin heavy chain comprising an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 260-277; and (b) a first immunoglobulin fusion light chain comprising an amino acid sequence that is based on or derived from SEQ ID NO: 278. The immunoglobulin fusion protein may comprise (a) a first immunoglobulin heavy chain comprising an amino acid sequence that is at least about 50% identical to any one of SEQ ID NOs: 260-277; and (b) a first immunoglobulin fusion light chain comprising an amino acid sequence that is at least about 50% identical to SEQ ID NO: 278. The first immunoglobulin heavy chain may comprise an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, or 97% identical to any one of SEQ ID NOs: 260-277. The first immunoglobulin fusion light chain comprising an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, or 97% identical to SEQ ID NO: 278.

The immunoglobulin fusion protein may comprise (a) a first immunoglobulin heavy chain encoded by a nucleotide sequence of any one of SEQ ID NOs: 279-296; and (b) a first immunoglobulin fusion light chain encoded by a nucleotide sequence of SEQ ID NOs: 297. The immunoglobulin fusion protein may comprise (a) a first immunoglobulin heavy chain encoded by a nucleotide sequence that is at least 50% or more homologous to a nucleotide sequence of any one of SEQ ID NOs: 279-296; and (b) a first immunoglobulin fusion light chain encoded by a nucleotide sequence that is at least 50% or more homologous to a nucleotide sequence of SEQ ID NO: 297. The first immunoglobulin heavy chain may be encoded by a nucleotide sequence that is at least 60%, 70%, 75%, 80%, 90%, 95%, or 97% or more homologous to a nucleotide sequence of any one of SEQ ID NOs: 279-296. The first immunoglobulin fusion light chain may be encoded by a nucleotide sequence that is at least 60%, 70%, 75%, 80%, 90%, 95%, or 97% or more homologous to a nucleotide sequence of SEQ ID NO: 297.

The immunoglobulin fusion proteins disclosed herein may comprise a therapeutic agent, wherein the therapeutic agent is a functional peptide. The immunoglobulin fusion protein may comprise a functional peptide grafted into an antibody scaffold. The functional peptide may be a linear peptide. The functional peptide may be a modified cyclic peptide. The functional peptide may comprise a peptide modified to comprise a β-hairpin structure. The β-hairpin structure may be locked into a β-hairpin conformation by one or more bonds between two or more amino acid residues of the β-hairpin structure. The N terminus and/or the C terminus of the functional peptide may be grafted to the extender fusion region of the immunoglobulin fusion protein. The N terminus of the functional peptide may be grafted to a first extender peptide of the extender fusion region and the C terminus of the functional peptide may be grafted to a second extender peptide of the extender fusion region. The functional peptide may comprise a peptide modified to comprise a conformationally constrained peptide. A conformationally constrained peptide may have a greatly improved binding affinity and/or specificity to a target relative to an endogenous or naturally-occurring binding partner of the target. An endogenous or naturally-occurring binding partner of the target may be a ligand or substrate of the target. By non-limiting example, the conformationally constrained peptide may be a peptide comprising a β-hairpin structure. The conformationally constrained peptide may comprise a region that binds to a binding site of a target. The target may be a receptor. By non-limiting example, the receptor may be a G protein coupled receptor (GPCR). By non-limiting example, the GPCR may be CXCR4. The target may be an enzyme. By non-limiting example, the enzyme may be a neutrophil elastase. The binding site of the target may be a deep pocket of a ligand binding domain or substrate binding domain. The functional peptide or portion thereof may bind the deep pocket of a ligand binding domain or substrate binding domain such that it blocks a target ligand and/or substrate from binding. The functional peptide or portion thereof may bind the deep pocket of a ligand binding domain or substrate binding domain such that it partially blocks the target ligand and/or substrate from binding. The functional peptide or portion thereof may bind the deep pocket of a ligand binding domain or substrate binding domain such that it completely blocks the target ligand or substrate from binding. The functional peptide or portion thereof may bind the surface of the ligand binding domain or substrate binding domain. The functional peptide may be an agonist. The functional peptide may be an antagonist. The functional peptide may be an inhibitor. The functional peptide may be a ligand. The functional peptide may be a substrate.

Dual Fusions

Disclosed herein are immunoglobulin dual fusion proteins comprising one or more antibody regions attached to a first extender fusion region and a second extender fusion region, wherein the first extender fusion region comprises a first therapeutic agent and a first extender peptide comprising a beta strand and the second extender fusion region comprises a second therapeutic agent and a second extender peptide selected from a beta strand and/or a linker peptide. The first extender fusion region may not comprise more than 7 consecutive amino acid from SEQ ID NO. 248.

Further disclosed herein are immunoglobulin dual fusion proteins comprising one or more antibody regions attached to a first extender fusion region and a second extender fusion region, wherein the first extender fusion region comprises a first therapeutic agent and a first extender peptide comprising a beta strand and the second extender fusion region comprises a second therapeutic agent and a second extender peptide selected from an alpha helix and/or a linker peptide. The first extender fusion region may not comprise more than 7 consecutive amino acid from SEQ ID NO. 248.

Disclosed herein are immunoglobulin dual fusion proteins comprising one or more antibody regions attached to a first extender fusion region and a second extender fusion region, wherein the first extender fusion region comprises a first therapeutic agent and a first extender peptide comprising a beta strand and the second extender fusion region comprises a second therapeutic agent and no extender peptide. The first extender fusion region may not comprise more than 7 consecutive amino acid from SEQ ID NO. 248.

Further disclosed herein are immunoglobulin dual fusion proteins comprising (a) an antibody region attached to a non-antibody region, wherein the non-antibody region comprises (i) an extender peptide, wherein the first extender peptide comprises an amino acid sequence comprising a beta strand secondary structure and wherein the first extender peptide does not comprise an ultralong CDR3; and (ii) a first therapeutic agent; and (b) a second therapeutic agent. Attachment of the antibody region to the non-antibody region may comprise insertion of the non-antibody region into the antibody region. The first therapeutic agent and the second therapeutic agent may be the same. The first therapeutic agent and the second therapeutic agent may be different.

Alternatively, the immunoglobulin dual fusion protein comprises (a) an antibody region attached to a non-antibody region, wherein the non-antibody region comprises (i) a first extender peptide, wherein the first extender peptide comprises an amino acid sequence comprising a beta strand secondary structure and wherein the first extender peptide comprises 7 or fewer amino acids based on or derived from an ultralong CDR3; and (ii) a first therapeutic agent; and (b) a second therapeutic agent. Attachment of the antibody region to the non-antibody region may comprise insertion of the non-antibody region into the antibody region. The first therapeutic agent and the second therapeutic agent may be the same. The first therapeutic agent and the second therapeutic agent may be different.

The immunoglobulin dual fusion protein may comprise an antibody region attached to (a) a first extender fusion region comprising a first extender peptide, wherein the first extender peptide comprises (i) an amino acid sequence comprising a beta strand secondary structure and wherein the first extender peptide comprises 7 or fewer amino acids based on or derived from an ultralong CDR3; and (ii) a first therapeutic agent; and (b) a second extender fusion region comprising (i) a second extender peptide, wherein the second extender peptide is selected from (1) a beta strand, an alpha helix and a linker or (2) no extender peptide; and (ii) a second therapeutic agent. The immunoglobulin dual fusion protein may further comprise one or more additional extender peptides. The first extender fusion region may comprise one or more additional extender peptides. The one or more additional extender peptides may comprise a beta strand secondary structure. The second extender fusion region may comprise one or more additional extender peptides, wherein the one or more additional extender peptides are selected from a beta strand, an alpha helix and a linker peptide. The second extender fusion region may not comprise an extender peptide.

The dual fusion antibody may comprise (a) a first immunoglobulin fusion protein comprising a first antibody region attached to a first extender fusion region, wherein the first extender fusion region comprises (i) a first extender peptide, wherein the first extender peptide comprises an amino acid sequence comprising a beta strand secondary structure and wherein the extender peptide comprises 7 or fewer amino acids based on or derived from an ultralong CDR3; and (ii) a first therapeutic agent; and (b) a second immunoglobulin fusion protein comprising a second antibody region attached to a second extender fusion region, wherein the second extender fusion region comprises (i) a second extender peptide and (ii) a second therapeutic agent. The second extender peptide may be a linker peptide. The linker may be flexible. The linker may be rigid.

The dual fusion antibody may comprise (a) a first immunoglobulin fusion protein comprising a first antibody region attached to a first extender fusion region, wherein the first extender fusion region comprises (i) a first extender peptide, wherein the first extender peptide comprises an amino acid sequence comprising a beta strand secondary structure and wherein the extender peptide comprises 7 or fewer amino acids based on or derived from an ultralong CDR3; and (ii) a first therapeutic agent; and (b) a second immunoglobulin fusion protein comprising a second antibody region attached to a second extender fusion region, wherein the second extender fusion region consists essentially of a second therapeutic agent.

The dual fusion antibody may comprise (a) a first immunoglobulin fusion protein comprising a first antibody region attached to a first extender fusion region, wherein the first extender fusion region comprises (i) a first extender peptide, wherein the first extender peptide comprises an amino acid sequence comprising a beta strand secondary structure and wherein the first extender peptide does not comprise an ultralong CDR3; and (ii) a first therapeutic agent; and (b) a second immunoglobulin fusion protein comprising a second antibody region attached to a second extender fusion region, wherein the second extender fusion region comprises (i) a second extender peptide comprising at least one secondary structure and (ii) a second therapeutic agent.

The dual fusion antibody may comprise (a) a first immunoglobulin fusion protein comprising a first antibody region attached to a first extender fusion region, wherein the first extender fusion region comprises (i) a first extender peptide, wherein the first extender peptide comprises an amino acid sequence comprising a beta strand secondary structure and wherein the extender peptide comprises 7 or fewer amino acids based on or derived from an ultralong CDR3; and (ii) a first therapeutic agent; and (b) a second immunoglobulin fusion protein comprising a second antibody region attached to a second extender fusion region, wherein the second extender fusion region comprises (i) a second extender peptide comprising at least one secondary structure and (ii) a second therapeutic agent.

The dual fusion antibody may comprise (a) a first immunoglobulin fusion protein comprising a first antibody region attached to a first extender fusion region, wherein the first extender fusion region comprises (i) a first extender peptide, wherein the first extender peptide comprises an amino acid sequence comprising a beta strand secondary structure and wherein the extender peptide comprises 7 or fewer amino acids based on or derived from an ultralong CDR3; and (ii) a first therapeutic agent; and (b) a second immunoglobulin fusion protein comprising a second antibody region attached to a second extender fusion region, wherein the second extender fusion region comprises (i) a second extender peptide comprises at least one alpha helix and (ii) a second therapeutic agent. The second extender fusion region may comprise one or more additional extender peptides, wherein the one or more additional extender peptides include an alpha helix. The second extender fusion region may comprise a coiled coil secondary structure.

The first therapeutic agent and the second therapeutic agent may be the same. The first therapeutic agent and the second therapeutic agent may be different. The immunoglobulin dual fusion protein may further comprise one or more additional therapeutic agents. The two or more therapeutic agents may be the same. Alternatively, or additionally, the two or more therapeutic agents may be different.

The first antibody region and the second antibody region may be the same. For example, the first antibody region and the second antibody region comprise an immunoglobulin heavy chain. Alternatively, the first antibody region and the second antibody region may comprise an immunoglobulin light chain. The first antibody region and the second antibody region may be different. For example, the first antibody region comprises an immunoglobulin heavy chain and the second antibody region comprises an immunoglobulin light chain or vice versa. The immunoglobulin dual fusion protein may further comprise one or more additional antibody regions. The two or more antibody regions may be the same. Alternatively, or additionally, the two or more antibody regions may be different.

The immunoglobulin dual fusion protein may further comprise one or more additional extender peptides. The two or more extender peptides may be the same. Alternatively, or additionally, the two or more extender peptides are different.

The immunoglobulin dual fusion protein may further comprise one or more additional antibody regions. The two or more antibody regions may be the same. Alternatively, or additionally, the two or more antibody regions are different.

The immunoglobulin dual fusion protein may further comprise one or more linkers. The immunoglobulin dual fusion protein may further comprise two or more linkers. The two or more linkers may be the same. Alternatively, or additionally, the two or more linkers are different.

The immunoglobulin dual fusion protein may further comprise one or more proteolytic cleavage sites. The immunoglobulin dual fusion protein may further comprise two or more proteolytic cleavage sites. The two or more proteolytic cleavage sites may be the same. Alternatively, or additionally, the two or more proteolytic cleavage sites are different.

Antibody Region

The immunoglobulin fusion proteins disclosed herein comprise one or more antibody regions. The antibody region may comprise an antibody or fragment thereof. The antibody region may comprise at least a portion of an immunoglobulin heavy chain, immunoglobulin light chain, or a combination thereof. The antibody region may comprise two or more immunoglobulin chains or portions thereof. The antibody region may comprise three or more immunoglobulin chains or portions thereof. The antibody region may comprise four or more immunoglobulin chains or portions thereof. The antibody region may comprise two immunoglobulin heavy chains and two immunoglobulin light chains.

The immunoglobulin fusion proteins disclosed herein may comprise one or more immunoglobulin regions. The immunoglobulin region may comprise an immunoglobulin or a fragment thereof. The immunoglobulin region may comprise at least a portion of an immunoglobulin heavy chain, immunoglobulin light chain, or a combination thereof. The immunoglobulin region may comprise two or more immunoglobulin chains or portions thereof. The immunoglobulin region may comprise three or more immunoglobulin chains or portions thereof. The immunoglobulin region may comprise four or more immunoglobulin chains or portions thereof. The immunoglobulin region may comprise five or more immunoglobulin chains or portions thereof. The immunoglobulin region may comprise two immunoglobulin heavy chains and two immunoglobulin light chains.

The immunoglobulin region may comprise an entire immunoglobulin molecule or any polypeptide comprising fragment of an immunoglobulin including, but not limited to, heavy chain, light chain, variable domain, constant domain, complementarity determining region (CDR), framework region, fragment antigen binding (Fab) region, Fab′, F(ab′)2, F(ab′)3, Fab′, fragment crystallizable (Fc) region, single chain variable fragment (scFV), di-scFv, single domain immunoglobulin, trifunctional immunoglobulin, chemically linked F(ab′)2, and any combination thereof. The immunoglobulin region may comprise one or more mutations. The Fc region may be a mutated Fc region. The mutated Fc region may comprise one or more mutations that eliminate an antibody-dependent cellular cytotoxicity (ADCC) effect of an Fc region. The mutated Fc region may comprise one or more mutations. The mutated Fc region may comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 mutations. The mutated Fc region may comprise about 6 mutations. The mutated Fc region may comprise about 1 deletion. The mutated Fc region may comprise about 6 mutations and 1 deletion.

In some embodiments, an immunoglobulin heavy chain may comprise an entire heavy chain or a portion of a heavy chain. For example, a variable domain or region thereof derived from a heavy chain may be referred to as a heavy chain or a region of a heavy chain. In some embodiments, an immunoglobulin light chain may comprise an entire light chain or a portion of a light chain. For example, a variable domain or region thereof derived from a light chain may be referred to as a light chain or a region of a light chain. A single domain immunoglobulin includes, but is not limited to, a single monomeric variable immunoglobulin domain, for example, a shark variable new antigen receptor immunoglobulin fragment (VNAR).

The immunoglobulin may be derived from any type known to one of skill in the art including, but not limited to, IgA, IgD, IgE, IgG, IgM, IgY, IgW. The immunoglobulin region may comprise one or more functional units, including but not limited to, 1, 2, 3, 4, and 5 functional units. Functional units may include, but are not limited to, non-immunoglobulin regions, heavy chain, light chain, variable domain, constant domain, complementarity determining region (CDR), framework region, fragment antigen binding (Fab) region, Fab′, F(ab′)2, F(ab′)3, Fab′, fragment crystallizable (Fc) region, single chain variable fragment (scFV), di-scFv, single domain immunoglobulin, trifunctional immunoglobulin, chemically linked F(ab′)2, and any combination or fragments thereof. Non-immunoglobulin regions include, but are not limited to, carbohydrates, lipids, small molecules and therapeutic peptides. The immunoglobulin region may comprise one or more units connected by one or more disulfide bonds. The immunoglobulin region may comprise one or more units connected by a peptide linker, for example, an scFv immunoglobulin. The immunoglobulin may be a recombinant immunoglobulin including immunoglobulins with amino acid mutations, substitutions, and/or deletions. The immunoglobulin may be a recombinant immunoglobulin comprising chemical modifications. The immunoglobulin may comprise a whole or part of an immunoglobulin-drug conjugate.

The antibody region may comprise at least a portion of an immunoglobulin heavy chain. The antibody region may comprise one or more immunoglobulin heavy chains or a portion thereof. The antibody region may comprise two or more immunoglobulin heavy chains or a portion thereof. The antibody region may comprise an amino acid sequence that is at least about 50% homologous to an immunoglobulin heavy chain. The antibody region may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more homologous to an immunoglobulin heavy chain. The antibody region may comprise an amino acid sequence that is at least about 70% homologous to an immunoglobulin heavy chain. The antibody region may comprise an amino acid sequence that is at least about 80% homologous to an immunoglobulin heavy chain. The antibody region may comprise an amino acid sequence that is at least about 90% homologous to an immunoglobulin heavy chain. The immunoglobulin heavy chain may comprise an amino acid sequence selected from any one of SEQ ID NOs: 24-27, 29-33 and 36-39.

The antibody region may comprise an amino acid sequence comprising 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or more amino acids of an immunoglobulin heavy chain. The antibody region may comprise an amino acid sequence comprising 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or more amino acids of an immunoglobulin heavy chain. The amino acids may be consecutive. Alternatively, or additionally, the amino acids are non-consecutive.

The immunoglobulin heavy chain may be encoded by a nucleotide sequence based on or derived from any one of SEQ ID NOs: 5-13 and 16-19. The immunoglobulin heavy chain may be encoded by a nucleotide sequence that is at least about 50% homologous to any one of SEQ ID NOs: 5-13 and 16-19. The immunoglobulin heavy chain may be encoded by a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more homologous to any one of SEQ ID NOs: 5-13 and 16-19. The immunoglobulin heavy chain may be encoded by a nucleotide sequence that is at least about 75% homologous to any one of SEQ ID NOs: 5-13 and 16-19. The immunoglobulin heavy chain may be encoded by a nucleotide sequence that is at least about 85% homologous to any one of SEQ ID NOs: 5-13 and 16-19.

The antibody region may comprise at least a portion of an immunoglobulin light chain. The antibody region may comprise one or more immunoglobulin light chains or a portion thereof. The antibody region may comprise two or more immunoglobulin light chains or a portion thereof. The antibody region may comprise an amino acid sequence that is at least about 50% homologous to an immunoglobulin light chain. The antibody region may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more homologous to an immunoglobulin light chain. The antibody region may comprise an amino acid sequence that is at least about 70% homologous to an immunoglobulin light chain. The antibody region may comprise an amino acid sequence that is at least about 80% homologous to an immunoglobulin light chain. The antibody region may comprise an amino acid sequence that is at least about 90% homologous to an immunoglobulin light chain. The immunoglobulin light chain may comprise an amino acid sequence selected from any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 278.

The antibody region may comprise an amino acid sequence comprising 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or more amino acids of an immunoglobulin light chain. The antibody region may comprise an amino acid sequence comprising 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or more amino acids of an immunoglobulin light chain. The amino acids may be consecutive. Alternatively, or additionally, the amino acids are non-consecutive.

The immunoglobulin light chain may be encoded by a nucleotide sequence based on or derived from any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence that is at least about 50% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence that is at least about 75% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259. The immunoglobulin light chain may be encoded by a nucleotide sequence that is at least about 85% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297 and 257-259.

The antibody region may comprise at least a portion of a complementarity-determining region (CDR). The antibody region may comprise one or more complementarity-determining regions (CDRs) or portions thereof. The antibody region may comprise 2, 3, 4, 5 or more complementarity-determining regions (CDRs) or portions thereof. The antibody region may comprise 6, 7, 8 or more complementarity-determining regions (CDRs) or portions thereof. The antibody region may comprise four or more complementarity-determining regions (CDRs) or portions thereof. The antibody region may comprise 9, 10, 11 or more complementarity-determining regions (CDRs) or portions thereof. The one or more CDRs may be CDR1, CDR2, CDR3 or a combination thereof. The one or more CDRs may be CDR1. The one or more CDRs may be CDR2. The one or more CDRs may be CDR3. The one or more CDRs may be a heavy chain CDR. The one or more CDRs may be a light chain CDR.

The antibody region may comprise an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids based on or derived from an amino acid sequence of a CDR. The antibody region may comprise an amino acid sequence comprising 3 or more amino acids based on or derived from an amino acid sequence of a CDR. The antibody region may comprise an amino acid sequence comprising 5 or more amino acids based on or derived from an amino acid sequence of a CDR. The antibody region may comprise an amino acid sequence comprising 10 or more amino acids based on or derived from an amino acid sequence of a CDR. The amino acids may be consecutive. The amino acids may be non-consecutive.

The antibody region may be based on or derived from at least a portion of an anti-T cell receptor antibody. The antibody region may be based on or derived from at least a portion of an anti-B cell receptor antibody.

The antibody region may be based on or derived from at least a portion of an anti-T cell co-receptor antibody. The antibody region may be based on or derived from at least a portion of an anti-CD3 antibody. The antibody region may be based on or derived from an anti-CD3 antibody. The anti-CD3 antibody may be UCHT1. The antibody region may be based on or derived from at least a portion of a Fab fragment of an anti-CD3 antibody. The antibody region may be based on or derived from an antibody fragment of an anti-CD3 antibody.

The antibody region may be based on or derived from an antibody or antibody fragment that binds to at least a portion of a receptor on a cell. The antibody region may be based on or derived from an antibody or antibody fragment that binds to at least a portion of a co-receptor on a cell. The antibody region may be based on or derived from an antibody or antibody fragment that binds to at least a portion of an antigen or cell surface marker on a cell. The cell may be a hematopoietic cell. The hematopoietic cell may be a myeloid cell. The myeloid cell may be an erythrocyte, thrombocyte, neutrophil, monocyte, macrophage, eosinophil, basophil, or mast cell. The hematopoietic cell may be a lymphoid cell. The lymphoid cell may be a B-cell, T-cell, or NK-cell. The hematopoietic cell may be a leukocyte. The hematopoietic cell may be a lymphocyte.

The antibody region may be based on or derived from an antibody or antibody fragment that binds to at least a portion of a receptor on a T-cell. The receptor may be a T-cell receptor (TCR). The TCR may comprise TCR alpha, TCR beta, TCR gamma and/or TCR delta. The receptor may be a T-cell receptor zeta.

The antibody region may be based on or derived from an antibody or antibody fragment that binds to at least a portion of a receptor on a lymphocyte, B-cell, macrophage, monocytes, neutrophils and/or NK cells. The receptor may be an Fc receptor. The Fc receptor may be an Fc-gamma receptor, Fc-alpha receptor and/or Fc-epsilon receptor. Fc-gamma receptors include, but are not limited to, FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a) and FcγRIIIB (CD16b). Fc-alpha receptors include, but are not limited to, FcαRI. Fc-epsilon receptors include, but are not limited to, FcεRI and FcεRII. The receptor may be CD89 (Fc fragment of IgA receptor or FCAR).

The antibody region may be based on or derived from an antibody or antibody fragment that binds at least a portion of a co-receptor on a T-cell. The co-receptor may be a CD3, CD4, and/or CD8. The antibody region may be based on or derived from an antibody fragment that binds to a CD3 co-receptor. The CD3 co-receptor may comprise CD3-gamma, CD3-delta and/or CD3-epsilon. CD8 may comprise CD8-alpha and/or CD8-beta chains.

The antibody region may be based on or derived from an anti-viral antibody. The anti-viral antibody may be directed against an epitope of a viral protein. The viral protein may be from a respiratory syncytial virus. The viral protein may be an F protein of the respiratory syncytial virus. The epitope may be in the A antigenic site of the F protein. The anti-viral antibody may be based on or derived from Palivizumab.

The antibody region may be based on or derived from an antiviral immunoglobulin G antibody. The antibody region may comprise at least a portion of an antiviral immunoglobulin G antibody. The antibody region may comprise an amino acid sequence that is at least about 50% homologous to at least a portion of an antiviral immunoglobulin G antibody. The antibody region may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more homologous to at least a portion of an antiviral immunoglobulin G antibody. The antibody region may comprise an amino acid sequence that is at least about 70% homologous to at least a portion of an antiviral immunoglobulin G antibody. The antibody region may comprise an amino acid sequence that is at least about 80% homologous to at least a portion of an antiviral immunoglobulin G antibody.

The antibody region may comprise an amino acid sequence that comprises 10, 20, 30, 40, 50, 60, 70, 80, 90 or more amino acids of an antiviral immunoglobulin G antibody sequence. The antibody region may comprise an amino acid sequence that comprises 100, 200, 300, 400, 500, 600, 700, 800, 900 or more amino acids of an antiviral immunoglobulin G antibody sequence. The antibody region may comprise an amino acid sequence that comprises 50 or more amino acids of an antiviral immunoglobulin G antibody sequence. The antibody region may comprise an amino acid sequence that comprises 100 or more amino acids of an antiviral immunoglobulin G antibody sequence. The antibody region may comprise an amino acid sequence that comprises 200 or more amino acids of an antiviral immunoglobulin G antibody sequence.

The antibody region may be based on or derived from a palivizumab antibody. The antibody region may be a wild type palivizumab antibody. The antibody region may be a mutated palivizumab antibody. The antibody region may be a palivizumab wild type IgG1 heavy chain. The antibody may be a palivizumab antibody comprising a heptad mutation in an IgG1 heavy chain. The antibody region may be a palivizumab antibody comprising a triple mutation in an hIgG4 heavy chain. The antibody region may be a light chain paired with a palivizumab antibody. The antibody region may comprise at least a portion of a palivizumab antibody. The antibody region may comprise an amino acid sequence that is at least about 50% homologous to at least a portion of a palivizumab antibody. The antibody region may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more homologous to at least a portion of a palivizumab antibody. The antibody region may comprise an amino acid sequence that is at least about 70% homologous to at least a portion of a palivizumab antibody. The antibody region may comprise an amino acid sequence that is at least about 80% homologous to at least a portion of a palivizumab antibody.

The antibody region may comprise an amino acid sequence that comprises 10, 20, 30, 40, 50, 60, 70, 80, 90 or more amino acids of a palivizumab antibody sequence. The antibody region may comprise an amino acid sequence that comprises 100, 200, 300, 400, 500, 600, 700, 800, 900 or more amino acids of a palivizumab antibody sequence. The antibody region may comprise an amino acid sequence that comprises 50 or more amino acids of a palivizumab antibody sequence. The antibody region may comprise an amino acid sequence that comprises 100 or more amino acids of a palivizumab antibody sequence. The antibody region may comprise an amino acid sequence that comprises 200 or more amino acids of a Palivizumab antibody sequence.

The antibody region may be a trastuzumab antibody or fragment thereof. The antibody region may be a trastuzumab wild type antibody. The antibody region may be a mutated trastuzumab antibody. The antibody region may be a trastuzumab antibody that comprises a heptad mutation in the IgG1 heavy chain. The antibody region may be a trastuzumab antibody that comprises a triple mutation in the IgG4 heavy chain. The antibody region may be a light chain paired with the trastuzumab antibody. The antibody region may be based on or derived from a trastuzumab immunoglobulin G antibody. The antibody region may comprise at least a portion of a trastuzumab immunoglobulin G antibody. The antibody region may comprise an amino acid sequence that is at least about 50% homologous to at least a portion of a trastuzumab immunoglobulin G antibody. The antibody region may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more homologous to at least a portion of a trastuzumab immunoglobulin G antibody. The antibody region may comprise an amino acid sequence that is at least about 70% homologous to at least a portion of a trastuzumab immunoglobulin G antibody. The antibody region may comprise an amino acid sequence that is at least about 80% homologous to at least a portion of a trastuzumab immunoglobulin G antibody.

The antibody region may comprise an amino acid sequence that comprises 10, 20, 30, 40, 50, 60, 70, 80, 90 or more amino acids of a trastuzumab immunoglobulin G antibody sequence. The antibody region may comprise an amino acid sequence that comprises 100, 200, 300, 400, 500, 600, 700, 800, 900 or more amino acids of a trastuzumab immunoglobulin G antibody sequence. The antibody region may comprise an amino acid sequence that comprises 50 or more amino acids of a trastuzumab immunoglobulin G antibody sequence. The antibody region may comprise an amino acid sequence that comprises 100 or more amino acids of a trastuzumab immunoglobulin G antibody sequence. The antibody region may comprise an amino acid sequence that comprises 200 or more amino acids of a trastuzumab immunoglobulin G antibody sequence.

The antibody region may be based on or derived from an anti-Her2 antibody. The antibody region may comprise at least a portion of an anti-Her2 antibody. The antibody region may comprise an amino acid sequence that is at least about 50% homologous to at least a portion of an anti-Her2 antibody. The antibody region may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more homologous to at least a portion of an anti-Her2 antibody. The antibody region may comprise an amino acid sequence that is at least about 70% homologous to at least a portion of an anti-Her2 antibody. The antibody region may comprise an amino acid sequence that is at least about 80% homologous to at least a portion of an anti-Her2 antibody.

The antibody region may comprise an amino acid sequence that comprises 10, 20, 30, 40, 50, 60, 70, 80, 90 or more amino acids of an anti-Her2 antibody sequence. The antibody region may comprise an amino acid sequence that comprises 100, 200, 300, 400, 500, 600, 700, 800, 900 or more amino acids of an anti-Her2 antibody sequence. The antibody region may comprise an amino acid sequence that comprises 50 or more amino acids of an anti-Her2 antibody sequence. The antibody region may comprise an amino acid sequence that comprises 100 or more amino acids of an anti-Her2 antibody sequence. The antibody region may comprise an amino acid sequence that comprises 200 or more amino acids of an anti-Her2 antibody sequence.

The antibody region may be based on or derived from an anti-CD47 antibody. The antibody region may comprise at least a portion of an anti-CD47 antibody. The antibody region may comprise an amino acid sequence that is at least about 50% homologous to at least a portion of an anti-CD47 antibody. The antibody region may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more homologous to at least a portion of an anti-CD47 antibody. The antibody region may comprise an amino acid sequence that is at least about 70% homologous to at least a portion of an anti-CD47 antibody. The antibody region may comprise an amino acid sequence that is at least about 80% homologous to at least a portion of an anti-CD47 antibody.

The antibody region may comprise an amino acid sequence that comprises 10, 20, 30, 40, 50, 60, 70, 80, 90 or more amino acids of an anti-CD47 antibody sequence. The antibody region may comprise an amino acid sequence that comprises 100, 200, 300, 400, 500, 600, 700, 800, 900 or more amino acids of an anti-CD47 antibody sequence. The antibody region may comprise an amino acid sequence that comprises 50 or more amino acids of an anti-CD47 antibody sequence. The antibody region may comprise an amino acid sequence that comprises 100 or more amino acids of an anti-CD47 antibody sequence. The antibody region may comprise an amino acid sequence that comprises 200 or more amino acids of an anti-CD47 antibody sequence.

The antibody region may be based on or derived from an anti-cancer antibody. Examples of anti-cancer antibody include, but are not limited to, abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab, canakinumab, certolizumab, cetuximab, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, golimumab, ibritumomab, infliximab, ipilimumab, muromonab-cd3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, tocilizumab, tositumomab, trastuzumab.

The antibody region may comprise at least a portion of a human antibody. The antibody region may comprise at least a portion of a humanized antibody. The antibody region may comprise at least a portion of a chimeric antibody. The antibody region may be based on or derived from a human antibody. The antibody region may be based on or derived from a humanized antibody. The antibody region may be based on or derived from a chimeric antibody. The antibody region may be based on or derived from a monoclonal antibody. The antibody region may be based on or derived from a polyclonal antibody. The antibody region may comprise at least a portion of an antibody from a mammal, avian, reptile, amphibian, or a combination thereof. The mammal may be a human. The mammal may be a non-human primate. The mammal may be a dog, cat, sheep, goat, cow, rabbit, or mouse. The antibody region may comprise an antibody to a non-naturally occurring peptide or non-naturally-occurring protein.

The antibody region may be a human antibody, wherein a portion of the human antibody is replaced with a non-human peptide. The antibody region may be a human antibody, wherein a non-human peptide is added to the human antibody. The non-human peptide may be a portion of a non-human antibody or non-human antibody fragment. The portion of the non-human antibody or non-human antibody fragment may be a portion of a bovine antibody or a fragment thereof. The portion of the non-human antibody or non-human antibody fragment may be a non-human CDR. The non-human CDR may be a non-human CDR3. The CDR may be a CDR2. The CDR may be an ultralong CDR. The CDR may be an ultralong bovine CDR. The CDR may be a bovine ultralong CDR3. The CDR may be an ultralong bovine CDR of an antibody heavy chain. The CDR may be a bovine ultralong CDR3 of an antibody heavy chain. The CDR may be an ultralong bovine CDR of an antibody light chain. The CDR may be a bovine ultralong CDR3 of an antibody light chain. The CDR may be a portion of an ultralong bovine CDR. The CDR may be a portion of a bovine ultralong CDR3. For example, some bovine antibodies have unusually long CDR3 sequences compared to other vertebrates. A typical CDR3 is about 8 to about 16 amino acids in length. An ultralong CDR3 sequence may be greater than about 35 amino acids in length. An ultralong CDR3 sequence may be greater than about 40 amino acids in length. An ultralong CDR3 sequence may be greater than about 50 amino acids in length. An ultralong CDR3 sequence may be greater than 60 amino acids in length. An ultralong CDR3 may be between about 50 and 61 amino acids in length. The ultralong CDR3 may comprise multiple cysteines. An ultralong CDR3 may comprise disulfide-bonded mini-domains as a result of the multiple cysteines. A significant proportion of the ultralong CDR3 may be encoded by a D-region of a VH gene formed through a process called V(D)J recombination. The ultralong CDR3 may comprise a stalk domain. The ultralong CDR3 may comprise a β-strand stalk domain and a knob domain. The ultralong CDR3 may comprise a β-strand stalk that supports a structurally diverse, disulfide-bonded, knob domain. The β-strand stalk may comprise a β-sheet. The immunoglobulin fusion proteins disclosed herein may comprise a β-strand stalk, wherein the β-strand stalk is not based on or derived from an ultralong CDR3. The immunoglobulin fusion proteins disclosed herein may comprise a β-strand stalk, wherein the β-strand stalk is encoded by a sequence that is based on or derived from a sequence of 35 or fewer consecutive amino acids of an ultralong CDR3. The immunoglobulin fusion proteins disclosed herein may comprise a β-strand stalk, wherein the β-strand stalk is encoded by a sequence that is based on or derived from a sequence of 15 or fewer consecutive amino acids of an ultralong CDR3. The immunoglobulin fusion proteins disclosed herein may comprise a β-strand stalk, wherein the β-strand stalk is encoded by a sequence that is based on or derived from a sequence of 7 or fewer consecutive amino acids of an ultralong CDR3. The portion of the bovine ultralong CDR3 may have a length of less than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 about 12, about 13, about 14, about 15, about 16, about 18, about 19 or about 20 amino acids. The immunoglobulin fusion protein may contain less than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 about 12, about 13, about 14, about 15, about 16, about 18, about 19 or about 20 amino acids based on, derived from, identical to or homologous to a bovine ultralong CDR3. The immunoglobulin fusion protein may contain 7 or fewer amino acids based on, derived from, identical to or homologous to a bovine ultralong CDR3. The immunoglobulin fusion protein may contain 15 or fewer amino acids based on, derived from, identical to or homologous to a bovine ultralong CDR3. The immunoglobulin fusion protein may contain less than about 20 amino acids based on, derived from, identical to or homologous to a bovine ultralong CDR3. The immunoglobulin fusion protein may contain less than about 35 amino acids based on, derived from, identical to or homologous to a bovine ultralong CDR3.

The bovine ultralong CDR3 may be selected from BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, and F18. The bovine ultralong CDR3 may be selected from BF4E9, B-L1 and B-L2. The bovine ultralong CDR3 may be BLV1H12. The bovine ultralong CDR3 may not be selected from BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, and F18. The bovine ultralong CDR3 not be selected from BF4E9, B-L1 and B-L2. The bovine ultralong CDR3 may not be BLV1H12. The bovine ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOs: 248-250. The bovine ultralong CDR3 may be based on or derived from a sequence selected from any one of SEQ ID NOs: 248-250. The bovine ultralong CDR3 may be about 50% homologous to a sequence selected from any one of SEQ ID NOs: 248-250. The bovine ultralong CDR3 may be about 50%, about 60%, about 70%, about 80% or about 90% homologous to a sequence selected from any one of SEQ ID NOs: 248-250. The bovine ultralong CDR3 may not comprise a sequence selected from any one of SEQ ID NOs: 248-250. The bovine ultralong CDR3 may not comprise a sequence based on or derived from any one of SEQ ID NOs: 248-250. The bovine ultralong CDR3 may not comprise a sequence based on or derived from a portion of any one of SEQ ID NOs: 248-250.

The human antibody may have a sequence that is about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous to a fully human antibody. The human antibody may have a sequence that is less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14% or less than about 15% homologous to a non-human peptide or non-human protein. The portion of the human antibody that may be replaced may be a CDR or a portion thereof. The portion of the human antibody that may be replaced may be at least a portion of a variable fragment of the human antibody. The portion of the human antibody replaced may be at least a portion of a Fab of the human antibody. The portion of the human antibody replaced may be a portion a light chain or heavy chain of the human antibody. The non-human peptide may be less than about 4, less than about 5, less than about 6, less than about 7, less than about 8, less than about 9, less than about 10, less than about 11, less than about 12, less than about 13, less than about 14, less than about 15, less than about 16, less than about 17, less than about 18, less than about 19, less than about 20, less than about 22, less than about 23, less than about 24, less than about 25, less than about 26, less than about 27, less than about 28, less than about 29 or less than about 30 amino acids.

The antibody region may comprise a sequence based on or derived from one or more antibodies and/or antibody fragment sequences. The antibody region may comprise a sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more homologous to a sequence based on or derived from one or more antibodies and/or antibody fragments. The antibody region may comprise a sequence that is at least about 70% homologous to a sequence based on or derived from one or more antibodies and/or antibody fragments. The antibody region may comprise a sequence that is at least about 80% homologous to a sequence based on or derived from one or more antibodies and/or antibody fragments. The antibody region may comprise a sequence that is at least about 90% homologous to a sequence based on or derived from one or more antibodies and/or antibody fragments. The antibody region may comprise a sequence that is at least about 95% homologous to a sequence based on or derived from one or more antibodies and/or antibody fragments. The sequence may be a peptide sequence. Alternatively, the sequence is a nucleotide sequence.

The antibody region may comprise a peptide sequence that differs from a peptide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 20, 17, 15, 12, 10, 8, 6, 5, 4 or fewer amino acids. The antibody region may comprise a peptide sequence that differs from a peptide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 4 or fewer amino acids. The antibody region may comprise a peptide sequence that differs from a peptide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 3 or fewer amino acids. The antibody region may comprise a peptide sequence that differs from a peptide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 2 or fewer amino acids. The antibody region may comprise a peptide sequence that differs from a peptide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 1 or fewer amino acids. The amino acids may be consecutive, nonconsecutive, or a combination thereof. For example, the antibody region may comprise a peptide sequence that differs from a peptide sequence based on or derived from one or more antibodies and/or antibody fragments by less than about 3 consecutive amino acids. Alternatively, or additionally, the antibody region may comprise a peptide sequence that differs from a peptide sequence based on or derived from one or more antibodies and/or antibody fragments by less than about 2 non-consecutive amino acids. In another example, the antibody region may comprise a peptide sequence that differs from a peptide sequence based on or derived from one or more antibodies and/or antibody fragments by less than about 5 amino acids, wherein 2 of the amino acids are consecutive and 2 of the amino acids are non-consecutive.

The antibody region may comprise a nucleotide sequence that differs from a nucleotide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or fewer nucleotides or base pairs. The antibody region may comprise a nucleotide sequence that differs from a nucleotide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 15 or fewer nucleotides or base pairs. The antibody region may comprise a nucleotide sequence that differs from a nucleotide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 12 or fewer nucleotides or base pairs. The antibody region may comprise a nucleotide sequence that differs from a nucleotide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 9 or fewer nucleotides or base pairs. The antibody region may comprise a nucleotide sequence that differs from a nucleotide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 6 or fewer nucleotides or base pairs. The antibody region may comprise a nucleotide sequence that differs from a nucleotide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 4 or fewer nucleotides or base pairs. The antibody region may comprise a nucleotide sequence that differs from a nucleotide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 3 or fewer nucleotides or base pairs. The antibody region may comprise a nucleotide sequence that differs from a nucleotide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 2 or fewer nucleotides or base pairs. The antibody region may comprise a nucleotide sequence that differs from a nucleotide sequence based on or derived from one or more antibodies and/or antibody fragments by less than or equal to about 1 or fewer nucleotides or base pairs. The nucleotides or base pairs may be consecutive, nonconsecutive, or a combination thereof. For example, the antibody region may comprise a nucleotide sequence that differs from a nucleotide sequence based on or derived from one or more antibodies and/or antibody fragments by less than about 3 consecutive nucleotides or base pairs. Alternatively, or additionally, the antibody region may comprise a nucleotide sequence that differs from a nucleotide sequence based on or derived from one or more antibodies and/or antibody fragments by less than about 2 non-consecutive nucleotides or base pairs. In another example, the antibody region may comprise a nucleotide sequence that differs from a nucleotide sequence based on or derived from one or more antibodies and/or antibody fragments by less than about 5 nucleotides or base pairs, wherein 2 of the nucleotides or base pairs are consecutive and 2 of the nucleotides or base pairs are non-consecutive.

The peptide sequence of the antibody region may differ from the peptide sequence of the antibody or antibody fragment that it is based on and/or derived from by one or more amino acid substitutions. The peptide sequence of the antibody region may differ from the peptide sequence of the antibody or antibody fragment that it is based on and/or derived from by two or more amino acid substitutions. The peptide sequence of the antibody region may differ from the peptide sequence of the antibody or antibody fragment that it is based on and/or derived from by three or more amino acid substitutions. The peptide sequence of the antibody region may differ from the peptide sequence of the antibody or antibody fragment that it is based on and/or derived from by four or more amino acid substitutions. The peptide sequence of the antibody region may differ from the peptide sequence of the antibody or antibody fragment that it is based on and/or derived from by five or more amino acid substitutions. The peptide sequence of the antibody region may differ from the peptide sequence of the antibody or antibody fragment that it is based on and/or derived from by six or more amino acid substitutions. The peptide sequence of the antibody region may differ from the peptide sequence of the antibody or antibody fragment that it is based on and/or derived from by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 25 or more amino acid substitutions.

The nucleotide sequence of the antibody region may differ from the nucleotide sequence of the antibody or antibody fragment that it is based on and/or derived from by one or more nucleotide and/or base pair substitutions. The nucleotide sequence of the antibody region may differ from the nucleotide sequence of the antibody or antibody fragment that it is based on and/or derived from by two or more nucleotide and/or base pair substitutions. The nucleotide sequence of the antibody region may differ from the nucleotide sequence of the antibody or antibody fragment that it is based on and/or derived from by three or more nucleotide and/or base pair substitutions. The nucleotide sequence of the antibody region may differ from the nucleotide sequence of the antibody or antibody fragment that it is based on and/or derived from by four or more nucleotide and/or base pair substitutions. The nucleotide sequence of the antibody region may differ from the nucleotide sequence of the antibody or antibody fragment that it is based on and/or derived from by five or more nucleotide and/or base pair substitutions. The nucleotide sequence of the antibody region may differ from the nucleotide sequence of the antibody or antibody fragment that it is based on and/or derived from by six or more nucleotide and/or base pair substitutions. The nucleotide sequence of the antibody region may differ from the nucleotide sequence of the antibody or antibody fragment that it is based on and/or derived from by nine or more nucleotide and/or base pair substitutions. The nucleotide sequence of the antibody region may differ from the nucleotide sequence of the antibody or antibody fragment that it is based on and/or derived from by twelve or more nucleotide and/or base pair substitutions. The nucleotide sequence of the antibody region may differ from the nucleotide sequence of the antibody or antibody fragment that it is based on and/or derived from by fifteen or more nucleotide and/or base pair substitutions. The nucleotide sequence of the antibody region may differ from the nucleotide sequence of the antibody or antibody fragment that it is based on and/or derived from by eighteen or more nucleotide and/or base pair substitutions. The nucleotide sequence of the antibody region may differ from the nucleotide sequence of the antibody or antibody fragment that it is based on and/or derived from by 20, 22, 24, 25, 27, 30 or more nucleotide and/or base pair substitutions.

The antibody region may comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids. The antibody region may comprise at least about 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700 or more amino acids. The antibody region may comprise at least about 100 amino acids. The antibody region may comprise at least about 200 amino acids. The antibody region may comprise at least about 400 amino acids. The antibody region may comprise at least about 500 amino acids. The antibody region may comprise at least about 600 amino acids.

The antibody region may comprise less than about 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200 or 1100 amino acids. The antibody region may comprise less than about 1000, 950, 900, 850, 800, 750, or 700 amino acids. The antibody region may comprise less than about 1500 amino acids. The antibody region may comprise less than about 1000 amino acids. The antibody region may comprise less than about 800 amino acids. The antibody region may comprise less than about 700 amino acids.

The IFP may further comprise an antibody region comprising 30 or fewer consecutive amino acids of a complementarity determining region 3 (CDR3). The antibody region may comprise 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 15 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 14 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 13 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 12 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 11 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 10 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 9 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 8 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 7 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 6 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 5 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 4 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 3 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 2 or fewer consecutive amino acids of a CDR3. The antibody region may comprise 1 or fewer consecutive amino acids of a CDR3. In some instances, the antibody region does not contain a CDR3.

The IFP may comprise a first antibody region comprising 6 or fewer consecutive amino acids of a complementarity determining region 3 (CDR3). The first antibody region may comprise 5 or fewer consecutive amino acids of a CDR3. The first antibody region may comprise 4 or fewer consecutive amino acids of a CDR3. The first antibody region may comprise 3 or fewer consecutive amino acids of a CDR3. The first antibody region may comprise 2 or fewer consecutive amino acids of a CDR3. The first antibody region may comprise 1 or fewer consecutive amino acids of a CDR3. In some instances, the first antibody region does not contain a CDR3.

The IFP may further comprise a second antibody region comprising 30 or fewer consecutive amino acids of a complementarity determining region 3 (CDR3). The second antibody region may comprise 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 15 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 14 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 13 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 12 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 11 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 10 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 9 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 8 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 7 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 6 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 5 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 4 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 3 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 2 or fewer consecutive amino acids of a CDR3. The second antibody region may comprise 1 or fewer consecutive amino acids of a CDR3. In some instances, the second antibody region does not contain a CDR3.

Non-Antibody Region

The immunoglobulin fusion proteins disclosed herein may comprise one or more non-antibody regions. The immunoglobulin fusion proteins disclosed herein may comprise two or more non-antibody regions. The immunoglobulin fusion proteins disclosed herein may comprise 3, 4, 5, 6, 7, 8, 9, 10 or more non-antibody regions.

The two or more non-antibody regions may be attached to one or more antibody regions. The two or more non-antibody regions may be attached to two or more antibody regions. The two or more non-antibody regions may be attached to one or more immunoglobulin chains. The two or more non-antibody regions may be attached to two or more immunoglobulin chains. The two or more non-antibody regions may be attached to one or more subunits within the one or more antibody regions. The two or more non-antibody regions may be attached to two or more subunits within the one or more antibody regions.

The non-antibody regions may comprise one or more therapeutic agents. The non-antibody regions may comprise two or more therapeutic agents. The non-antibody regions may comprise 3, 4, 5, 6, 7 or more therapeutic agents. The therapeutic agents may be different. The therapeutic agents may be the same.

The non-antibody regions may comprise one or more extender peptides. The non-antibody regions may comprise two or more extender peptides. The non-antibody regions may comprise 3, 4, 5, 6, 7 or more extender peptides. The extender peptides may be different. The extender peptides may be the same. The non-antibody region comprising one or more extender peptides may be referred to as an extender fusion region.

The non-antibody regions may comprise one or more linkers. The non-antibody regions may comprise two or more linkers. The non-antibody regions may comprise 3, 4, 5, 6, 7 or more linkers. The linkers may be different. The linkers may be the same. The linker may directly connect the therapeutic agent to the antibody region. In some instances, the non-antibody region does not comprise a linker.

The non-antibody region may be inserted into the antibody region. Insertion of the non-antibody region into the antibody region may comprise removal or deletion of a portion of the antibody from which the antibody region is based on or derived from. The non-antibody region may replace at least a portion of a heavy chain. The non-antibody region may replace at least a portion of a light chain. The non-antibody region may replace at least a portion of a V region. The non-antibody region may replace at least a portion of a D region. The non-antibody region may replace at least a portion of a J region. The non-antibody region may replace at least a portion of a variable region. The non-antibody region may replace at least a portion of a constant region. The non-antibody region may replace at least a portion of a complementarity determining region (CDR). The non-antibody region may replace at least a portion of a CDR1. The non-antibody region may replace at least a portion of a CDR2. The non-antibody region may replace at least a portion of a CDR3. The non-antibody region may replace at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the antibody or portion thereof. For example, the non-antibody region may replace at least about 50% of a CDR. The non-antibody region may replace at least about 70% of a CDR. The non-antibody region may replace at least about 80% of a CDR. The non-antibody region may replace at least about 90% of a CDR. The non-antibody region may replace at least about 95% of a CDR.

Extender Fusion Region

The immunoglobulin fusion proteins disclosed herein may comprise one or more extender fusion regions. The extender fusion region may be a non-antibody region disclosed herein. The immunoglobulin fusion proteins may comprise two or more extender fusion regions. The immunoglobulin fusion proteins may comprise 3, 4, 5, 6, 7, 8, 9, 10 or more extender fusion regions.

The two or more extender fusion regions may be attached to one or more antibody regions. The two or more extender fusion regions may be attached to two or more antibody regions. The two or more extender fusion regions may be attached to one or more immunoglobulin chains. The two or more extender fusion regions may be attached to two or more immunoglobulin chains. The two or more extender fusion regions may be attached to one or more subunits within the one or more antibody regions. The two or more extender fusion regions may be attached to two or more subunits within the one or more antibody regions.

The extender fusion regions may comprise one or more extender peptides. The extender fusion regions may comprise two or more extender peptides. The extender fusion regions may comprise 3, 4, 5, 6 or more extender peptides.

The extender fusion regions may comprise one or more therapeutic agents. The extender fusion regions may comprise two or more therapeutic agents. The extender fusion regions may comprise 3, 4, 5, 6, 7 or more therapeutic agents. The therapeutic agents may be different. The therapeutic agents may be the same.

The immunoglobulin fusion proteins disclosed herein may comprise an antibody region attached to an extender fusion region. The extender fusion region may be attached to the N-terminus, C-terminus, or N- and C-terminus of the antibody region. The antibody region may be directly attached to the extender fusion region. Alternatively, or additionally, the antibody region may be indirectly attached to the non-antibody sequence. Attachment of the extender fusion region to the antibody region may comprise covalent attachment. Attachment may comprise fusion of the extender fusion region to the antibody region. Attachment may comprise chemical conjugation.

Alternatively, or additionally, attachment comprises insertion of the extender fusion region into the antibody region. The extender fusion region may be inserted into a heavy chain of the antibody region. The extender fusion region may be inserted into a light chain of the antibody region. The extender fusion region may be inserted into a variable domain of the antibody region. The extender fusion region may be inserted into a constant domain of the antibody region. The extender fusion region may be inserted into a complementarity-determining region (CDR) of the antibody region.

The extender fusion region may replace at least a portion of an antibody from which the antibody region is based on or derived. The extender fusion region may replace at least a portion of a heavy chain of an antibody from which the antibody region may be based on or derived. The extender fusion region may replace at least a portion a light chain of an antibody from which the antibody region may be based on or derived. The extender fusion region may replace at least a portion of a variable domain of an antibody from which the antibody region may be based on or derived. The extender fusion region may replace at least a portion of a variable domain of an antibody from which the antibody region may be based on or derived. The extender fusion region may replace at least a portion of a complementarity-determining region (CDR) of an antibody from which the antibody region may be based on or derived. The extender fusion region may replace at least a portion of a CDR1, CDR2, CDR3, or a combination thereof of an antibody from which the antibody or fragment thereof may be based on or derived. The extender fusion region may replace at least a portion of a CDR3 of an antibody from which the antibody region may be based on or derived.

The extender fusion region may comprise a CDR or portion thereof. The extender fusion region may comprise a human CDR or portion thereof. The extender fusion region may comprise a non-human CDR or portion thereof. The CDR or portion thereof may be a CDR3 or portion thereof. The CDR or portion thereof may be a CDR2 or portion thereof. The CDR or portion thereof may be an ultralong CDR or portion thereof. The CDR or portion thereof may be an ultralong bovine CDR or portion thereof. The CDR or portion thereof may be a bovine ultralong CDR3 or portion thereof. The CDR or portion thereof may be an ultralong bovine CDR of an antibody heavy chain or portion thereof. The CDR or portion thereof may be a bovine ultralong CDR3 of an antibody heavy chain or portion thereof. The CDR or portion thereof may be an ultralong bovine CDR of an antibody light chain or portion thereof. The CDR or portion thereof may be a bovine ultralong CDR3 of an antibody light chain or portion thereof. The CDR or portion thereof may be a portion of an ultralong bovine CDR. The CDR or portion thereof may be a portion of a bovine ultralong CDR3. For example, some bovine antibodies have unusually long CDR3 sequences compared to other vertebrates. A typical CDR3 is about 8 to about 16 amino acids in length. An ultralong CDR3 sequence may be greater than about 35 amino acids in length. An ultralong CDR3 sequence may be greater than about 40 amino acids in length. An ultralong CDR3 sequence may be greater than about 50 amino acids in length. An ultralong CDR3 sequence may be greater than 60 amino acids in length. An ultralong CDR3 may be between about 50 and 61 amino acids in length. The ultralong CDR3 may comprise multiple cysteines. An ultralong CDR3 may comprise disulfide-bonded mini-domains as a result of the multiple cysteines. A significant proportion of the ultralong CDR3 may be encoded by a D-region of a VH gene formed through a process called V(D)J recombination. The ultralong CDR3 may comprise a stalk domain. The ultralong CDR3 may comprise a β-strand stalk domain and a knob domain. The ultralong CDR3 may comprise a β-strand stalk that supports a structurally diverse, disulfide-bonded, knob domain. The β-strand stalk may comprise a β-sheet. The immunoglobulin fusion proteins disclosed herein may comprise a β-strand stalk, wherein the β-strand stalk is not based on or derived from an ultralong CDR3. The extender fusion regions disclosed herein may comprise a β-strand stalk, wherein the β-strand stalk is encoded by a sequence that is based on or derived from a sequence of 35 or fewer consecutive amino acids of an ultralong CDR3. The extender fusion regions disclosed herein may comprise a β-strand stalk, wherein the β-strand stalk is encoded by a sequence that is based on or derived from a sequence of 15 or fewer consecutive amino acids of an ultralong CDR3. The extender fusion regions disclosed herein may comprise a β-strand stalk, wherein the β-strand stalk is encoded by a sequence that is based on or derived from a sequence of 7 or fewer consecutive amino acids of an ultralong CDR3. The portion of the bovine ultralong CDR3 may have a length of less than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 about 12, about 13, about 14, about 15, about 16, about 18, about 19 or about 20 amino acids. The immunoglobulin fusion protein may contain less than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 about 12, about 13, about 14, about 15, about 16, about 18, about 19 or about 20 amino acids based on, derived from, identical to or homologous to a bovine ultralong CDR3. The extender fusion regions may contain 7 or fewer amino acids based on, derived from, identical to or homologous to a bovine ultralong CDR3. The immunoglobulin fusion protein may contain 15 or fewer amino acids based on, derived from, identical to or homologous to a bovine ultralong CDR3. The extender fusion regions may contain less than about 20 amino acids based on, derived from, identical to or homologous to a bovine ultralong CDR3. The extender fusion regions may contain less than about 35 amino acids based on, derived from, identical to or homologous to a bovine ultralong CDR3. The bovine ultralong CDR3 may be selected from BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, and F18. The bovine ultralong CDR3 may be selected from BF4E9, B-L1 and B-L2. The bovine ultralong CDR3 may be BLV1H12. The bovine ultralong CDR3 may not be selected from BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, and F18. The bovine ultralong CDR3 not be selected from BF4E9, B-L1 and B-L2. The bovine ultralong CDR3 may not be BLV1H12. The bovine ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOs: 248-250. The bovine ultralong CDR3 may be based on or derived from a sequence selected from any one of SEQ ID NOs: 248-250. The bovine ultralong CDR3 may be about 50% homologous to a sequence selected from any one of SEQ ID NOs: 248-250. The bovine ultralong CDR3 may be about 50%, about 60%, about 70%, about 80% or about 90% homologous to a sequence selected from any one of SEQ ID NOs: 248-250. The bovine ultralong CDR3 may not comprise a sequence selected from any one of SEQ ID NOs: 248-250. The bovine ultralong CDR3 may not comprise a sequence based on or derived from SEQ ID NOs: 248-250. The bovine ultralong CDR3 may not comprise a sequence based on or derived from a portion of any one of SEQ ID NOs: 248-250.

The extender fusion region may replace at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acids of an antibody from which the antibody region is based on or derived. The extender fusion region may replace at least about 1 or more amino acids of an antibody from which the antibody region is based on or derived. The extender fusion region may replace at least about 3 or more amino acids of an antibody from which the antibody region is based on or derived. The extender fusion region may replace at least about 5 or more amino acids of an antibody from which the antibody region is based on or derived.

The extender fusion region may comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. The extender fusion region may comprise at least about 150, 200, 250, 300, 350, 400, 450, 500 or more amino acids. The extender fusion region may comprise at least about 10 or more amino acids. The extender fusion region may comprise at least about 25 or more amino acids. The extender fusion region may comprise at least about 50 or more amino acids. The extender fusion region may comprise at least about 75 or more amino acids. The extender fusion region may comprise at least about 100 or more amino acids.

The extender fusion region may comprise less than about 1000, 900, 800, 700, 600, or 500 amino acids. The extender fusion region may comprise less than about 450, 400, 350, 300, 275, 250, 225, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50 amino acids. The extender fusion region may comprise less than about 400 amino acids. The extender fusion region may comprise less than about 300 amino acids. The extender fusion region may comprise less than about 250 amino acids.

The extender fusion region may comprise between about 10 to about 500 amino acids. The extender fusion region may comprise between about 10 to about 400 amino acids. The extender fusion region may comprise between about 10 to about 300 amino acids. The extender fusion region may comprise between about 10 to about 250 amino acids. The extender fusion region may comprise between about 20 to about 500 amino acids. The extender fusion region may comprise between about 20 to about 400 amino acids. The extender fusion region may comprise between about 20 to about 300 amino acids.

Extender fusion regions may comprise (a) one or more extender peptides; (b) one or more therapeutic agents; (c) optionally, one or more linkers; and (d) optionally, one or more proteolytic cleavage sites. Exemplary extender fusion regions are depicted in FIG. 3A-G. For example, as shown in FIG. 3A, an extender fusion region comprises an extender peptide (210) and a therapeutic agent (220). As shown in FIG. 3B, an extender fusion region comprises two extender peptides (210, 230) and a therapeutic agent (220). As shown in FIG. 3C, an extender fusion region comprises an extender peptide (210) and a therapeutic agent (220) connected by a linker (240). As shown in FIG. 3D, an extender fusion region comprises an extender peptide (210), and therapeutic agent (220) flanked by two linkers (240, 250). As shown in FIG. 3E, an extender fusion region comprises an extender peptide (210), a therapeutic agent (220) and a proteolytic cleavage site (260), wherein the proteolytic cleavage site (260) is inserted between the extender peptide and therapeutic agent. As shown on FIG. 3F, an extender fusion region comprises two extender peptides (210, 230), two linkers (240, 250) and a therapeutic agent (220). As shown on FIG. 3G, an extender fusion region comprises two extender peptides (210, 230), two linkers (240, 250), a proteolytic cleavage site (260) and a therapeutic agent (220).

The extender fusion regions may comprise (a) a first extender peptide, wherein the first extender peptide comprises (i) an amino acid sequence comprising a beta strand secondary structure; and (ii) 7 or fewer amino acids based on or derived from an ultralong CDR3; and (b) a therapeutic agent. The extender fusion regions may further comprise one or more additional extender peptides comprising a beta strand secondary structure. The extender fusion regions may further comprise one or more linkers. The extender fusion regions may further comprise one or more proteolytic cleavage sites.

The extender fusion regions may comprise (a) a first extender peptide, wherein the first extender peptide comprises (i) an amino acid sequence comprising a beta strand secondary structure; and (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a first therapeutic agent. The extender fusion regions may further comprise one or more additional extender peptides comprising a beta strand secondary structure. The extender fusion regions may further comprise one or more linkers. The extender fusion regions may further comprise one or more proteolytic cleavage sites.

Extender Peptide

The immunoglobulin fusion proteins disclosed herein may comprise two or more extender peptides. The two or more extender peptides may be attached to the N-terminus, C-terminus, or N- and C-terminus of a therapeutic agent. The two or more extender peptides may be attached to each end of a therapeutic agent. The two or more extender peptides may be attached to different ends of a therapeutic agent.

The extender fusion region of the immunoglobulin fusion proteins disclosed herein may comprise one or more extender peptides. The extender fusion region may comprise 2 or more extender peptides. The extender fusion region may comprise 3 or more extender peptides. The extender fusion region may comprise 4 or more extender peptides. The extender fusion region may comprise 5 or more extender peptides. The extender fusion region may comprise a first extender peptide and a second extender peptide.

The extender peptide may comprise a secondary structure region, wherein the secondary structure region comprises a secondary structure and one or more additional amino acids. The secondary structure region may comprise a secondary structure and about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16 about 18 about 20, about 22, about 24, about 26, about 28, about 30, about 32, about 34, about 36, about 38 or about 40 additional amino acids. The secondary structure region may comprise a secondary structure and about 1 to about 100 additional amino acids. The secondary structure region may be a beta strand secondary structure region. The secondary structure region may comprise a secondary structure and a peptide. The extender peptide may comprise one or more secondary structures. The extender peptide may comprise two or more secondary structures. The extender peptide may comprise 3, 4, 5, 6, 7 or more secondary structures. The two or more extender peptide may comprise one or more secondary structures. The two or more extender peptides may comprise two or more secondary structures. The two or more extender peptides may comprise 3, 4, 5, 6, 7 or more secondary structures. Each extender peptide may comprise at least one secondary structure. The secondary structures of the two or more extender peptides may be the same. Alternatively, the secondary structures of the two or more extender peptides may be different.

The one or more secondary structures may comprise one or more beta strands. The extender peptides may comprise two or more beta strands. For example, the first extender peptide comprises a first beta strand and the second extender peptide comprises a second beta strand. The extender peptides may comprise 3, 4, 5, 6, 7 or more beta strands. The two or more beta strands may be anti-parallel. The two or more beta strands may be parallel. The two or more beta strands may form a beta sheet.

The one or more extender peptides may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. The one or more extender peptides may comprise at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more amino acids. The one or more extender peptides may comprise at least about 35, 40, 45, 50 or more amino acids.

The one or more extender peptides may comprise less than about 100 amino acids. The one or more extender peptides may comprise less than about 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 amino acids. The one or more extender peptides may comprise less than about 90 amino acids. The one or more extender peptides may comprise less than about 80 amino acids. The one or more extender peptides may comprise less than about 70 amino acids.

The two or more extender peptides may be the same length. For example, the first extender peptide and the second extender peptide are the same length. Alternatively, the two or more extender peptides are different lengths. In another example, the first extender peptide and the second extender peptide are different lengths. The two or more extender peptides may differ in length by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. The two or more extender peptides may differ in length by at least about 1 or more amino acids. The two or more extender peptides may differ in length by at least about 3 or more amino acids. The two or more extender peptides may differ in length by at least about 5 or more amino acids.

The extender peptide may be adjacent to an antibody region. The extender peptide may be attached to the N-terminus, C-terminus, or N- and C-terminus of the antibody region. The extender peptide may be adjacent to a non-antibody region. The extender peptide may be attached to the N-terminus, C-terminus, or N- and C-terminus of the non-antibody region. The extender peptide may be adjacent to a therapeutic agent. The extender peptide may be attached to the N-terminus, C-terminus, or N- and C-terminus of the therapeutic agent. The extender peptide may be adjacent to a linker. The extender peptide may be attached to the N-terminus, C-terminus, or N- and C-terminus of the linker. The extender peptide may be adjacent to a proteolytic cleavage site. The extender peptide may be attached to the N-terminus, C-terminus, or N- and C-terminus of the proteolytic cleavage site.

The extender peptide may connect the therapeutic agent to the antibody region. The extender peptide may be between the antibody region and the therapeutic agent, linker, and/or proteolytic cleavage site. The extender peptide may be between two or more antibody regions, therapeutic agents, linkers, proteolytic cleavage sites or a combination thereof. The extender peptide may be N-terminal to the antibody region, therapeutic agent, the linker, the proteolytic cleavage site, or a combination thereof. The extender peptide may be C-terminal to the antibody region, therapeutic agent, the linker, the proteolytic cleavage site, or a combination thereof. The extender peptide may comprise a linker. The linker may be rigid. The linker may be flexible. The linker may comprise one or more amino acids.

The extender peptide may comprise an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 109-128, 305 and 308. The extender peptide may comprise an amino acid sequence that is at least about 50% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 109-128, 305 and 308. The extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or more homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 109-128, 305 and 308. The extender peptide may comprise an amino acid sequence that is at least about 70% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 109-128, 305 and 308. The extender peptide may comprise an amino acid sequence that is at least about 80% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 109-128, 305 and 308. The extender peptide may comprise an amino acid sequence that is at least about 85% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 109-128, 305 and 308.

The first extender peptide may comprise an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 109-114 and 305. The first extender peptide may comprise an amino acid sequence that is at least about 50% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 109-114 and 305. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or more homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 109-114 and 305. The first extender peptide may comprise an amino acid sequence that is at least about 75% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 109-114 and 305. The first extender peptide may comprise an amino acid sequence that is at least about 80% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 109-114 and 305.

The second extender peptide may comprise an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 115-128 and 308. The second extender peptide may comprise an amino acid sequence that is at least about 50% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 115-128 and 308. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or more homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 115-128 and 308. The second extender peptide may comprise an amino acid sequence that is at least about 70% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 115-128 and 308. The second extender peptide may comprise an amino acid sequence that is at least about 80% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 115-128 and 308.

The immunoglobulin fusion protein may comprise a first extender peptide. The first extender peptide may comprise a first beta strand. The therapeutic agent may comprise a second extender peptide. The second extender peptide may comprise a second beta strand. The first beta strand and the second beta strand may form a beta sheet.

The extender peptide may comprise an amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 109), wherein X1 is a negatively charged amino acid; X2 is a polar, uncharged amino acid; X3 is a positively charged amino acid; X4 is a positively charged amino acid; X5 is a hydrophobic amino acid; X6 is a polar, uncharged amino acid; and X7 is a polar, uncharged amino acid. A negatively charged amino acid may be D or E. A polar, uncharged amino acid may be S, T, C, Y, N, or Q. A positively charged amino acid may be K, R, or H. A hydrophobic amino acid may be G, A, V, L, I, M, W, F or P. X1 may be E or D. X2 may be T. X3 may be K. X4 may be K. X5 may be Y. X6 may be Q. X7 may be S. One or more additional amino acids may be inserted into SEQ ID NO: 109. One to ten amino acids may be inserted into SEQ ID NO: 109. The two or more additional amino acids may be inserted into one or more sites within SEQ ID NO: 109. The two or more additional amino acids may be inserted into two or more sites within SEQ ID NO: 109. The one or more additional amino acids may be inserted between X6 and X7 of SEQ ID NO: 109. The two or more additional amino acids may be contiguous. Alternatively, or additionally, the two or more amino acids are not contiguous. Alternatively, or additionally, one or more amino acids are added to one or more ends of SEQ ID NO: 109.

The extender peptide may comprise an amino acid sequence of ETKKYQXnS (SEQ ID NO: 305). N may be between 1 and 8. Xn may be independently selected from a charged amino acid. Xn may be independently selected from a basic amino acid. Xn may be independently selected from an acidic amino acid. Xn may be independently selected from a polar amino acid. Xn may be independently selected from K, R, H, T and E.

The extender peptide may comprise an amino acid sequence of X1TX2NX3 (SEQ ID NO: 115). X1, X2 or X3 may be a polar amino acid. The polar amino acid may be S, T, C, Y, N, or Q. The polar amino acid may be Y. X1, X2 or X3 may be Y. X1, X2 or X3 may be the same amino acid. X1, X2 or X3 may be different amino acids. One or more additional amino acids may be inserted into SEQ ID NO: 115. One to ten amino acids may be inserted into SEQ ID NO: 115. The two or more additional amino acids may be inserted into one or more sites within SEQ ID NO: 115. The two or more additional amino acids may be inserted into two or more sites within SEQ ID NO: 115. The two or more additional amino acids may be contiguous. Alternatively, or additionally, the two or more amino acids are not contiguous. Alternatively, or additionally, one or more amino acids are added to one or more ends of SEQ ID NO: 115. The one or more additional amino acids may be N-terminal to X1 of SEQ ID NO: 115. The one or more additional amino acids N-terminal to X1 may be S. The one or more additional amino acids may be C-terminal to X3 of SEQ ID NO: 115. The one or more additional amino acids C-terminal to X3 may be E.

The extender peptide may comprise an amino acid sequence of YX1YX2Y (SEQ ID NO: 128). X1 or X1 may be a polar amino acid. The polar amino acid may be S, T, C, Y, N, or Q. The polar amino acid may be T or N. X1 or X1 may be independently selected from T or N. X1 or X1 may be the same amino acid. X1 or X1 may be different amino acids. One or more additional amino acids may be inserted into SEQ ID NO: 128. One to ten amino acids may be inserted into SEQ ID NO: 128. The two or more additional amino acids may be inserted into one or more sites within SEQ ID NO: 128. The two or more additional amino acids may be inserted into two or more sites within SEQ ID NO: 128. The two or more additional amino acids may be contiguous. Alternatively, or additionally, the two or more amino acids are not contiguous. Alternatively, or additionally, one or more amino acids are added to one or more ends of SEQ ID NO: 128. The N-terminus of SEQ ID NO: 128 may further comprise a S residue. The C-terminus of SEQ ID NO: 128 may further comprise an E residue.

The extender peptide may comprise an amino acid sequence of SXnX1TX2NX3X4 (SEQ ID NO: 308). N may be between 1 and 8. Xn may be one or more polar amino acids. Xn may 2, 3, 4, 5, 6, 7 or more polar amino acids. Xn may comprise one or more non-polar amino acids.

The extender peptide may comprise 5 or more polar amino acids. The extender peptide may comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more polar amino acids. The polar amino acids may be consecutive. Alternatively, or additionally, the polar amino acids may be non-consecutive.

The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 111; and (b) a second extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 119. The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 111; and (b) a second extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 119. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 111. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 119. The first extender peptide may comprise an amino acid sequence comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 111. The first extender peptide may comprise an amino acid sequence comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 119. The second extender peptide may comprise an amino acid sequence comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 119. The second extender peptide may comprise an amino acid sequence comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 119.

The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 111; and (b) a second extender peptide comprising an amino acid sequence based on or derived from any one of SEQ ID NOs: 120-124. The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 111; and (b) a second extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of any one of SEQ ID NOs: 120-124. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 111. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of any one of SEQ ID NOs: 120-124. The first extender peptide may comprise an amino acid sequence comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 111. The first extender peptide may comprise an amino acid sequence comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 111. The second extender peptide may comprise an amino acid sequence comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of any one of SEQ ID NOs: 120-124. The second extender peptide may comprise an amino acid sequence comprising 5 or more amino acids based on or derived from an amino acid sequence of any one of SEQ ID NOs: 120-124.

The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence based on or derived from any one of SEQ ID NOS: 118-119 and 308; and (b) a second extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 126. The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of any one of SEQ ID NOS: 118-119 and 308; and (b) a second extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 126. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NOS: 118-119 and 308. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 126. The first extender peptide may comprise an amino acid sequence comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of any one of SEQ ID NOS: 118-119 and 308. The first extender peptide may comprise an amino acid sequence comprising 5 or more amino acids based on or derived from an amino acid sequence of any one of SEQ ID NOS: 118-119 and 308. The second extender peptide may comprise an amino acid sequence comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 126. The second extender peptide may comprise an amino acid sequence comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 126.

The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 114; and (b) a second extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 127. The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 114; and (b) a second extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 127. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 172. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 127. The first extender peptide may comprise an amino acid sequence comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 114. The first extender peptide may comprise an amino acid sequence comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 114. The second extender peptide may comprise an amino acid sequence comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 127. The second extender peptide may comprise an amino acid sequence comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 127.

The extender peptides disclosed herein may be based on or derived from a CDR. The extender peptides disclosed herein may be based on or derived from a CDR3. The extender peptide may be based on or derived from a human CDR. The extender peptide may not be based on or derived from any CDR. The extender peptide may be synthetic. The extender peptide may comprise a beta strand secondary structure. The extender fusion region may comprise a first extender peptide comprising a beta strand and a second extender peptide comprising a second beta strand, wherein the first beta strand and the second beta strand form a beta sheet. The extender peptide may not comprise a beta strand. The extender peptide may form a rigid structure. The rigid structure may not comprise a beta strand. The rigid structure may not comprise an alpha helix. The CDR3 may be an ultralong CDR3. An “ultralong CDR3” or an “ultralong CDR3 sequence”, used interchangeably herein, may comprise a CDR3 that is not derived from a human antibody sequence. An ultralong CDR3 may be 35 amino acids in length or longer, for example, 40 amino acids in length or longer, 45 amino acids in length or longer, 50 amino acids in length or longer, 55 amino acids in length or longer, or 60 amino acids in length or longer. The ultralong CDR3 may be a heavy chain CDR3 (CDR-H3 or CDRH3). The ultralong CDR3 may comprise a sequence derived from or based on a ruminant (e.g., bovine) sequence. An ultralong CDR3 may comprise one or more cysteine motifs. An ultralong CDR3 may comprise at least 3 or more cysteine residues, for example, 4 or more cysteine residues, 6 or more cysteine residues, or 8 or more cysteine residues. Additional details on ultralong CDR3 sequences can be found in Saini S S, et al. (Exceptionally long CDR3H region with multiple cysteine residues in functional bovine IgM antibodies, European Journal of Immunology, 1999), Zhang Y, et al. (Functional antibody CDR3 fusion proteins with enhanced pharmacological properties, Angew Chem Int Ed Engl, 2013), Wang F, et al. (Reshaping antibody diversity, Cell, 2013) and U.S. Pat. No. 6,740,747.

The extender peptides may comprise 7 or fewer amino acids based on or derived from a CDR. The extender peptides may comprise 6, 5, 4, 3, 2, 1 or fewer amino acids based on or derived from a CDR. The amino acids may be consecutive. The amino acids may be non-consecutive. The CDR may be CDR1. The CDR may be CDR2. The CDR may be CDR3. The CDR may be an ultralong CDR. The CDR may be bovine. The CDR may be human. The CDR may be non-human.

The extender peptides may be based on or derived from a CDR, wherein the CDR is not an ultralong CDR3. The extender peptides may comprise 10 or fewer amino acids based on or derived from a CDR3. The extender peptides may comprise 9, 8, 7, 6, 5, 4, 3, 2, 1 or fewer amino acids based on or derived from a CDR3. The extender peptides may comprise 8 or fewer amino acids based on or derived from a CDR3. The extender peptides may comprise 7 or fewer amino acids based on or derived from a CDR3. The extender peptides may comprise 5 or fewer amino acids based on or derived from a CDR3. The extender peptide may be based on or derived from a human sequence. The extender peptide may be based on or derived from a non-human sequence. The extender peptide may be a synthetic sequence. The extender peptide may be based on or derived from a human sequence encoding a protein secondary structure. The extender peptide may be based on or derived from a human sequence encoding a beta strand. The first extender peptide may be based on or derived from a human sequence encoding a beta strand and the second extender peptide may be based on or derived from a human sequence encoding a beta strand, wherein the first beta strand and the second beta strand form a beta sheet.

The extender peptides may comprise an amino acid sequence that is less than about 50% identical to an amino acid sequence comprising an ultralong CDR3. The extender peptides may comprise an amino acid sequence that is less than about 45%, 40%, 35%, 30%, 25%, 20%, 25%, or 10% identical to an amino acid sequence comprising an ultralong CDR3. The extender peptides may comprise an amino acid sequence that is less than about 30% identical to an amino acid sequence comprising an ultralong CDR3. The extender peptides may comprise an amino acid sequence that is less than about 25% identical to an amino acid sequence comprising an ultralong CDR3. The extender peptides may comprise an amino acid sequence that is less than about 20% identical to an amino acid sequence comprising an ultralong CDR3.

The extender peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids attached to or inserted into an ultralong CDR3-based portion of the extender peptide. The extender peptide may comprise 1 or more amino acids attached to or inserted into an ultralong CDR3-based portion of the extender peptide. The extender peptide may comprise 3 or more amino acids attached to or inserted into an ultralong CDR3-based portion of the extender peptide. The extender peptide may comprise 5 or more amino acids attached to or inserted into an ultralong CDR3-based portion of the extender peptide. The two or more amino acids attached to or inserted into the ultralong CDR3 may be contiguous. Alternatively, or additionally, the two or more amino acids attached to or inserted into the ultralong CDR3 are not contiguous.

The extender peptide may comprise 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or fewer amino acids attached to or inserted into an ultralong CDR3-based portion of the extender peptide. The extender peptide may comprise 20 or fewer amino acids attached to or inserted into an ultralong CDR3-based portion of the extender peptide. The extender peptide may comprise 15 or fewer amino acids attached to or inserted into an ultralong CDR3-based portion of the extender peptide. The extender peptide may comprise 10 or fewer amino acids attached to or inserted into an ultralong CDR3-based portion of the extender peptide. The amino acids attached to or inserted into the ultralong CDR3 may be contiguous. Alternatively, or additionally, the amino acids attached to or inserted into the ultralong CDR3 are not contiguous.

Various extender peptides comprising anti-parallel beta strands and their thermostability are depicted in FIG. 4.

Therapeutic Agent

The immunoglobulin fusion proteins disclosed herein may comprise one or more therapeutic agents. The therapeutic agent may be a protein. The therapeutic agent may be a polypeptide. The therapeutic agent may be a peptide. The therapeutic agent may be a functional peptide. The therapeutic agent may be a synthetic and/or non-naturally occurring peptide. The therapeutic agent may be an agonist and/or an activator. The therapeutic agent may be an antagonist and/or an inhibitor. The therapeutic agent may be a substrate. The therapeutic agent may be a binding partner. The therapeutic agent may be a small molecule. The therapeutic agent may be a co-factor and/or co-regulator. The immunoglobulin fusion proteins disclosed herein may comprise two or more therapeutic agents. The immunoglobulin fusion proteins disclosed herein may comprise 3, 4, 5, 6 or more therapeutic agents. The two or more therapeutic agents may be the same. The two or more therapeutic agents may be different.

The one or more therapeutic agents may be based on or derived from a protein. The protein may be a growth factor, cytokine, hormone or toxin. The growth factor, by non-limiting example, may be GCSF, GMCSF or GDF11. The GCSF may be a bovine GCSF (bGCSF). The GCSF may be a human GCSF. The GMCSF may be a bovine GMCSF or a human GMCSF. The therapeutic agent may be grafted into an ultralong CDR3H of an antibody region, as shown in FIG. 36. FIG. 36 shows an exemplary scheme for grafting bGCSF onto the ‘knob’ domain of a bovine antibody with an ultralong CDR3H region.

The cytokine may be an interferon or interleukin. The cytokine, by non-limiting example, may be stromal cell-derived factor 1 (SDF-1). The interferon may be interferon-beta. The interferon may be interferon-alpha. The interleukin may be interleukin 11 (IL-11). The interleukin may be interleukin 8 (IL-8) or interleukin 21 (IL-21).

The hormone, by non-limiting example, may be exendin-4, GLP-1, relaxin, oxyntomodulin, leptin, betatrophin, bovine growth hormone (bGH), human growth hormone (hGH), erythropoietin (EPO), or parathyroid hormone. The hormone may be somatostatin. The parathyroid hormone may be a human parathyroid hormone. The erythropoietin may be a human erythropoietin. The therapeutic agent may be grafted into an ultra-long CDR3H of an antibody region, as shown in FIG. 23. In some embodiments, the therapeutic agent replaces a portion of an ultralong CDR3H. In some embodiments, the therapeutic agent replaces a knob domain of an ultralong CDR3H. FIG. 28 depicts an exemplary schematic representation of engineering glucagon-like peptide 1 (GLP-1) or Exendin-4 (Ex-4) into an ultralong CDR3H. The resulting GLP-1 and/or Ex-4 fusion proteins comprise an N-terminal proteolytic cleavage site. The fusion proteins may be cleaved to release the N-terminus of GLP-1 and/or Ex-4.

The toxin, by non-limiting example, may be Moka1, or Vm-24. The toxin may be ziconotide or chlorotoxin. The therapeutic agent may be grafted into an ultra-long CDR3H of an antibody region, as shown in FIG. 17. In some embodiments, the therapeutic agent replaces a knob domain of an ultralong CDR3H. FIG. 17 depicts the replacement of a knob domain of an ultralong CDR3H on a bovine antibody (PDB ID: 4K3D) with a toxin.

The protein may be angiogenic. The protein may be anti-angiogenic. The protein, by non-limiting example, may be angiopoeitin-like 3 (ANGPTL3). The angiopoeitin-like 3 may be a human angiopoeitin-like 3.

The one or more therapeutic agents may be based on or derived from a peptide. The peptide, by non-limiting example, may be a neutrophil elastase inhibitor (EI).

The therapeutic agent may be based on or derived from an amino acid sequence selected from any one of SEQ ID NOs: 263-298. The therapeutic agent may be based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 263-298. The therapeutic agent may be based on or derived from an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to any one of SEQ ID NOs: 263-298.

The therapeutic agent may comprise an amino acid sequence comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more amino acids of any one of SEQ ID NOs: 200-235. The therapeutic agent may comprise an amino acid sequence comprising at least about 10 or more amino acids of any one of SEQ ID NOs: 200-235. The therapeutic agent may comprise an amino acid sequence comprising at least about 15 or more amino acids of any one of SEQ ID NOs: 200-235. The therapeutic agent may comprise an amino acid sequence comprising at least about 20 or more amino acids of any one of SEQ ID NOs: 200-235. The therapeutic agent may comprise an amino acid sequence comprising at least about 30 or more amino acids of any one of SEQ ID NOs: 200-235. The amino acids may be consecutive. The amino acids may be non-consecutive.

The therapeutic agent may be based on or derived from an amino acid sequence selected from any one of SEQ ID NOs: 200-235. The therapeutic agent may be based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 200-235. The therapeutic agent may be based on or derived from an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to any one of SEQ ID NOs: 200-235. The therapeutic agent may be based on or derived from an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 200-235. The therapeutic agent may be based on or derived from an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 200-235.

The therapeutic agent may be encoded by a nucleic acid sequence based on or derived from any one of SEQ ID NOs: 167-199. The therapeutic agent may be encoded by a nucleic acid sequence that may be at least about 50% homologous to any one of SEQ ID NOs: 167-199. The therapeutic agent may be encoded by a nucleic acid sequence that may be at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to any one of SEQ ID NOs: 167-199.

The therapeutic agent may be encoded by a nucleic acid sequence based on or derived from any one of SEQ ID NOs: 167-199. The therapeutic agent may be encoded by a nucleic acid sequence that may be at least about 50% homologous to any one of SEQ ID NOs: 167-199. The therapeutic agent may be encoded by a nucleic acid sequence that may be at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to any one of SEQ ID NOs: 167-199. The therapeutic agent may be encoded by a nucleic acid sequence that may be at least about 70% homologous to any one of SEQ ID NOs: 167-199. The therapeutic agent may be encoded by a nucleic acid sequence that may be at least about 80% homologous to any one of SEQ ID NOs: 167-199.

The therapeutic agents may be inserted into the antibody region. Insertion of the therapeutic agent into the antibody region may comprise removal or deletion of one or more amino acids from the antibody region.

The one or more extender peptides may be attached to the N-terminus, C-terminus or both the N- and C-termini of a therapeutic agent. The one or more linkers may be attached to the N-terminus, C-terminus or both the N- and C-termini of a therapeutic agent. The one or more proteolytic cleavage sites may be attached to the N-terminus, C-terminus or both the N- and C-termini of a therapeutic agent. Alternatively, the therapeutic agent may be connected to the antibody region without the aid of an extender peptide. The therapeutic agent may be connected to the antibody via one or more linkers.

The therapeutic agent may be an agonist. The therapeutic agent may be an antagonist. The therapeutic agent may be a peptide. The therapeutic agent may be a cyclic peptide. The cyclic peptide may comprise a polypeptide chain wherein the amino termini and carboxyl termini, amino termini and side chain, carboxyl termini and side chain, or side chain and side chain are linked with a covalent bond that generates a ring. The cyclic peptide may comprise a 2 or more amino acids. The cyclic peptide may be selected from a cyclic isopeptide, a cyclic depsipeptide, a bicyclic peptide and a homodetic cyclic peptide. The cyclic peptide may comprise a naturally occurring cyclic peptide. The cyclic peptide may comprise a synthetic cyclic peptide. The cyclic peptide may comprise a modified naturally occurring peptide. The peptide may comprise a conformationally constrained peptide. The peptide may comprise a peptide modified to comprise a conformationally constrained peptide. The conformationally constrained peptide may have a reduced conformational entropy relative to a respective peptide that is not conformationally constrained. The conformationally constrained peptide may comprise a rigid feature and/or a rigid region and/or a rigid domain. The conformationally constrained peptide may be locked into a conformation by one or more bonds between non-consecutive amino acids. The one or more bonds may be a disulfide bond. A conformationally constrained peptide may have a greatly improved binding affinity and/or specificity to a target relative to endogenous or naturally-occurring binding partners of the target. By non-limiting example, the conformationally constrained peptide may be a peptide comprising a β-hairpin structure. The conformationally constrained peptide may comprise a region that is U-shaped, rigid, stalk-like, knob-like, pointed, angular or shaped to fit into a specific region of a target or binding partner. The therapeutic agent may further comprise one or more turn sequences. The turn sequence may comprise one or more amino acids. The turn sequence comprise about 1, about 2, about 3, about 4 or about 5 amino acids. The turn sequence may comprise one or more amino acids selected from glycine, asparagine and proline. The turn sequence may provide the therapeutic agent with a target binding conformation. The therapeutic agent may be a non-cyclic peptide. The therapeutic agent may be less than about 30 peptides, less than about 25 peptides, less than 20 peptides, less than about 15 peptides or less than about 10 peptides. The peptide may be synthesized. The peptide may be genetically encoded. The therapeutic agent may be naturally occurring. The therapeutic agent may be synthetic. The therapeutic agent may be a naturally occurring peptide comprising a modification. The modification can be an addition of one or more amino acids. The modification can be a deletion of one or more amino acids. The modification may be a re-arrangement of two or more amino acids.

The therapeutic agent may bind a target. The target may be a cell surface molecule. The target may be a circulating molecule. The cell surface molecule may be a receptor. By non-limiting example, the receptor may be a G protein coupled receptor. The receptor may be CXCR4. The target may be an enzyme. By non-limiting example, the enzyme may be a neutrophil elastase. The therapeutic agent may be about 80%, about 85%, about 90%, about 95% or about 100% effective at blocking a competing target-binding molecule as measured by a competitive binding assay. The therapeutic agent may be about 80%, about 85%, about 90%, about 95% or about 100% effective at blocking a competing target-binding molecule as measured by a target activity assay. The therapeutic agent may have a greater binding affinity for a target than a competing target-binding molecule. The competing target-binding molecule may be ligand. The competing target-binding molecule may be a substrate. The competing target-binding molecule may be endogenous. The competing target-binding molecule may be exogenous. The competing target-binding molecule may be naturally occurring. The competing target-binding molecule may be synthetic. The competing target-binding molecule may be organic. The competing target-binding molecule may be inorganic. The competing target-binding molecule may be a toxin. The competing target-binding molecule may be molecule on another cell. By way of non-limiting examples, the competing target-binding molecule may be selected from a chemokine, a cytokine, a growth factor, an integrin, a cell adhesion molecule, a biomolecule and a hormone. The therapeutic agent may bind a deep pocket, a cavity, an active site or a fold of a ligand binding domain or a catalytic domain of a target and block endogenous or exogenous ligands or substrates from binding to the target. The therapeutic agent may bind the surface of a ligand binding domain or catalytic domain of a target to effectively block endogenous or exogenous ligand or substrate binding to the target.

Linkers

The immunoglobulin fusion proteins, antibody regions, and/or extender fusion regions may further comprise one or more linkers. The immunoglobulin fusion proteins, antibody regions, and/or extender fusion region may further comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more linkers. The extender fusion region may further comprise one or more linkers. The extender fusion region may further comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more linkers. The one or more linkers may be rigid. The one or more linkers may be flexible. The linker may comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 amino acids. The linker may comprise about 10, about 15, about 20, about 25, about 30, about 35 or about 40 amino acids.

The one or more linkers are attached to the N terminus, C terminus or both N and C termini of a therapeutic agent. The one or more linkers are attached to the N terminus, C terminus or both N and C termini of the extender peptide. The one or more linkers are attached to the N-terminus, C-terminus or both N and C termini of a proteolytic cleavage site. The one or more linkers may be attached to a therapeutic agent, extender peptide, proteolytic cleavage site, extender fusion region, antibody region, or a combination thereof.

The one or more linkers may comprise the sequence (XeXfXgXh)n(SEQ ID NO: 309). N may be 1 to 5. Xe, Xf and Xg are independently selected from a hydrophobic amino acid. Xh may be a polar, uncharged amino acid. The linker sequence may further comprise one or more cysteine (C) residues. The one or more cysteine residues are at the N-terminus, C-terminus, or a combination thereof.

The one or more linkers may comprise an amino acid sequence selected from any one of SEQ ID NOs: 161-166 and 309. The one or more linkers may comprise an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 161-166 and 309. The one or more linkers may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to any one of SEQ ID NOs: 161-166 and 309. The one or more linkers may comprise an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 161-166 and 309. The one or more linkers may comprise an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 161-166 and 309.

Proteolytic Cleavage Site

The immunoglobulin fusion proteins disclosed herein may further comprise one or more proteolytic cleavage sites. The immunoglobulin fusion protein comprising one or more proteolytic cleavage sites may be referred to herein as a clip fusion protein and/or a clipped version (of the fusion protein). The immunoglobulin fusion protein may comprise a connecting peptide, wherein the connecting peptide comprises a proteolytic cleavage site. The connecting peptide comprising the proteolytic cleavage site may be located between the therapeutic agent and the antibody region. The connecting peptide comprising the proteolytic cleavage site may be located between the extender fusion region and the antibody region. The connecting peptide comprising the proteolytic cleavage site may be located between the extender peptide and the therapeutic agent. The connecting peptide comprising the proteolytic cleavage site may be located within the extender peptide. The connecting peptide comprising the proteolytic cleavage site may be located within the antibody region. The connecting peptide comprising the proteolytic cleavage site may be located within the extender fusion region. The connecting peptide comprising the proteolytic cleavage site may be located within the therapeutic agent. The proteolytic cleavage site may be selected from any one of SEQ ID NOs: 236-239. The immunoglobulin fusion proteins disclosed herein may further comprise 2 or more proteolytic cleavage sites. The immunoglobulin fusion proteins disclosed herein may further comprise 3 or more proteolytic cleavage sites. The immunoglobulin fusion proteins disclosed herein may further comprise 4, 5, 6, 7 or more proteolytic cleavage sites.

The immunoglobulin fusion proteins disclosed herein may comprise a sequence with one or more cleavage sites between (a) the antibody region and the extender fusion region; (b) the antibody region and the extender peptide; (c) the extender peptide and therapeutic agent; or (d) a combination of a-c. The extender fusion region may comprise one or more proteolytic cleavage sites. The one or more cleavage sites may be on the C-terminus, N-terminus, and/or N- and C-terminus of a therapeutic agent. The one or more cleavage sites may be on the C-terminus, N-terminus, and/or N- and C-terminus of the extender peptide. The one or more cleavage sites may be on the C-terminus, N-terminus, and/or N- and C-terminus of the antibody region. The proteolytic cleavage site may be on the N- or C-terminus of the therapeutic agent. Digestion of the proteolytic cleavage site may result in release of the N- or C-terminus of the therapeutic agent from the immunoglobulin fusion protein. For example, an immunoglobulin fusion protein which may be cleaved to release the amino-terminus of a therapeutic agent is referred to as RN, for released N-terminus. The proteolytic cleavage site may be on the N- and C-termini of the therapeutic agent. Digestion of the proteolytic cleavage site may result in release of the therapeutic agent from the immunoglobulin fusion protein.

Alternatively, or additionally, the proteolytic cleavage site is located within the amino acid sequence of the therapeutic agent, extender peptide, antibody region, or a combination thereof. The therapeutic agent may comprise one or more proteolytic cleavage sites within its amino acid sequence. For example, a clip fusion protein may be SEQ ID NO: 78, which discloses an exendin-4 fusion protein comprising two internal proteolytic cleavage sites. Digestion of the proteolytic cleavage sites within the relaxin protein may result in release of an internal fragment of the relaxin protein.

Two or more proteolytic cleavage sites may surround a therapeutic agent, extender peptide, linker, antibody region, or combination thereof. Digestion of the proteolytic cleavage site may result in release of a peptide fragment located between the two or more proteolytic cleavage sites. For example, the proteolytic cleavage sites may flank a therapeutic agent-linker peptide. Digestion of the proteolytic cleavage sites may result in release of the therapeutic agent-linker.

The proteolytic cleavage site may be recognized by one or more proteases. The one or more proteases may be a serine protease, threonine protease, cysteine protease, aspartate protease, glutamic protease, metalloprotease, exopeptidases, endopeptidases, or a combination thereof. The proteases may be selected from the group comprising Factor VII or Factor Xa. Additional examples of proteases include, but are not limited to, aminopeptidases, carboxypeptidases, trypsin, chymotrypsin, pepsin, papain, and elastase.

Dual Fusion Proteins

Further disclosed herein are dual fusion proteins. As used herein, dual fusion proteins may also be referred to as immunoglobulin dual fusion proteins and immunoglobulin fusion proteins. Dual fusion proteins disclosed herein may comprise one or more immunoglobulin fusion proteins disclosed herein or one or more portions thereof. The dual fusion protein may comprise two or more therapeutic agents and one or more antibody regions. The dual fusion protein may comprise one or more immunoglobulin fusion proteins disclosed herein. The antibody region may comprise a heavy chain and a light chain. At least one therapeutic agent may be inserted into the heavy chain. At least one therapeutic agent may be inserted into the light chain. The two or more therapeutic agents may be inserted the heavy chain. The two or more therapeutic agents may be inserted into the light chain. By non-limiting example, the dual fusion protein may comprise GCSF and EPO as the therapeutic agents. Also by non-limiting example, the dual fusion protein may comprise leptin and exendin-4 as the therapeutic agents.

The dual fusion protein may comprise one antibody region and two or more extender fusion regions. The dual fusion protein may comprise a first extender fusion region and a second extender fusion region. The first extender fusion region may comprise a beta strand. The first extender fusion region may comprise at least two beta strands, wherein the two beta strands form a beta sheet. The two beta strands may be parallel. The two beta strands may be anti-parallel. The second extender fusion region may comprise a beta strand. The second antibody region may comprise at least two beta strands, wherein the two beta strands form a beta sheet. The two beta strands may be parallel. The two beta strands may be anti-parallel. The second extender fusion region may comprise an alpha helix. The second extender fusion may comprise two alpha helices, wherein the alpha helices form a coiled coil. The alpha helices may be parallel. The alpha helices may be anti-parallel. The second extender fusion region may comprise a linker. The linker may be less than about 20 amino acids. The linker may be less than about 10 amino acids. The second extender fusion region may comprise two or more linkers. The linker may be less than about 7 amino acids. The first extender fusion region and/or the second extender fusion region may be a non-antibody region.

The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 24-27, 29-33, and 36-39. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 24-27, 29-33, and 36-39. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 24-27, 29-33, and 36-39. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 24-27, 29-33, and 36-39. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 90% homologous to any one of SEQ ID NOs: 24-27, 29-33, and 36-39. The dual fusion protein may comprise a light chain fusion based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The dual fusion protein may comprise a light chain fusion based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The dual fusion protein may comprise a light chain fusion based on or derived from an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The dual fusion protein may comprise a light chain fusion based on or derived from an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250. The dual fusion protein may comprise a light chain fusion based on or derived from an amino acid sequence that is at least about 90% homologous to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 248-250.

The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 76, 240 and 244. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 76, 240 and 244. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 70% homologous to SEQ ID NOs: 76, 240 and 244. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 76, 240 and 244. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 90% homologous to SEQ ID NOs: 76, 240 and 244.

The dual fusion protein may comprise a light chain fusion based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 77, 241 and 245. The dual fusion protein may comprise a light chain fusion based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 77, 241 and 245. The dual fusion protein may comprise a light chain fusion based on or derived from an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 77, 241 and 245. The dual fusion protein may comprise a light chain fusion based on or derived from an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 77, 241 and 245. The dual fusion protein may comprise a light chain fusion based on or derived from an amino acid sequence that is at least about 90% homologous to any one of SEQ ID NOs: 77, 241 and 245.

The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 81, 242 and 246. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 81, 242 and 246. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 81, 242 and 246. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 81, 242 and 246. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 90% homologous to any one of SEQ ID NOs: 81, 242 and 246. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 81, 242 and 246. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 81, 242 and 246. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 81, 242 and 246. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 81, 242 and 246. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 90% homologous to any one of SEQ ID NOs: 81, 242 and 246.

The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 83, 243 and 247. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 83, 243 and 247. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 83, 243 and 247. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 83, 243 and 247. The dual fusion protein may comprise a heavy chain fusion based on or derived from an amino acid sequence that is at least about 90% homologous to any one of SEQ ID NOs: 83, 243 and 247.

The dual fusion protein may comprise a light chain fusion based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 90% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297. The light chain fusion may further comprise a therapeutic agent based on or derived from an amino acid sequence that is at least about 50% homologous to SEQ ID NO: 201. The light chain fusion may further comprise a therapeutic agent based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NO: 201. The light chain fusion may further comprise a therapeutic agent based on or derived from an amino acid sequence that is at least about 70% homologous to SEQ ID NO: 201. The light chain fusion may further comprise a therapeutic agent based on or derived from an amino acid sequence that is at least about 80% homologous to SEQ ID NO: 201. The light chain fusion may further comprise a therapeutic agent based on or derived from an amino acid sequence that is at least about 90% homologous to SEQ ID NO: 201.

The dual fusion protein may comprise a light chain fusion based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 80% homologous to SEQ ID NOs: 1-4, 14, 15, 20 and 297. The dual fusion protein may a light chain fusion based on or derived from an amino acid sequence that is at least about 90% homologous to any one of SEQ ID NOs: 1-4, 14, 15, 20 and 297. The light chain fusion may further comprise a therapeutic agent based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NO: 210. The light chain fusion may further comprise a therapeutic agent based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to SEQ ID NO: 210. The light chain fusion may further comprise a therapeutic agent based on or derived from an amino acid sequence that is at least about 70% homologous to SEQ ID NO: 210. The light chain fusion may further comprise a therapeutic agent based on or derived from an amino acid sequence that is at least about 80% homologous to SEQ ID NO: 210. The light chain fusion may further comprise a therapeutic agent based on or derived from an amino acid sequence that is at least about 90% homologous to SEQ ID NO: 210.

At least a portion of the dual fusion protein may be encoded by one or more nucleic acid sequences that are at least about 50% homologous to any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304, and 240-247. At least a portion of the dual fusion protein may be encoded by one or more nucleic acid sequences that are at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304, and 240-247. At least a portion of the dual fusion protein may be encoded by one or more nucleic acid sequences that are at least about 70% homologous to any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304, and 240-247. At least a portion of the dual fusion protein may be encoded by one or more nucleic acid sequences that are at least about 80% homologous to any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304, and 240-247. At least a portion of the dual fusion protein may be encoded by one or more nucleic acid sequences that are at least about 90% homologous to any one of SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304, and 240-247.

The dual fusion protein may be comprise an antibody region that is encoded by one or more nucleotide sequences that are at least about 50% homologous to any one of SEQ ID NOs: 1-20 and 254-259. The dual fusion protein may be comprise an antibody region that is encoded by one or more nucleotide sequences that are at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 1-20 and 254-259. The dual fusion protein may be comprise an antibody region that is encoded by one or more nucleotide sequences that are at least about 70% homologous to any one of SEQ ID NOs: 1-20 and 254-259. The dual fusion protein may be comprise an antibody region that is encoded by one or more nucleotide sequences that are at least about 80% homologous to any one of SEQ ID NOs: 1-20 and 254-259. The dual fusion protein may be comprise an antibody region that is encoded by one or more nucleotide sequences that are at least about 90% homologous to any one of SEQ ID NOs: 1-20 and 254-259.

The dual fusion protein may comprise two or more therapeutic agents, wherein at least one of the therapeutic agents are encoded by a nucleotide sequence that is at least about 50% homologous to any one of SEQ ID NOs: 167-199. The therapeutic agent may be encoded by a nucleotide sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 167-199. The therapeutic agent may be encoded by a nucleotide sequence that is at least about 70% homologous to any one of SEQ ID NOs: 167-199. The therapeutic agent may be encoded by a nucleotide sequence that is at least about 80% homologous to any one of SEQ ID NOs: 167-199. The therapeutic agent may be encoded by a nucleotide sequence that is at least about 90% homologous to any one of SEQ ID NOs: 167-199.

The dual fusion protein may be comprise an antibody region that is based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 21-40 and 248-253. The dual fusion protein may be comprise an antibody region that is based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 21-40 and 248-253. The dual fusion protein may be comprise an antibody region that is based on or derived from an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 21-40 and 248-253. The dual fusion protein may be comprise an antibody region that is based on or derived from an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 21-40 and 248-253. The dual fusion protein may be comprise an antibody region that is based on or derived from an amino acid sequence that is at least about 90% homologous to any one of SEQ ID NOs: 21-40 and 248-253. The dual fusion protein may comprise two or more therapeutic agents, wherein at least one of the therapeutic agents are based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 200-235. The therapeutic agent may be based on or derived from an amino acid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% homologous to any one of SEQ ID NOs: 200-235. The therapeutic agent may be based on or derived from an amino acid sequence that is at least about 70% homologous to any one of SEQ ID NOs: 200-235. The therapeutic agent may be based on or derived from an amino acid sequence that is at least about 80% homologous to any one of SEQ ID NOs: 200-235. The therapeutic agent may be based on or derived from an amino acid sequence that is at least about 90% homologous to any one of SEQ ID NOs: 200-235.

Exemplary immunoglobulin dual fusion proteins are depicted in FIG. 2, Formula IIIA and Formula VIIA. As shown in Formula IIIA of FIG. 2, the immunoglobulin dual fusion protein may comprise (a) a first antibody region (A1) attached to a first extender fusion region comprising a first therapeutic agent (T1) attached to two extender peptides (E1, E2); and (b) a second antibody region (A2) attached to a second extender fusion region comprising a second therapeutic agent (T2) attached to two extender peptides (E3, E4). The immunoglobulin dual fusion proteins may further comprise one or more linkers and one or more proteolytic cleavage sites. The one or more proteolytic cleavage sites may be attached to the N- and/or C-terminus of a therapeutic agent. Proteolytic cleavage of the proteolytic cleavage site may release the N- and/or C-terminus of the therapeutic agent from the immunoglobulin fusion protein. Formula VIIA of FIG. 2 depicts an exemplary immunoglobulin dual fusion protein in which the N-terminus of the second therapeutic agent (T2) has been released. E3 may comprise a peptide selected from a beta strand, an alpha helix and a linker E4 may comprise a peptide selected from a beta strand, an alpha helix and a linker. An immunoglobulin dual fusion protein may comprise (a) a first antibody region (A1) attached to a first extender fusion region comprising a first therapeutic agent (T1) attached to two extender peptides (E1, E2); and (b) a second antibody region (A2) attached directly to a second therapeutic agent (T2). The therapeutic agent may comprise one or more secondary structures. The therapeutic agent may comprise a beta strand. The therapeutic agent may comprise an alpha helix. The therapeutic agent may comprise a linker. The therapeutic agent may comprise a rigid domain and/or region. The therapeutic agent may comprise a stalk-like feature. The therapeutic agent may comprise a knob-like feature. The therapeutic agent may comprise a beta sheet. The therapeutic agent may comprise a coiled coil. The therapeutic agent may comprise a hairpin structure. The therapeutic agent may comprise a beta hairpin structure.

The second extender fusion region may comprise one or more secondary structures, wherein the secondary structure comprises one or more alpha helices. The one or more alpha helices may form a coiled coil secondary structure. The extender peptides may comprise two or more alpha helices. By non-limiting example, the second extender fusion region may comprise a first extender peptide, wherein the first extender peptide comprises a first alpha helix and the second extender peptide comprises a second alpha helix. The extender peptides may comprise 3, 4, 5, 6, 7 or more alpha helices. The two or more alpha helices may be anti-parallel. The two or more alpha helices may be parallel. The two or more alpha helices may form one or more coiled-coil domains. The one or more alpha helices may comprise an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 135-138 and 145-160. The extender peptide may comprise an amino acid sequence that is at least about 50% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 135-138 and 145-160. The extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or more homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 135-138 and 145-160. The extender peptide may comprise an amino acid sequence that is at least about 70% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 135-138 and 145-160. The extender peptide may comprise an amino acid sequence that is at least about 80% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 135-138 and 145-160. The extender peptide may comprise an amino acid sequence that is at least about 85% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 135-138 and 145-160.

The first extender peptide may comprise an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 135-138, 149, 151, 153, 155, 157 and 159. The first extender peptide may comprise an amino acid sequence that is at least about 50% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 135-138, 149, 151, 153, 155, 157 and 159. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or more homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 135-138, 149, 151, 153, 155, 157 and 159. The first extender peptide may comprise an amino acid sequence that is at least about 75% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 135-138, 149, 151, 153, 155, 157 and 159. The first extender peptide may comprise an amino acid sequence that is at least about 80% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 135-138, 149, 151, 153, 155, 157 and 159.

The second extender peptide may comprise an amino acid sequence that is based on or derived from any one of SEQ ID NOs: 145-148, 150, 152, 154, 156, 158, and 160. The second extender peptide may comprise an amino acid sequence that is at least about 50% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 145-148, 150, 152, 154, 156, 158, and 160. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or more homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 145-148, 150, 152, 154, 156, 158, and 160. The second extender peptide may comprise an amino acid sequence that is at least about 70% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 145-148, 150, 152, 154, 156, 158, and 160. The second extender peptide may comprise an amino acid sequence that is at least about 80% homologous to an amino acid sequence based on or derived from any one of SEQ ID NOs: 145-148, 150, 152, 154, 156, 158, and 160.

The extender peptide may comprise the sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 129). X1-X14 may be independently selected from a positively charged amino acid or a hydrophobic amino acid. X1-X14 may be independently selected from the group comprising alanine (A), asparagine (N), isoleucine (I) leucine (L), valine (V), glutamine (Q), glutamic acid (E) and lysine (K). X1-X14 may be independently selected from the group comprising alanine (A), leucine (L) and lysine (K). Alanine may comprise at least about 30% of the total amino acid composition Alanine may comprise less than about 70% of the total amino acid composition. Leucine may comprise at least about 20% of the total amino acid composition. Leucine may comprise less than about 50% of the total amino acid composition. Lysine may comprise at least about 20% of the total amino acid composition. Lysine may comprise less than about 50% of the total amino acid composition. The hydrophobic amino acids may comprise at least about 50% of the total amino acid composition. The hydrophobic amino acids may comprise at least about 60% of the total amino acid composition. The hydrophobic amino acids may comprise at least about 70% of the total amino acid composition. The hydrophobic amino acids may comprise less than about 90% of the total amino acid composition.

The extender peptide may comprises the sequence (X1X2X3X4X5X6X7)n (SEQ ID NO. 310). N may be 1-5. N may be 1-3. X1-X7 may be independently selected from a positively charged amino acid or a hydrophobic amino acid. X1-X7 may be independently selected from the group comprising alanine (A), asparagine (N), isoleucine, (I), leucine (L), valine (V), glutamine (Q), glutamic acid (E) and lysine (K). Alanine (A) may comprise at least about 30% of the total amino acid composition Alanine (A) may comprise less than about 70% of the total amino acid composition. Leucine may comprise at least about 20% of the total amino acid composition. Leucine may comprise less than about 50% of the total amino acid composition. Lysine may comprise at least about 20% of the total amino acid composition. Lysine may comprise less than about 50% of the total amino acid composition. Asparagine may comprise about 50% of the total amino acid composition. Isoleucine may comprise about 50% of the total amino acid composition. Valine may comprise about 50% of the total amino acid composition. Glutamine may comprise about 50% of the total amino acid composition. Glutamic acid may comprise about 50% of the total amino acid composition. The hydrophobic amino acids may comprise at least about 50% of the total amino acid composition. The hydrophobic amino acids may comprise at least about 60% of the total amino acid composition. The hydrophobic amino acids may comprise at least about 70% of the total amino acid composition. The hydrophobic amino acids may comprise less than about 90% of the total amino acid composition.

The first extender peptide may comprise the sequence XaXbXcXd(X1X2X3X4X5X6X7)n (SEQ ID NO: 311). n may be equal to a number selected from any one of the numbers selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. n may be equal to 1. n may be equal to 2. n may be equal to 3. Xa, Xb and Xd may be independently selected from a hydrophobic amino acid. Xc may be a polar, uncharged amino acid. Xa, Xb and Xd may be the same amino acid. Xa, Xb and Xd may be different amino acids.

The first extender peptide may comprise the sequence XaXbXcXd(AKLAALK)n (SEQ ID NO. 312). n may be equal to a number selected from any one of the numbers selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. n may be equal to 1. n may be equal to 2. n may be equal to 3. X1-X7 may be independently selected from a positively charged amino acid or a hydrophobic amino acid. X1-X7 may be independently selected from the group comprising A, L and K. A may comprise at least about 30% of the total amino acid composition. A may comprise less than about 70% of the total amino acid composition. L may comprise at least about 20% of the total amino acid composition. L may comprise less than about 50% of the total amino acid composition. K may comprise at least about 20% of the total amino acid composition. K may comprise less than about 50% of the total amino acid composition. The hydrophobic amino acids may comprise at least about 50% of the total amino acid composition. The hydrophobic amino acids may comprise at least about 60% of the total amino acid composition. The hydrophobic amino acids may comprise at least about 70% of the total amino acid composition. The hydrophobic amino acids may comprise less than about 90% of the total amino acid composition. Xa, Xb and Xd may be independently selected from a hydrophobic amino acid. Xc may be a polar, uncharged amino acid. Xa, Xb and Xd may be the same amino acid. Xa, Xb and Xd may different amino acids. Xa, Xb and Xd may be Glycine (G). Xc may be Serine (S).

The first extender peptide may comprise the sequence (AKLAALK)n (SEQ ID NO. 313). n may be equal to a number selected from any one of the numbers selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. n may be equal to 1. n may be equal to 2. n may be equal to 3. The first extender peptide may comprise the sequence GGSG(AKLAALK)n (SEQ ID NO: 314). n may be equal to a number selected from any one of the numbers selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. n may be equal to 1. n may be equal to 2. n may be equal to 3. The second extender peptide may comprise the sequence XaXbXcXd (X1X2X3X4X5X6X7)n (SEQ ID NO: 311). n may be equal to a number selected from any one of the numbers selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. n may be equal to 1. n may be equal to 2. n may be equal to 3. X1-X7 may be independently selected from a positively charged amino acid or a hydrophobic amino acid. X1-X7 may be independently selected from the group comprising alanine (A), leucine (L) and lysine (K). A may comprise at least about 30% of the total amino acid composition. A may comprise less than about 70% of the total amino acid composition. L may comprise at least about 20% of the total amino acid composition. L may comprise less than about 50% of the total amino acid composition. K may comprise at least about 20% of the total amino acid composition. K may comprise less than about 50% of the total amino acid composition. The hydrophobic amino acids may comprise at least about 50% of the total amino acid composition. The hydrophobic amino acids may comprise at least about 60% of the total amino acid composition. The hydrophobic amino acids may comprise at least about 70% of the total amino acid composition. The hydrophobic amino acids may comprise less than about 90% of the total amino acid composition. Xa, Xb and Xd may be independently selected from a hydrophobic amino acid. Xc may be a polar, uncharged amino acid. Xa, Xb and Xd may be the same amino acid. Xa, Xb and Xd may different amino acids. Xa, Xb and Xd may be Glycine (G). Xc may be Serine (S).

The second extender peptide may comprise the sequence (ELAALEA)n XaXbXcXd (SEQ ID NO: 315). n may be equal to a number selected from any one of the numbers selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. n may be equal to 1. n may be equal to 2. n may be equal to 3. Xa, Xb and Xd may be independently selected from a hydrophobic amino acid. Xc may be a polar, uncharged amino acid. Xa, Xb and Xd may be the same amino acid. Xa, Xb and Xd may be different amino acids. Xa, Xb and Xd may be Glycine (G). Xc may be Serine (S).

The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 31; and (b) a second extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 39. The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 31; and (b) a second extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 39. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 31. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 39. The first extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 31. The first extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 31. The second extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 39. The second extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 39.

The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 136; and (b) a second extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 146. The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 136; and (b) a second extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 146. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 136. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 146. The first extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 136. The first extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 136. The second extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 146. The second extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 146.

The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 149; and (b) a second extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 150. The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 149; and (b) a second extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 150. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 149. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 150. The first extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 149. The first extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 149. The second extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 150. The second extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 150.

The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 151; and (b) a second extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 152. The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 151; and (b) a second extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 152. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 151. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 152. The first extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 151. The first extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 151. The second extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 152. The second extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 152.

The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 153; and (b) a second extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 154. The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 153; and (b) a second extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 154. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 153. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 154. The first extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 153. The first extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 153. The second extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 154. The second extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 154.

The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 155; and (b) a second extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 156. The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 155; and (b) a second extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 156. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 155. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 156. The first extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 155. The first extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 155. The second extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 156. The second extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 156.

The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 157; and (b) a second extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 158. The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 157; and (b) a second extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 158. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 157. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 158. The first extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 157. The first extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 215. The second extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 158. The second extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 158.

The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 159; and (b) a second extender peptide comprising an amino acid sequence based on or derived from SEQ ID NO: 160. The immunoglobulin fusion protein may comprise (a) a first extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 159; and (b) a second extender peptide comprising an amino acid sequence that is at least about 50% homologous to an amino acid sequence of SEQ ID NO: 160. The first extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 159. The second extender peptide may comprise an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to an amino acid sequence of SEQ ID NO: 160. The first extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 159. The first extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 159. The second extender peptide may comprise an amino acid sequencing comprising 3, 4, 5, 6, 7 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 160. The second extender peptide may comprise an amino acid sequencing comprising 5 or more amino acids based on or derived from an amino acid sequence of SEQ ID NO: 160.

The aliphatic amino acids may comprise at least about 20% of the total amino acids of the extender peptides. The aliphatic amino acids may comprise at least about 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45% or more of the total amino acids of the extender peptides. The aliphatic amino acids may comprise at least about 22% of the total amino acids of the extender peptides. The aliphatic amino acids may comprise at least about 27% of the total amino acids of the extender peptides.

The aliphatic amino acids may comprise less than about 50% of the total amino acids of the extender peptides. The aliphatic amino acids may comprise less than about 47%, 45%, 43%, 40%, 38%, 35%, 33% or 30% of the total amino acids of the extender peptides.

The aliphatic amino acids may comprise between about 20% to about 45% of the total amino acids of the extender peptides. The aliphatic amino acids may comprise between about 23% to about 45% of the total amino acids of the extender peptides. The aliphatic amino acids may comprise between about 23% to about 40% of the total amino acids of the extender peptides.

The aromatic amino acids may comprise less than about 20% of the total amino acids of the extender peptides. The aromatic amino acids may comprise less than about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% or 10% of the total amino acids of the extender peptides. The aromatic amino acids may comprise between 0% to about 20% of the total amino acids of the extender peptides.

The non-polar amino acids may comprise at least about 30% of the total amino acids of the extender peptides. The non-polar amino acids may comprise at least about 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% of the total amino acids of the extender peptides. The non-polar amino acids may comprise at least about 32% of the total amino acids of the extender peptides.

The non-polar amino acids may comprise less than about 80% of the total amino acids of the extender peptides. The non-polar amino acids may comprise less than about 77%, 75%, 72%, 70%, 69%, or 68% of the total amino acids of the extender peptides.

The non-polar amino acids may comprise between about 35% to about 80% of the total amino acids of the extender peptides. The non-polar amino acids may comprise between about 38% to about 80% of the total amino acids of the extender peptides. The non-polar amino acids may comprise between about 38% to about 75% of the total amino acids of the extender peptides. The non-polar amino acids may comprise between about 35% to about 70% of the total amino acids of the extender peptides.

The polar amino acids may comprise at least about 20% of the total amino acids of the extender peptides. The polar amino acids may comprise at least about 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35% or more of the total amino acids of the extender peptides. The polar amino acids may comprise at least about 23% of the total amino acids of the extender peptides.

The polar amino acids may comprise less than about 80% of the total amino acids of the extender peptides. The polar amino acids may comprise less than about 77%, 75%, 72%, 70%, 69%, or 68% of the total amino acids of the extender peptides. The polar amino acids may comprise less than about 77% of the total amino acids of the extender peptides. The polar amino acids may comprise less than about 75% of the total amino acids of the extender peptides. The polar amino acids may comprise less than about 72% of the total amino acids of the extender peptides.

The polar amino acids may comprise between about 25% to about 70% of the total amino acids of the extender peptides. The polar amino acids may comprise between about 27% to about 70% of the total amino acids of the extender peptides. The polar amino acids may comprise between about 30% to about 70% of the total amino acids of the extender peptides.

Alternatively, the immunoglobulin fusion proteins disclosed herein do not comprise an extender peptide.

Vectors, Host Cells and Recombinant Methods

Immunoglobulin fusion proteins, as disclosed herein, may be expressed by recombinant methods. Generally, a nucleic acid encoding an immunoglobulin fusion protein may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the immunoglobulin fusion protein may be prepared by PCR amplification and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to nucleotides encoding immunoglobulin fusion proteins). In an exemplary embodiment, nucleic acid encoding an immunoglobulin fusion protein is PCR amplified, restriction enzyme digested and gel purified. The digested nucleic acid may be inserted into a replicable vector. The replicable vector containing the digested immunoglobulin fusion protein insertion may be transformed or transduced into a host cell for further cloning (amplification of the DNA) or for expression. Host cells may be prokaryotic or eukaryotic cells.

Polynucleotide sequences encoding polypeptide components (e.g., antibody region, extender peptide, therapeutic agent) of the immunoglobulin fusion proteins may be obtained by PCR amplification. Polynucleotide sequences may be isolated and sequenced from cells containing nucleic acids encoding the polypeptide components. Alternatively, or additionally, polynucleotides may be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptide components may be inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic and/or eukaryotic hosts.

In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism may be used as transforming vectors in connection with these hosts. For example, bacteriophage such as λGEM™-11 may be utilized in making a recombinant vector which may be used to transform susceptible host cells such as E. coli LE392.

Immunoglobulin fusion proteins may be expressed intracellularly (e.g., cytoplasm) or extracellularly (e.g., secretion). For extracellular expression, the vector may comprise a secretion signal which enables translocation of the immunoglobulin fusion proteins to the outside of the cell.

Suitable host cells for cloning or expression of immunoglobulin fusion proteins-encoding vectors include prokaryotic or eukaryotic cells. The host cell may be a eukaryotic. Examples of eukaryotic cells include, but are not limited to, Human Embryonic Kidney (HEK) cell, Chinese Hamster Ovary (CHO) cell, fungi, yeasts, invertebrate cells (e.g., plant cells and insect cells), lymphoid cell (e.g., YO, NSO, Sp20 cell). Other examples of suitable mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); baby hamster kidney cells (BHK); mouse sertoli cells; monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells; MRC 5 cells; and FS4 cells. The host cell may be a prokaryotic cell (e.g., E. coli).

Host cells may be transformed with vectors containing nucleotides encoding an immunoglobulin fusion proteins. Transformed host cells may be cultured in media. The media may be supplemented with one or more agents for inducing promoters, selecting transformants, or amplifying or expressing the genes encoding the desired sequences. Methods for transforming host cells are known in the art and may include electroporation, calcium chloride, or polyethylene glycol/DMSO.

Alternatively, host cells may be transfected or transduced with vectors containing nucleotides encoding an immunoglobulin fusion proteins. Transfected or transduced host cells may be cultured in media. The media may be supplemented with one or more agents for inducing promoters, selecting transfected or transduced cells, or expressing genes encoding the desired sequences.

Additionally, host cells may express a protease that cleaves the proteolytic site of the immunoglobulin fusion protein. The host cells may be transfected or transduced with a polynucleotide, wherein the polynucleotide or portion thereof encodes the protease. The protease may be Factor Xa.

The expressed immunoglobulin fusion proteins may be secreted into and recovered from the periplasm of the host cells or transported into the culture media. Protein recovery from the periplasm may involve disrupting the host cell. Disruption of the host cell may comprise osmotic shock, sonication or lysis. Centrifugation or filtration may be used to remove cell debris or whole cells. The immunoglobulin fusion proteins may be further purified, for example, by affinity resin chromatography.

Alternatively, immunoglobulin fusion proteins that are secreted into the culture media may be isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides may be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

Immunoglobulin fusion proteins production may be conducted in large quantity by a fermentation process. Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins. Large-scale fermentations have at least 1000 liters of capacity, preferably about 1,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (a preferred carbon/energy source). Small scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase. A variety of inducers may be used, according to the vector construct employed, as is known in the art and described herein. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction time may be used.

To improve the production yield and quality of the immunoglobulin fusion proteins disclosed herein, various fermentation conditions may be modified. For example, to improve the proper assembly and folding of the secreted immunoglobulin fusion proteins polypeptides, additional vectors overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis,trans-isomerase with chaperone activity) may be used to co-transform the host prokaryotic cells. The chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells.

To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient for proteolytic enzymes may be used for the present disclosure. For example, host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some E. coli protease-deficient strains are available.

Standard protein purification methods known in the art may be employed. The following procedures are exemplary of suitable purification procedures: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography and gel filtration using, for example, Sephadex G-75.

Immunoglobulin fusion proteins may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon® ultrafiltration unit.

Protease inhibitors or protease inhibitor cocktails may be included in any of the foregoing steps to inhibit proteolysis of the immunoglobulin fusion proteins.

In some cases, an immunoglobulin fusion protein may not be biologically active upon isolation. Various methods for “refolding” or converting a polypeptide to its tertiary structure and generating disulfide linkages, may be used to restore biological activity. Such methods include exposing the solubilized polypeptide to a pH usually above 7 and in the presence of a particular concentration of a chaotrope. The selection of chaotrope is very similar to the choices used for inclusion body solubilization, but usually the chaotrope is used at a lower concentration and is not necessarily the same as chaotropes used for the solubilization. In most cases the refolding/oxidation solution will also contain a reducing agent or the reducing agent plus its oxidized form in a specific ratio to generate a particular redox potential allowing for disulfide shuffling to occur in the formation of the protein's cysteine bridge(s). Some of the commonly used redox couples include cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride, dithiothreitol(DTT)/dithiane DTT, and 2-mercaptoethanol(bME)/di-thio-b(ME). In many instances, a cosolvent may be used to increase the efficiency of the refolding, and common reagents used for this purpose include glycerol, polyethylene glycol of various molecular weights, arginine and the like.

Compositions

Disclosed herein are compositions comprising an immunoglobulin fusion protein and/or component of an immunoglobulin fusion protein disclosed herein. The compositions may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more immunoglobulin fusion proteins. The immunoglobulin fusion proteins may be different. Alternatively, the immunoglobulin fusion proteins may be the same or similar. The immunoglobulin fusion proteins may comprise different antibody regions, extender fusion regions, extender peptides, therapeutic agents or a combination thereof.

The compositions may further comprise one or more pharmaceutically acceptable salts, excipients or vehicles. Pharmaceutically acceptable salts, excipients, or vehicles for use in the present pharmaceutical compositions include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.

Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate carriers. The pharmaceutical compositions may include antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter ions such as sodium; and/or nonionic surfactants such as Tween, pluronics, or polyethylene glycol (PEG). Also by way of example, suitable tonicity enhancing agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol, and the like. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen peroxide also may be used as preservative. Suitable cosolvents include glycerin, propylene glycol, and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, and the like. The buffers may be conventional buffers such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be about pH 4-5.5, and Tris buffer may be about pH 7-8.5. Additional pharmaceutical agents are set forth in Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990.

The composition may be in liquid form or in a lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and/or bulking agents (see, for example, U.S. Pat. Nos. 6,685,940, 6,566,329, and 6,372,716). In one embodiment, a lyoprotectant is included, which is a non-reducing sugar such as sucrose, lactose or trehalose. The amount of lyoprotectant generally included is such that, upon reconstitution, the resulting formulation will be isotonic, although hypertonic or slightly hypotonic formulations also may be suitable. In addition, the amount of lyoprotectant should be sufficient to prevent an unacceptable amount of degradation and/or aggregation of the protein upon lyophilization. Exemplary lyoprotectant concentrations for sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilized formulation are from about 10 mM to about 400 mM. In another embodiment, a surfactant is included, such as for example, nonionic surfactants and ionic surfactants such as polysorbates (e.g., polysorbate 20, polysorbate 80); poloxamers (e.g., poloxamer 188); poly(ethylene glycol) phenyl ethers (e.g., Triton); sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl ofeyl-taurate; the MONAQUAT™ series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68 etc). Exemplary amounts of surfactant that may be present in the pre-lyophilized formulation are from about 0.001-0.5%. High molecular weight structural additives (e.g., fillers, binders) may include for example, acacia, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragacanth microcrystalline cellulose, starch, and zein. Exemplary concentrations of high molecular weight structural additives are from 0.1% to 10% by weight. In other embodiments, a bulking agent (e.g., mannitol, glycine) may be included.

Compositions may be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes. A parenteral formulation typically will be a sterile, pyrogen-free, isotonic aqueous solution, optionally containing pharmaceutically acceptable preservatives.

Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringers' dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, anti-microbials, anti-oxidants, chelating agents, inert gases and the like. See generally, Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.

Compositions described herein may be formulated for controlled or sustained delivery in a manner that provides local concentration of the product (e.g., bolus, depot effect) and/or increased stability or half-life in a particular local environment. The compositions may comprise the formulation of immunoglobulin fusion proteins, polypeptides, nucleic acids, or vectors disclosed herein with particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., as well as agents such as a biodegradable matrix, injectable microspheres, microcapsular particles, microcapsules, bioerodible particles beads, liposomes, and implantable delivery devices that provide for the controlled or sustained release of the active agent which then may be delivered as a depot injection. Techniques for formulating such sustained- or controlled-delivery means are known and a variety of polymers have been developed and used for the controlled release and delivery of drugs. Such polymers are typically biodegradable and biocompatible. Polymer hydrogels, including those formed by complexation of enantiomeric polymer or polypeptide segments, and hydrogels with temperature or pH sensitive properties, may be desirable for providing drug depot effect because of the mild and aqueous conditions involved in trapping bioactive protein agents. See, for example, the description of controlled release porous polymeric microparticles for the delivery of pharmaceutical compositions in WO 93/15722.

Suitable materials for this purpose include polylactides (see, e.g., U.S. Pat. No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids), such as poly-D-(−)-3-hydroxybutyric acid (EP 133,988A), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyric acid. Other biodegradable polymers include poly(lactones), poly(acetals), poly(orthoesters), and poly(orthocarbonates). Sustained-release compositions also may include liposomes, which may be prepared by any of several methods known in the art (see, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The carrier itself, or its degradation products, should be nontoxic in the target tissue and should not further aggravate the condition. This may be determined by routine screening in animal models of the target disorder or, if such models are unavailable, in normal animals.

The immunoglobulin fusion proteins disclosed herein may be microencapsulated.

A pharmaceutical composition disclosed herein can be administered to a subject by any suitable administration route, including but not limited to, parenteral (intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravitreal, infusion, or local), topical, oral, or nasal administration.

Formulations suitable for intramuscular, subcutaneous, peritumoral, or intravenous injection can include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles including water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for subcutaneous injection also contain optional additives such as preserving, wetting, emulsifying, and dispensing agents.

For intravenous injections, an active agent can be optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.

Parenteral injections optionally involve bolus injection or continuous infusion. Formulations for injection are optionally presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. The pharmaceutical composition described herein can be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of an active agent in water soluble form. Additionally, suspensions are optionally prepared as appropriate oily injection suspensions.

Alternatively or additionally, the compositions may be administered locally via implantation into the affected area of a membrane, sponge, or other appropriate material on to which an immunoglobulin fusion protein disclosed herein has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of an immunoglobulin fusion protein, nucleic acid, or vector disclosed herein may be directly through the device via bolus, or via continuous administration, or via catheter using continuous infusion.

A pharmaceutical composition comprising an immunoglobulin fusion protein disclosed herein may be formulated for inhalation, such as for example, as a dry powder. Inhalation solutions also may be formulated in a liquefied propellant for aerosol delivery. In yet another formulation, solutions may be nebulized. Additional pharmaceutical composition for pulmonary administration include, those described, for example, in WO 94/20069, which discloses pulmonary delivery of chemically modified proteins. For pulmonary delivery, the particle size should be suitable for delivery to the distal lung. For example, the particle size may be from 1 μm to 5 μm; however, larger particles may be used, for example, if each particle is fairly porous.

Certain formulations comprising an immunoglobulin fusion protein disclosed herein may be administered orally. Formulations administered in this fashion may be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of a selective binding agent. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders also may be employed.

Another preparation may involve an effective quantity of an immunoglobulin fusion protein in a mixture with non-toxic excipients which are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions may be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

Suitable and/or preferred pharmaceutical formulations may be determined in view of the present disclosure and general knowledge of formulation technology, depending upon the intended route of administration, delivery format, and desired dosage. Regardless of the manner of administration, an effective dose may be calculated according to patient body weight, body surface area, or organ size.

Further refinement of the calculations for determining the appropriate dosage for treatment involving each of the formulations described herein are routinely made in the art and is within the ambit of tasks routinely performed in the art. Appropriate dosages may be ascertained through use of appropriate dose-response data.

The compositions disclosed herein may be useful for providing prognostic or providing diagnostic information.

“Pharmaceutically acceptable” may refer to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

“Pharmaceutically acceptable salt” may refer to a salt of a compound that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.

“Pharmaceutically acceptable excipient, carrier or adjuvant” may refer to an excipient, carrier or adjuvant that may be administered to a subject, together with at least one antibody of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

“Pharmaceutically acceptable vehicle” may refer to a diluent, adjuvant, excipient, or carrier with which at least one antibody of the present disclosure is administered.

Kits

Further disclosed herein are kits which comprise one or more immunoglobulin fusion proteins or components thereof. The immunoglobulin fusion proteins may be packaged in a manner which facilitates their use to practice methods of the present disclosure. For example, a kit comprises an immunoglobulin fusion protein described herein packaged in a container with a label affixed to the container or a package insert that describes use of the immunoglobulin fusion protein in practicing the method. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kit may comprise a container with an immunoglobulin fusion protein contained therein. The kit may comprise a container with (a) an antibody region of an immunoglobulin fusion protein; (b) an extender fusion region of an immunoglobulin fusion protein; (c) an extender peptide of the extender fusion region; (d) a therapeutic agent of the extender fusion region; or (e) a combination of a-d. The kit may further comprise a package insert indicating that the first and second compositions may be used to treat a particular condition. Alternatively, or additionally, the kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer (e.g., bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution). It may further comprise other materials desirable from a commercial and user standpoint, including, but not limited to, other buffers, diluents, filters, needles, and syringes. The immunoglobulin fusion protein may be packaged in a unit dosage form. The kit may further comprise a device suitable for administering the immunoglobulin fusion protein according to a specific route of administration or for practicing a screening assay. The kit may contain a label that describes use of the immunoglobulin fusion protein composition.

The composition comprising the immunoglobulin fusion protein may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to mammals, such as humans, bovines, felines, canines, and murines. Typically, compositions for intravenous administration comprise solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and/or a local anaesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients may be supplied either separately or mixed together in unit dosage form. For example, the immunoglobulin fusion protein may be supplied as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the immunoglobulin fusion protein. Where the composition is to be administered by infusion, it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

The amount of the composition described herein which will be effective in the treatment, inhibition and/or prevention of a disease or disorder associated with aberrant expression and/or activity of a therapeutic agent may be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation may also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro, animal model test systems or clinical trials.

Therapeutic Use

Further disclosed herein are immunoglobulin fusion proteins for and methods of treating, alleviating, inhibiting and/or preventing one or more diseases and/or conditions. The method may comprise administering to a subject in need thereof a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one secondary structure; and (b) a therapeutic agent. The composition may further comprise a pharmaceutically acceptable carrier. The subject may be a mammal. The mammal may be a human. Alternatively, the mammal is a bovine. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The therapeutic agent may be GCSF, bovine GCSF, human GCSF, Moka1, Vm24, Mamba1, neutrophil elastase inhibitor, human GLP-1, Exendin-4, human EPO, human GMCSF, human interferon-beta, human interferon-alpha, relaxin, oxyntomodulin, leptin, betatrophin, growth differentiation factor 11 (GDF11), parathyroid hormone, angiopoietin-like 3 (ANGPTL3), IL-11, human growth hormone (hGH), BCCX2, CVX15, elafin or derivative or variant thereof. Alternatively, or additionally, therapeutic agent is interleukin 8 (IL-8), IL-21, ziconotide, somatostatin, chlorotoxin, SDF1 alpha or derivative or variation thereof. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The disease or condition may be an autoimmune disease, heteroimmune disease or condition, inflammatory disease, pathogenic infection, thromboembolic disorder, respiratory disease or condition, metabolic disease, central nervous system (CNS) disorder, bone disease or cancer. In other instances, the disease or condition is a blood disorder. In some instances, the disease or condition is obesity, diabetes, osteoporosis, anemia, or pain. The therapeutic agent may be hGCSF or a derivative thereof and the disease or condition may be neutropenia. The therapeutic agent may be leptin or a derivative thereof and the disease or condition may be diabetes. The therapeutic agent may be hGH or a derivative thereof and the disease or condition may be a growth disorder. The therapeutic agent may be IFN-alpha or a derivative thereof and the disease or condition may be a viral infection. The therapeutic agent may be Mamba1 or a derivative thereof and the disease or condition may be pain. The therapeutic agent may be BCCX2 and the disease or condition may be cancer. The therapeutic agent may be CVX15 or a derivative thereof and the disease or condition may be cancer. The therapeutic agent may be elafin and the disease or condition may be inflammation. The therapeutic agent may be a neutrophil elastase inhibitor and the condition may be pain.

The disease and/or condition may be a chronic disease or condition. Alternatively, the disease and/or condition is an acute disease or condition. The disease or condition may be recurrent, refractory, accelerated, or in remission. The disease or condition may affect one or more cell types. The one or more diseases and/or conditions may be an autoimmune disease, inflammatory disease, cardiovascular disease, metabolic disorder, pregnancy, and cell proliferative disorder.

The disease or condition may be an autoimmune disease. In some cases, the autoimmune disease may be scleroderma, diffuse scleroderma or systemic scleroderma.

The disease or condition may be an inflammatory disease. In some cases, the inflammatory disease may be hepatitis, fibromyalgia or psoriasis.

The disease or condition may be a rheumatic disease. In some cases, the rheumatic disease may be Ankylosing spondylitis, back pain, bursitis, tendinitis, shoulder pain, wrist pain, bicep pain, leg pain, knee pain, ankle pain, hip pain, Achilles pain, Capsulitis, neck pain, osteoarthritis, systemic lupus, erythematosus, rheumatoid arthritis, juvenile arthritis, Sjögren syndrome, Polymyositis, Behcet's disease, Reiter's syndrome, or Psoriatic arthritis. The rheumatic disease may be chronic. Alternatively, the rheumatic disease is acute.

The disease or condition may be a cardiovascular disease. In some cases, the cardiovascular disease may be acute heart failure, congestive heart failure, compensated heart failure, decompensated heart failure, hypercholesterolemia, atherosclerosis, coronary heart disease or ischemic stroke. The cardiovascular disease may be cardiac hypertrophy.

The disease or condition may be a metabolic disorder. In some cases, the metabolic disorder may be hypercholesterolemia, hypobetalipoproteinemia, hypertriglyceridemia, hyperlipidemia, dyslipidemia, ketosis, hypolipidemia, refractory anemia, appetite control, gastric emptying, non-alcoholic fatty liver disease, obesity, type I diabetes mellitus, type II diabetes mellitus, gestational diabetes mellitus, metabolic syndrome. The metabolic disorder may be type I diabetes. The metabolic disorder may be type II diabetes.

The disease or condition may be pregnancy. The immunoglobulin fusion proteins may be used to treat preeclampsia or induce labor.

The disease or condition may be a cell proliferative disorder. The cell proliferative disorder may be a leukemia, lymphoma, carcinoma, sarcoma, or a combination thereof. The cell proliferative disorder may be a myelogenous leukemia, lymphoblastic leukemia, myeloid leukemia, myelomonocytic leukemia, neutrophilic leukemia, myelodysplastic syndrome, B-cell lymphoma, burkitt lymphoma, large cell lymphoma, mixed cell lymphoma, follicular lymphoma, mantle cell lymphoma, hodgkin lymphoma, recurrent small lymphocytic lymphoma, hairy cell leukemia, multiple myeloma, basophilic leukemia, eosinophilic leukemia, megakaryoblastic leukemia, monoblastic leukemia, monocytic leukemia, erythroleukemia, erythroid leukemia, hepatocellular carcinoma, solid tumors, lymphoma, leukemias, liposarcoma (advanced/metastatic), myeloid malignancy, breast cancer, lung cancer, ovarian cancer, uterine cancer, kidney cancer, pancreatic cancer, and malignant glioma of brain.

Disclosed herein are methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition comprising an immunoglobulin fusion protein disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises an extender peptide and a therapeutic agent, wherein the therapeutic agent is oxyntomodulin. The disease or condition may be a metabolic disorder. The metabolic disorder may be diabetes. Diabetes may be type II diabetes mellitus. Diabetes may be type I diabetes. The metabolic disorder may be obesity. Additional metabolic disorders include, but are not limited to, metabolic syndrome, appetite control or gastric emptying.

Disclosed herein are methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition comprising an immunoglobulin fusion protein, wherein the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises an extender peptide and a therapeutic agent, wherein the therapeutic agent is relaxin. The disease or condition may be a cardiovascular disease. The cardiovascular disease may be acute heart failure. Additional cardiovascular diseases include, but are not limited to, congestive heart failure, compensated heart failure or decompensated heart failure. The disease or condition may be an autoimmune disorder. The autoimmune disorder may be scleroderma, diffuse scleroderma or systemic scleroderma. The disease or condition may be an inflammatory disease. The inflammatory disease may be fibromyalgia. The disease or condition may be fibrosis. Alternatively, the disease or condition is pregnancy. The immunoglobulin fusion protein may be used to treat preeclampsia or induce labor.

Disclosed herein are methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition comprising an immunoglobulin fusion protein disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises an extender peptide and a therapeutic agent, wherein the therapeutic agent is betatrophin. The disease or condition may be a metabolic disorder. The metabolic disorder may be obesity. Alternatively, the metabolic disorder is diabetes. Diabetes may be type I diabetes mellitus or type II diabetes mellitus.

Disclosed herein are methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition comprising an immunoglobulin fusion protein disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise GDF11. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises an extender peptide and a therapeutic agent, wherein the therapeutic agent is GDF11. The disease or condition may be a cell proliferative disorder. The cell proliferative disorder may be acute, chronic, recurrent, refractory, accelerated, in remission, stage I, stage II, stage III, stage IV, juvenile or adult. The cell proliferative disorder may be a myelogenous leukemia, lymphoblastic leukemia, myeloid leukemia, myelomonocytic leukemia, neutrophilic leukemia, myelodysplastic syndrome, B-cell lymphoma, burkitt lymphoma, large cell lymphoma, mixed cell lymphoma, follicular lymphoma, mantle cell lymphoma, hodgkin lymphoma, recurrent small lymphocytic lymphoma, hairy cell leukemia, multiple myeloma, basophilic leukemia, eosinophilic leukemia, megakaryoblastic leukemia, monoblastic leukemia, monocytic leukemia, erythroleukemia, erythroid leukemia, hepatocellular carcinoma, solid tumors, lymphoma, leukemias, liposarcoma (advanced/metastatic), myeloid malignancy, breast cancer, lung cancer, ovarian cancer, uterine cancer, kidney cancer, pancreatic cancer, and malignant glioma of brain. The disease or condition may be a cardiovascular disease. The cardiovascular disease may be age-related cardiac disease. The disease or condition may be cardiac hypertrophy.

Disclosed herein are methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition comprising an immunoglobulin fusion protein disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises an extender peptide and a therapeutic agent, wherein the therapeutic agent is angiopoietin-like 3. The metabolic disorder may be hypercholesterolemia, hypobetalipoproteinemia, hypertriglyceridemia, hyperlipidemia, dyslipidemia, hypolipidemia or ketosis. The disease or condition may be a cardiovascular disease. The cardiovascular disease may be atherosclerosis, coronary heart disease or ischemic stroke. The disease or condition may be a rheumatic disease. The rheumatic disease may be ankylosing spondylitis, back pain, bursitis, tendinitis, shoulder pain, wrist pain, bicep pain, leg pain, knee (patellar) pain, ankle pain, hip pain, Achilles pain, Capsulitis, Neck pain, osteoarthritis, systemic lupus, erythematosus, rheumatoid arthritis, juvenile arthritis, Sjögren syndrome, scleroderma, Polymyositis, Behcet's disease, Reiter's syndrome, Psoriatic arthritis. In some cases, the disease or condition may be a cell proliferative disorder. The cell proliferative disorder may be hepatocellular carcinoma or ovarian cancer. The disease or condition may be an inflammatory disease. The inflammatory disease may be hepatitis.

Disclosed herein may be a method of preventing or treating a disease or condition in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The immunoglobulin fusion protein may comprise one or more immunoglobulin heavy chains, light chains, or a combination thereof. The immunoglobulin fusion protein sequence may share 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more amino acid sequence identity to a heavy chain sequence provided by SEQ ID NOs: 76-108, 260-277, 298, 300, 302, and 304. The antibody region may comprise an immunoglobulin heavy chain. The immunoglobulin heavy chain polypeptide sequence may share 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more amino acid sequence identity to a heavy chain sequence provided by SEQ ID NOs: 24-27, 29-33, 36-39 and 251-253. The antibody region may comprise an immunoglobulin light chain. The immunoglobulin light chain polypeptide sequence may share 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more amino acid sequence identity to a light chain sequence provided by SEQ ID NOs: 21-23, 28, 34, 35 or 40. The immunoglobulin fusion protein may be encoded by a nucleotide sequence that is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more homologous to a nucleotide sequence of any one of SEQ ID NOs: 41-75 279-296, 299, 301, and 303. The immunoglobulin heavy chain may be encoded by a nucleotide sequence that is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more homologous to SEQ ID NOs: 5-13 or 16-19. The immunoglobulin light chain may be encoded by a nucleotide sequence that is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more homologous to SEQ ID NOs: 1-4, 14, 15 or 20. The immunoglobulin fusion protein may further comprise one or more linkers. The immunoglobulin fusion protein may further comprise one or more proteolytic cleavage sites. The disease or condition may be an autoimmune disease, heteroimmune disease or condition, inflammatory disease, pathogenic infection, thromboembolic disorder, respiratory disease or condition, metabolic disease, central nervous system (CNS) disorder, bone disease or cancer. The disease or condition may be a blood disorder. In some instances, the disease or condition may be obesity, diabetes, osteoporosis, anemia, or pain.

Disclosed herein is a method of preventing or treating an autoimmune disease in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The composition may further comprise a pharmaceutically acceptable carrier. The subject may be a mammal. The mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be Moka1 or a derivative or variant thereof. The therapeutic agent may be VM-24 or a derivative or variant thereof. The therapeutic agent may be beta-interferon or a derivative or variant thereof. The immunoglobulin fusion protein or antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. The immunoglobulin domain may be from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. The mammalian antibody may be a murine antibody. The antibody, antibody region or extender fusion region may further comprise a linker. The linker may attach Moka1, VM-24, beta-interferon, or a derivative or variant thereof to the extender peptide. The linker may attach the antibody region to the extender fusion region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The autoimmune disease may be a T-cell mediated autoimmune disease. T-cell mediated autoimmune diseases include, but are not limited to, multiple sclerosis, type-1 diabetes, and psoriasis. In other instances, the autoimmune disease lupus, Sjogren's syndrome, scleroderma, rheumatoid arthritis, dermatomyositis, Hasmimoto's thyroiditis, Addison's disease, celiac disease, Crohn's disease, pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, Ord's thyroiditis, Graves' disease, Guillain-Barre syndrome, acute disseminated encephalomyelitis, opsoclonus-myoclonus syndrome, ankylosing spondylitisis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hepatitis, Goodpasture's syndrome, Reiter's syndrome, Takayasu's arteritis, temporal arteritis, Wegener's granulomatosis, alopecia universalis, Behcet's disease, chronic fatigue, dysautonomia, endometriosis, interstitial cystitis, neuromyotonia, scleroderma, and vulvodynia. Lupus can include, but may be not limited to, acute cutaneous lupus erythematosus, subacute cutaneous lupus erythematosus, chronic cutaneous lupus erythematosus, discoid lupus erythematosus, childhood discoid lupus erythematosus, generalized discoid lupus erythematosus, localized discoid lupus erythematosus, chilblain lupus erythematosus (hutchinson), lupus erythematosus-lichen planus overlap syndrome, lupus erythematosus panniculitis (lupus erythematosus profundus), tumid lupus erythematosus, verrucous lupus erythematosus (hypertrophic lupus erythematosus), complement deficiency syndromes, drug-induced lupus erythematosus, neonatal lupus erythematosus, and systemic lupus erythematosus. The disease or condition may be multiple sclerosis. The disease or condition may be diabetes.

Further disclosed herein is a method of preventing or treating a disease or condition which would benefit from the modulation of a potassium voltage-gated channel in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The composition may further comprise a pharmaceutically acceptable carrier. The potassium voltage-gated channel may be a KCNA3 or Kv1.3 channel. The subject may be a mammal. The mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be Moka1 or a derivative or variant thereof. The therapeutic agent may be VM24 or a derivative or variant thereof. The immunoglobulin fusion protein or antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. The immunoglobulin domain may be from a mammalian antibody. Alternatively, the immunoglobulin domain may be from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody may be a murine antibody. The immunoglobulin fusion protein, antibody region, and/or extender fusion region may further comprise one or more linkers. The linker may attach Moka1, VM-24, or a derivative or variant thereof to the extender peptide. The linker may attach the antibody region to the extender fusion region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The disease or condition may be an autoimmune disease. The autoimmune disease may be a T-cell mediated autoimmune disease. The disease or condition may be episodic ataxia, seizure, or neuromyotonia. Modulating a potassium voltage-gated channel may comprise inhibiting or blocking a potassium voltage-gated channel. Modulating a potassium voltage-gated channel may comprise activating a potassium voltage-gated channel.

Disclosed herein are methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition comprising an immunoglobulin fusion protein disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises an extender peptide and a therapeutic agent, wherein the therapeutic agent is an antagonist of CXCR4. The antagonist of CXCR4 may be a 16 residue cyclic peptide analogue e of a horseshoe crab peptide polyphemusin (CVX15). The antagonist of CXCR4 may be a modified C VX15 (BCCX2). The CXCR4 antagonist may be encoded by an amino acid sequence selected from SEQ ID NOs: 231-234. The disease or condition may be an immunodeficiency. The disease or condition may be a viral infection and/or due to a viral infection. The disease or condition may be HIV and/or AIDS. The disease or condition may be whim syndrome. The disease or condition may be hypogammaglobulinemia. The disease or condition may be a tumor. The disease or condition may be a cancer. The disease or condition may be a metastatic cancer. The disease or condition may be hematopoietic stem cell mobilization. The disease or condition may be a cardiovascular disease.

Further disclosed herein are methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition comprising an immunoglobulin fusion protein disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises an extender peptide and a therapeutic agent, wherein the therapeutic agent is a neutrophil elastase inhibitor. The neutrophil elastase inhibitor may be encoded by an amino acid of SEQ ID NO. 235. The disease or condition may be selected from a neutropenia, a severe congenital neutropenia, pulmonary emphysema, cystic fibrosis, inflammation, postperfusion syndrome. The disease or condition may be a respiratory disorder. The disease or condition may be bronchoalveolar lavage fluid. The disease or condition may be an alpha 1-antitrypsin deficiency. The disease or condition may be asthma. The disease or condition may be chronic obstructive pulmonary disorder.

Provided herein is a method of preventing or treating a metabolic disease or condition in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The composition may further comprise a pharmaceutically acceptable carrier. The subject may be a mammal. The mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be GLP-1, Exendin-4, or a derivative or variant thereof. The GLP-1 may be a human GLP-1. The antibody or antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. The immunoglobulin domain may be from a mammalian antibody. Alternatively, the immunoglobulin domain may be from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody may be a murine antibody. The immunoglobulin fusion protein, antibody region, and/or extender fusion region may further comprise one or more linkers. The linker may attach GLP-1, Exendin-4, or a derivative or variant thereof to the extender peptide. The linker may attach the antibody region to the extender fusion region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. Metabolic diseases and/or conditions may include disorders of carbohydrate metabolism, amino acid metabolism, organic acid metabolism (organic acidurias), fatty acid oxidation and mitochondrial metabolism, porphyrin metabolism, purine or pyrimidine metabolism, steroid metabolism, mitochondrial function, peroxisomal function, urea cycle disorder, urea cycle defects or lysosomal storage disorders. The metabolic disease or condition may be diabetes. In other instances, the metabolic disease or condition may be glycogen storage disease, phenylketonuria, maple syrup urine disease, glutaric acidemia type 1, Carbamoyl phosphate synthetase I deficiency, alcaptonuria, Medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD), acute intermittent porphyria, Lesch-Nyhan syndrome, lipoid congenital adrenal hyperplasia, congenital adrenal hyperplasia, Kearns-Sayre syndrome, Zellweger syndrome, Gaucher's disease, or Niemann Pick disease.

Provided herein is a method of preventing or treating a central nervous system (CNS) disorder in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The composition may further comprise a pharmaceutically acceptable carrier. The subject may be a mammal. The mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be GLP-1, Exendin-4 or a derivative or variant thereof. The GLP-1 may be a human GLP-1. The antibody may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. The immunoglobulin domain may be from a mammalian antibody. Alternatively, the immunoglobulin domain may be from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody may be a murine antibody. The immunoglobulin fusion protein, antibody region, and/or extender fusion region may further comprise one or more linkers. The linker may attach GLP-1, Exendin-4, or a derivative or variant thereof to the immunoglobulin domain or fragment thereof. The linker may attach the antibody region to the extender fusion region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The CNS disorder may be Alzheimer's disease (AD). Additional CNS disorders include, but are not limited to, encephalitis, meningitis, tropical spastic paraparesis, arachnoid cysts, Huntington's disease, locked-in syndrome, Parkinson's disease, Tourette's, and multiple sclerosis.

Provided herein is a method of preventing or treating a disease or condition which benefits from a GLP-1R and/or glucagon receptor (GCGR) agonist in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The composition may further comprise a pharmaceutically acceptable carrier. The subject may be a mammal. The mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be GLP-1, Exendin-4 or a derivative or variant thereof. The GLP-1 may be a human GLP-1. The immunoglobulin fusion protein or antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. The immunoglobulin domain may be from a mammalian antibody. Alternatively, the immunoglobulin domain may be from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody may be a murine antibody. The immunoglobulin fusion protein, antibody region, and/or extender fusion region may further comprise one or more linkers. The linker may attach GLP-1, Exendin-4, or a derivative or variant thereof to the extender peptide. In other instances, the linker attaches the extender fusion region to the antibody region. The disease or condition may be a metabolic disease or disorder. The disease or condition may be diabetes. In other instances, the disease or condition may be obesity. Additional diseases and/or conditions which benefit from a GLP-1R and/or GCGR agonist include, but are not limited to, dyslipidemia, cardiovascular and fatty liver diseases.

Provided herein is a method of preventing or treating a blood disorder in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The composition may further comprise a pharmaceutically acceptable carrier. The subject may be a mammal. The mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be erythropoietin, GMCSF or a derivative or variant thereof. The erythropoietin may be a human erythropoietin. The GMCSF may be a human GMCSF. The antibody may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. The immunoglobulin domain may be from a mammalian antibody. Alternatively, the immunoglobulin domain may be from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody may be a murine antibody. The immunoglobulin fusion protein, antibody region, and/or extender fusion region may further comprise one or more linkers. The linker may attach erythropoietin, GMCSF, or a derivative or variant thereof to the extender peptide. The linker may attach the antibody region to the extender fusion region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The blood disorder may be anemia. Examples of anemia include, but are not limited to, hereditary xerocytosis, congenital dyserythropoietic anemia, Rh null disease, infectious mononucleosis related anemia, drugs-related anemia, aplastic anemia, microcytic anemia, macrocytic anemia, normocytic anemia, hemolytic anemia, poikilocytic anemia, spherocytic anemia, drepanocytic anemia, normochromic anemia, hyperchromic anemia, hypochromic anemia, macrocytic-normochromic anemia, microcytic-hypochromic anemia, normocytic-normochromic anemia, iron-deficiency anemia, pernicious anemia, folate-deficiency anemia, thalassemia, sideroblastic anemia, posthemorrhagic anemia, sickle cell anemia, chronic anemia, achrestic anemia, autoimmune haemolytic anemia, Cooley's anemia, drug-induced immune haemolytic anemia, erythroblastic anemia, hypoplastic anemia, Diamond-Blackfan anemia, Pearson's anemia, transient anemia, Fanconi's anemia, Lederer's anemia, myelpathic anemia, nutritional anemia, spur-cell anemia, Von Jaksh's anemia, sideroblatic anemia, sideropenic anemia, alpha thalassemia, beta thalassemia, hemoglobin h disease, acute acquired hemolytic anemia, warm autoimmune hemolytic anemia, cold autoimmune hemolytic anemia, primary cold autoimmune hemolytic anemia, secondary cold autoimmune hemolytic anemia, secondary autoimmune hemolytic anemia, primary autoimmune hemolytic anemia, x-linked sideroblastic anemia, pyridoxine-responsive anemia, nutritional sideroblastic anemia, pyridoxine deficiency-induced sideroblastic anemia, copper deficiency-induced sideroblastic anemia, cycloserine-induced sideroblastic anemia, chloramphenicol-induced sideroblastic anemia, ethanol-induced sideroblastic anemia, isoniazid-induced sideroblastic anemia, drug-induced sideroblastic anemia, toxin-induced sideroblastic anemia, microcytic hyperchromic anemia, macrocytic hyperchromic anemia, megalocytic-normochromic anemia, drug-induced immune hemolytic anemia, non-hereditary spherocytic anemia, inherited spherocytic anemia, and congenital spherocytic anemia. In other instances, the blood disorder may be malaria. Alternatively, the blood disorder may be lymphoma, leukemia, multiple myeloma, or myelodysplastic syndrome. The blood disorder may be neutropenia, Shwachmann-Daimond syndrome, Kostmann syndrome, chronic granulomatous disease, leukocyte adhesion deficiency, meyloperoxidase deficiency, or Chediak Higashi syndrome.

Provided herein is a method of preventing or treating a disease or disorder which benefits from stimulating or increasing white blood cell production in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The composition may further comprise a pharmaceutically acceptable carrier. The subject may be a mammal. The mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be GMCSF or a derivative or variant thereof. The GMCSF may be a human GMCSF. The immunoglobulin fusion protein or antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. The immunoglobulin domain may be from a mammalian antibody. Alternatively, the immunoglobulin domain may be from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody may be a murine antibody. The immunoglobulin fusion protein, antibody region, and/or extender fusion region may further comprise one or more linkers. The linker may attach the antibody region to the extender fusion region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The disease or disorder may be neutropenia, Shwachmann-Daimond syndrome, Kostmann syndrome, chronic granulomatous disease, leukocyte adhesion deficiency, meyloperoxidase deficiency, or Chediak Higashi syndrome.

Provided herein is a method of preventing or treating a disease or disorder which benefits from stimulating or increasing red blood cell production in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The composition may further comprise a pharmaceutically acceptable carrier. The subject may be a mammal. The mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be erythropoietin or a derivative or variant thereof. The erythropoietin may be a human erythropoietin. The antibody may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. The immunoglobulin domain may be from a mammalian antibody. Alternatively, the immunoglobulin domain may be from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody may be a murine antibody. The immunoglobulin fusion protein, antibody region, and/or extender fusion region may further comprise one or more linkers. The linker may attach erythropoietin, or a derivative or variant thereof to the extender peptide. The linker may attach the antibody region to the extender fusion region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The disease or disorder may be anemia.

Provided herein is a method of preventing or treating obesity in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The composition may further comprise a pharmaceutically acceptable carrier. The subject may be a mammal. The mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be GLP-1 or a derivative or variant thereof. The GLP-1 may be a human GLP-1. The therapeutic agent may be Exendin-4 or a derivative or variant thereof. The antibody may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. The immunoglobulin domain may be from a mammalian antibody. Alternatively, the immunoglobulin domain may be from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody may be a murine antibody. The immunoglobulin fusion protein, antibody region, and/or extender fusion region may further comprise one or more linkers. The linker may attach GLP-1, Exendin-4, or a derivative or variant thereof to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent.

Provided herein is a method of preventing or treating a pain in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The subject may be a mammal. In certain instances, the mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be a Mamba1 or a derivative or variant thereof. The immunoglobulin fusion proteins, antibody regions, and/or extender fusion regions may further comprise one or more linkers. The linker may attach the Mamba1 or a derivative or variant thereof to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent.

Provided herein is a method of preventing or treating a disease or condition which benefits from modulating a sodium ion channel in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The subject may be a mammal. In certain instances, the mammal may be a human. Alternatively, the mammal may be a bovine. The one or more antibodies, antibody fragments, or immunoglobulin constructs further comprise a linker. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. Modulating a sodium ion channel may comprise inhibiting or blocking a sodium ion channel. Modulating a sodium ion channel may comprise activating a sodium ion channel. The disease or condition may be Dravet Syndrome, generalized epilepsy with febrile seizures plus (GEFS+), paramyotonia congenital or erythromelalgia. The disease or condition may be pain.

Provided herein is a method of preventing or treating a disease or condition which benefits from modulating an acid sensing ion channel (ASIC) in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The subject may be a mammal. In certain instances, the mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be Mamba 1 or a derivative or variant thereof. The one or more antibodies, antibody fragments, or immunoglobulin constructs further comprise a linker. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. Modulating an ASIC may comprise inhibiting or blocking the ASIC. Modulating an ASIC may comprise activating the ASIC. The disease or condition may be a central nervous system disorder. In other instances, the disease or condition is pain.

Provided herein is a method of preventing or treating a pathogenic infection in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The composition may further comprise a pharmaceutically acceptable carrier. The subject may be a mammal. The mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be alpha-interferon or a derivative or variant thereof. The therapeutic agent may be beta-interferon or a derivative or variant thereof. The antibody may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. The immunoglobulin domain may be from a mammalian antibody. Alternatively, the immunoglobulin domain may be from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody may be a murine antibody. The immunoglobulin fusion protein, antibody region, and/or extender fusion region may further comprise one or more linkers. The linker may attach alpha-interferon, beta-interferon, or a derivative or variant thereof to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The pathogenic infection may be a bacterial infection. The pathogenic infection may be a fungal infection. The pathogenic infection may be a parasitic infection. The pathogenic infection may be a viral infection. The viral infection may be a herpes virus.

Provided herein is a method of preventing or treating a cancer in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The composition may further comprise a pharmaceutically acceptable carrier. The subject may be a mammal. The mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be beta-interferon or a derivative or variant thereof. The therapeutic agent may be CVX15 or a derivative or variant thereof. The therapeutic agent may be BCCX2 or a derivative or variant thereof. The antibody may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. The immunoglobulin domain may be from a mammalian antibody. Alternatively, the immunoglobulin domain may be from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody may be a murine antibody. The immunoglobulin fusion protein, antibody region, and/or extender fusion region may further comprise one or more linkers. The linker may attach beta-interferon, CVX15, BCCX2 or a derivative or variant thereof to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The cancer may be a hematological malignancy. The hematological malignancy may be a leukemia or lymphoma. The hematological malignancy may be a B-cell lymphoma, T-cell lymphoma, follicular lymphoma, marginal zone lymphoma, hairy cell leukemia, chronic myeloid leukemia, mantle cell lymphoma, nodular lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, chronic lymphocytic leukemia, or small lymphocytic leukemia.

Provided herein is a method of preventing or treating pain in a subject in need thereof comprising administering to the subject a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising an alpha helix and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The subject may be a mammal. In certain instances, the mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be a neutrophil elastase inhibitor or a derivative or variant thereof. The immunoglobulin fusion proteins, antibody regions, and/or extender fusion regions may further comprise one or more linkers. The linker may attach the neutrophil elastase inhibitor or a derivative or variant thereof to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent.

Provided herein is a method of preventing or treating a disease or condition which would benefit from modulation of a receptor in a subject in need thereof comprising administering to the subject a composition disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises one or more immunoglobulin fusion proteins comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The subject may be a mammal. In certain instances, the mammal may be a human. Alternatively, the mammal may be a bovine. The therapeutic agent may be hGCSF or a derivative or variant thereof and the receptor may be GCSFR. The therapeutic agent may be erythropoeitin or a derivative or variant thereof and the receptor may be EPOR. The therapeutic agent may be Exendin-4 or a derivative or variant thereof and the receptor may be GLP1R. The therapeutic agent may be GLP-1 or a derivative or variant thereof and the receptor may be GLP1R. The therapeutic agent may be leptin or a derivative or variant thereof and the receptor may be LepR. The therapeutic agent may be hGH or a derivative or variant thereof and the receptor may be GHR. The therapeutic agent may be interferon-alpha or a derivative or variant thereof and the receptor may be IFNR. The therapeutic agent may be interferon-beta or a derivative or variant thereof and the receptor may be IFNR. The therapeutic agent may be relaxin or a derivative or variant thereof and the receptor may be LGR7. The therapeutic agent may be CVX15 or a derivative or variant thereof and the receptor may be CXCR4. The therapeutic agent may be BCCX2 or a derivative or variant thereof and the receptor may be CXCR4. The therapeutic agent may be a neutrophil elastase inhibitor or a derivative or variant thereof. The therapeutic agent may be GMCSF or a derivative or variant thereof and the receptor may be GMCSFR. The one or more antibodies, antibody fragments, or immunoglobulin constructs further comprise a linker. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The disease or condition may be an autoimmune disease. The autoimmune disease may be a T-cell mediated autoimmune disease. The disease or condition may be a metabolic disorder. The metabolic disorder may be diabetes. The disease or condition may be an inflammatory disorder. The inflammatory disorder may be multiple sclerosis. The disease or condition may be a cell proliferative disorder. The disease or condition may be a blood disorder. The blood disorder may be neutropenia. The blood disorder may be anemia. The disease or condition may be a pathogenic infection. The pathogenic infection may be a viral infection. The disease or condition may be a growth disorder. The disease or condition may be a cardiovascular condition. The cardiovascular condition may be acute heart failure. Modulating the receptor may comprise inhibiting or blocking the receptor. Modulating the receptor may comprise activating the receptor. The therapeutic agent may act as a receptor agonist. The therapeutic agent may act as a receptor antagonist.

Provided herein is a method of preventing or treating a disease in a mammal in need thereof comprising administering a pharmaceutical composition described herein to said mammal. In some embodiments, the disease may be an infectious disease. In certain embodiments, the infectious disease may be mastitis. In some embodiments, the infectious disease may be a respiratory disease. In certain embodiments, the respiratory disease may be bovine respiratory disease of shipping fever. In certain embodiments, the mammal in need may be a dairy animal selected from a list comprising cow, camel, donkey, goat, horse, reindeer, sheep, water buffalo, moose and yak. In some embodiments, the mammal in need may be bovine.

Provided may be a method of preventing or treating mastitis in a dairy animal, comprising providing to said dairy animal an effective amount of a composition comprising one or more immunoglobulin fusion proteins disclosed herein. The immunoglobulin fusion protein may comprise an antibody region attached to a non-antibody region. The non-antibody region may comprise a therapeutic agent. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising an amino acid sequence comprising a beta strand and (i) an amino acid sequence comprising 7 or fewer amino acids based on or derived from an ultralong CDR3 or (ii) an amino acid sequence that does not comprise an ultralong CDR3; and (b) a therapeutic agent. The antibody region may comprise a heavy chain polypeptide based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 77 and 81. The antibody region may further comprise a light chain polypeptide based on or derived from an amino acid sequence that is at least about 50% homologous to any one of SEQ ID NOs: 21-23, 28, 34, 35, 40 and 278. The therapeutic agent may be GCSF. The GCSF may be a bovine GCSF. The GCSF may be a human GCSF. In some embodiments, the dairy animal may be a cow or a water buffalo.

Provided are methods of treatment, inhibition and prevention of a disease or condition in a subject in need thereof by administration to the subject of an effective amount of an immunoglobulin fusion protein or pharmaceutical composition described herein. The immunoglobulin fusion protein may be substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject may be an animal, including but not limited to animals such as cows, pigs, sheep, goats, rabbits, horses, chickens, cats, dogs, mice, etc. The subject may be a mammal. The subject may be a human. The subject may be a non-human primate. Alternatively, the subject may be a bovine. The subject may be an avian, reptile or amphibian.

Additional Uses

Further disclosed herein are uses of an immunoglobulin fusion protein (IFP) in the manufacture of a medicament for the treatment of a disease or condition. The IFP may be any of the IFPs disclosed herein. Disclosed herein is the use of an immunoglobulin fusion protein in the manufacture of a medicament for the treatment of a disease or condition, the immunoglobulin fusion protein comprising an antibody region attached to a non-antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. Further disclosed herein is the use of an immunoglobulin fusion protein in the manufacture of a medicament for the treatment of a disease or condition, the IFP comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one secondary structure; and (b) a therapeutic agent. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may comprise GCSF. The GCSF may be a human GCSF. The therapeutic agent may be Moka1. The therapeutic agent may be VM24. The therapeutic agent may be Exendin-4. The therapeutic agent may be erythropoietin. The erythropoietin may be a human erythropoeitin. The therapeutic agent may be leptin. The therapeutic agent may be a growth hormone (GH). The growth hormone may be a human growth hormone (hGH). The therapeutic agent may be inteferon-alpha. The therapeutic agent may be interferon-beta. The therapeutic agent may be GLP-1. The therapeutic agent may be relaxin. The therapeutic agent may be Mamba1. The therapeutic agent may be CVX15. The therapeutic agent may be BCCX2. The therapeutic agent may be a neutrophil elastase inhibitor. The therapeutic agent may be elafin. The therapeutic agent may be betatrophin. The therapeutic agent may be GDF11. The therapeutic agent may be GMCSF. The disease or condition may be an autoimmune disease, heteroimmune disease or condition, inflammatory disease, pathogenic infection, thromboembolic disorder, respiratory disease or condition, metabolic disease, central nervous system (CNS) disorder, bone disease or cancer. In other instances, the disease or condition is a blood disorder. In some instances, the disease or condition is obesity, diabetes, osteoporosis, anemia, or pain. The disease or condition may be a growth disorder.

Disclosed herein is the use of an immunoglobulin fusion protein in the manufacture of a medicament for the treatment of a cell proliferative disorder. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one secondary structure; and (b) a therapeutic agent. The cell proliferative disorder may be cancer. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule.

Disclosed herein is the use of an immunoglobulin fusion protein in the manufacture of a medicament for the treatment of a metabolic disorder. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one secondary structure; and (b) a therapeutic agent. The metabolic disorder may be diabetes. Diabetes may be type I diabetes. Diabetes may be type II diabetes. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be Exendin-4. The therapeutic agent may be GLP-1. The therapeutic agent may be leptin. The therapeutic agent may be betatrophin.

Disclosed herein is the use of an immunoglobulin fusion protein in the manufacture of a medicament for the treatment of an autoimmune disease or condition. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one secondary structure; and (b) a therapeutic agent. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be Moka1. The therapeutic agent may be VM24.

Disclosed herein is the use of an immunoglobulin fusion protein in the manufacture of a medicament for the treatment of an inflammatory disease or condition. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The inflammatory disease or condition may be multiple sclerosis. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be elafin. The therapeutic agent may be interferon-beta.

Disclosed herein is the use of an immunoglobulin fusion protein in the manufacture of a medicament for the treatment of a disease or condition of the central nervous system. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The disease or condition of the central nervous system may be pain. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be Mamba1.

Disclosed herein is the use of an immunoglobulin fusion protein in the manufacture of a medicament for the treatment of a cardiovascular disease or condition. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The cardiovascular disease or condition may be acute heart failure. The cardiovascular disease or condition may be cardiac hypertrophy. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be relaxin. The therapeutic agent may be GDF11.

Disclosed herein is the use of an immunoglobulin fusion protein in the manufacture of a medicament for the treatment of a hematological disease or condition. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The hematological disease or condition may be anemia. The hematological disease or condition may be neutropenia. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be GCSF. The GCSF may be a human GCSF. The therapeutic agent may be erythropoietin. The erythropoietin may be a human erythropoietin. The therapeutic agent may be GMCSF.

Disclosed herein is the use of an immunoglobulin fusion protein in the manufacture of a medicament for the treatment of a pathogenic infection. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The pathogenic infection may be a viral infection. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be interferon-alpha.

Disclosed herein is the use of an immunoglobulin fusion protein in the manufacture of a medicament for the treatment of a growth disorder. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprising an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. Examples of growth disorders included, but are not limited to, achondroplasia, achondroplasia in children, acromegaly, adiposogenital dystrophy, dwarfism, gigantism, Brooke Greenberg, hemihypertrophy, hypochondroplasia, Jansen's metaphy seal chondrodysplasia, Kowarski syndrome, Léri-Weill dyschondrosteosis, local gigantism, macrodystrophia lipomatosa, Majewski's polydactyly syndrome, microcephalic osteodysplastic primordial dwarfism type II, midget, overgrowth syndrome, parastremmatic dwarfism, primordial dwarfism, pseudoachondroplasia, psychosocial short stature, Seckel syndrome, short rib-polydactyly syndrome and Silver-Russell syndrome. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be a growth hormone. The growth hormone may be a human growth hormone (hGH).

Further disclosed herein are uses of an immunoglobulin fusion protein for the treatment of a disease or condition. Disclosed herein is the use of an immunoglobulin fusion protein for the treatment of a disease or condition in a subject in need thereof. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one secondary structure; and (b) a therapeutic agent. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may comprise GCSF. The GCSF may be a human GCSF. The therapeutic agent may be Moka1. The therapeutic agent may be VM24. The therapeutic agent may be Exendin-4. The therapeutic agent may be erythropoietin. The erythropoietin may be a human erythropoietin. The therapeutic agent may be leptin. The therapeutic agent may be a growth hormone (GH). The growth hormone may be a human growth hormone (hGH). The therapeutic agent may be interferon-alpha. The therapeutic agent may be interferon-beta. The therapeutic agent may be GLP-1. The therapeutic agent may be relaxin. The therapeutic agent may be Mamba1. The therapeutic agent may be CVX15. The therapeutic agent may be BCCX2. The therapeutic agent may be a neutrophil elastase inhibitor. The therapeutic agent may be elafin. The therapeutic agent may be betatrophin. The therapeutic agent may be GDF11. The therapeutic agent may be GMCSF. The disease or condition may be an autoimmune disease, heteroimmune disease or condition, inflammatory disease, pathogenic infection, thromboembolic disorder, respiratory disease or condition, metabolic disease, central nervous system (CNS) disorder, bone disease or cancer. In other instances, the disease or condition is a blood disorder. In some instances, the disease or condition is obesity, diabetes, osteoporosis, anemia, or pain. The disease or condition may be a growth disorder.

Disclosed herein is the use of an immunoglobulin fusion protein for the treatment of a cell proliferative disorder in a subject in need thereof. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The cell proliferative disorder may be cancer. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be CVX15. The therapeutic agent may be BCCX2.

Disclosed herein is the use of an immunoglobulin fusion protein for the treatment of a respiratory disorder in a subject in need thereof. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The respiratory disorder may be selected from asthma, bronchitis, emphysema, pneumonia, pulmonary hypertension, pulmonary edema, pleural mesothelioma, a respiratory tract infection, tuberculosis, pulmonary hyperplasia and a common cold. The respiratory disorder may be chronic obstructive pulmonary disorder. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof.

The therapeutic agent may be a neutrophil elastase inhibitor.

Disclosed herein is the use of an immunoglobulin fusion protein for the treatment of a metabolic disorder in a subject in need thereof. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The metabolic disorder may be diabetes. Diabetes may be type I diabetes. Diabetes may be type II diabetes. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be Exendin-4. The therapeutic agent may be GLP-1. The therapeutic agent may be leptin. The therapeutic agent may be betatrophin.

Disclosed herein is the use of an immunoglobulin fusion protein for the treatment of an autoimmune disease or condition in a subject in need thereof. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be Moka1. The therapeutic agent may be VM24.

Disclosed herein is the use of an immunoglobulin fusion protein for the treatment of an inflammatory disease or condition in a subject in need thereof. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The inflammatory disease or condition may be multiple sclerosis. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be elafin. The therapeutic agent may be interferon-beta.

Disclosed herein is the use of an immunoglobulin fusion protein for the treatment of a disease or condition of the central nervous system in a subject in need thereof. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The disease or condition of the central nervous system may be pain. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be Mamba1.

Disclosed herein is the use of an immunoglobulin fusion protein for the treatment of a cardiovascular disease or condition in a subject in need thereof. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The cardiovascular disease or condition may be acute heart failure. The cardiovascular disease or condition may be cardiac hypertrophy. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be relaxin. The therapeutic agent may be GDF11.

Disclosed herein is the use of an immunoglobulin fusion protein for the treatment of a hematological disease or condition in a subject in need thereof. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The hematological disease or condition may be anemia. The hematological disease or condition may be neutropenia. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be GCSF. The GCSF may be a human GCSF. The therapeutic agent may be erythropoietin. The erythropoietin may be a human erythropoietin. The therapeutic agent may be GMCSF.

Disclosed herein is the use of an immunoglobulin fusion protein for the treatment of a pathogenic infection in a subject in need thereof. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. The pathogenic infection may be a viral infection. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be interferon-alpha.

Disclosed herein is the use of an immunoglobulin fusion protein for the treatment of a growth disorder in a subject in need thereof. The IFP may be any of the IFPs disclosed herein. The IFP may comprise a non-antibody region attached to an antibody region, wherein the antibody region comprises 6 or fewer amino acids of an ultralong CDR3. The non-antibody region may comprise one or more therapeutic agents. In some instances, the immunoglobulin fusion protein comprises an antibody region attached to an extender fusion region, wherein the extender fusion region comprises (a) an extender peptide comprising at least one beta strand secondary structure; and (b) a therapeutic agent. The beta strand secondary structure may not comprise more than 7 consecutive amino acids from an ultralong CDR3 of SEQ ID NO. 248. Examples of growth disorders included, but are not limited to, achondroplasia, achondroplasia in children, acromegaly, adiposogenital dystrophy, dwarfism, gigantism, Brooke Greenberg, hemihypertrophy, hypochondroplasia, Jansen's metaphyseal chondrodysplasia, Kowarski syndrome, Léri-Weill dyschondrosteosis, local gigantism, macrodystrophia lipomatosa, Majewski's polydactyly syndrome, microcephalic osteodysplastic primordial dwarfism type II, midget, overgrowth syndrome, parastremmatic dwarfism, primordial dwarfism, pseudoachondroplasia, psychosocial short stature, Seckel syndrome, short rib-polydactyly syndrome and Silver-Russell syndrome. The extender fusion region may be inserted within the antibody region. The extender fusion region may be inserted within an immunoglobulin heavy chain of the antibody region. The extender fusion region may be inserted within an immunoglobulin light chain of the antibody region. The extender fusion region may be conjugated to the antibody region. The extender fusion region may be conjugated to a position within the antibody region. The antibody region may comprise one or more immunoglobulin domains. The immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain may be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody. Alternatively, the immunoglobulin domain is from a chimeric antibody. The immunoglobulin domain may be from an engineered antibody or recombinant antibody. The immunoglobulin domain may be from a humanized, human engineered or fully human antibody. The mammalian antibody may be a bovine antibody. The mammalian antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody. The immunoglobulin fusion protein, antibody region and/or extender fusion region may further comprise one or more linkers. The linker may attach therapeutic agent to the extender peptide. The linker may attach the extender fusion region to the antibody region. The linker may attach a proteolytic cleavage site to the antibody region, extender fusion region, extender peptide, or therapeutic agent. The therapeutic agent may be a peptide or derivative or variant thereof. Alternatively, therapeutic agent is a small molecule. The therapeutic agent may be a growth hormone. The growth hormone may be a human growth hormone (hGH).

Pharmacological Properties

Further disclosed herein are methods of improving one or more pharmacological properties of a therapeutic agent. The method may comprise producing an immunoglobulin fusion protein disclosed herein. Examples of pharmacological properties may include, but are not limited to, half-life, stability, solubility, immunogenicity, toxicity, bioavailability, absorption, liberation, distribution, metabolization, and excretion. Liberation may refer to the process of releasing of a therapeutic agent from the pharmaceutical formulation. Absorption may refer to the process of a substance entering the blood circulation. Distribution may refer to the dispersion or dissemination of substances throughout the fluids and tissues of the body. Metabolization (or biotransformation, or inactivation) may refer to the recognition by an organism that a foreign substance is present and the irreversible transformation of parent compounds into daughter metabolites. Excretion may refer to the removal of the substances from the body.

The half-life of a therapeutic agent may greater than the half-life of the non-conjugated therapeutic agent. The half-life of the therapeutic agent may be greater than 4 hours, greater than 6 hours, greater than 12 hours, greater than 24 hours, greater than 36 hours, greater than 2 days, greater than 3 days, greater than 4 days, greater than 5 days, greater than 6 days, greater than 7 days, greater than 8 days, greater than 9 days, greater than 10 days, greater than 11 days, greater than 12 days, greater than 13 days, or greater than 14 days when administered to a subject. The half-life of the therapeutic agent may be greater than 4 hours when administered to a subject. The half-life of the therapeutic agent may be greater than 6 hours when administered to a subject.

The half-life of the therapeutic agent may increase by at least about 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 or more hours. The half-life of the therapeutic agent may increase by at least about 2 hours. The half-life of the therapeutic agent may increase by at least about 4 hours. The half-life of the therapeutic agent may increase by at least about 6 hours. The half-life of the therapeutic agent may increase by at least about 8 hours.

The half-life of a therapeutic agent may be at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold greater than the half-life of the non-conjugated therapeutic peptide. The half-life of a therapeutic agent an antibody described herein may be at least about 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50-fold greater than the half-life of the non-conjugated therapeutic peptide. The half-life of a therapeutic agent an antibody described herein may be at least about 2-fold greater than the half-life of the non-conjugated therapeutic peptide. The half-life of a therapeutic agent an antibody described herein may be at least about 5-fold greater than the half-life of the non-conjugated therapeutic peptide. The half-life of a therapeutic agent an antibody described herein may be at least about 10-fold greater than the half-life of the non-conjugated therapeutic peptide.

The half-life of a therapeutic agent an antibody described herein may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% greater than the half-life of the non-conjugated therapeutic peptide. The half-life of a therapeutic agent an antibody described herein may be at least about 10% greater than the half-life of the non-conjugated therapeutic peptide. The half-life of a therapeutic agent an antibody described herein may be at least about 20% greater than the half-life of the non-conjugated therapeutic peptide. The half-life of a therapeutic agent an antibody described herein may be at least about 30% greater than the half-life of the non-conjugated therapeutic peptide. The half-life of a therapeutic agent an antibody described herein may be at least about 40% greater than the half-life of the non-conjugated therapeutic peptide. The half-life of a therapeutic agent an antibody described herein may be at least about 50% greater than the half-life of the non-conjugated therapeutic peptide.

EXAMPLES

Example 1

Constructing Vectors of Trastuzumab-Beta-Strand Based Fusion Proteins for Expression in Mammalian Cells

Genes encoding bovine GCSF (bGCSF), Moka1, Vm24, Exendin-4 (Ex-4), human growth hormone (hGH), human GCSF (hGCSF) and human erythropoietin (hEPO) were synthesized by Genscript or IDT, and amplified by polymerase chain reaction (PCR). To optimize the folding and stability of fusion proteins, flexible linkers of GGGGS (SEQ ID NO: 164) were added on both ends of the bGCSF, Moka1 and Vm24 fragments. A cleavage site of Factor Xa was placed in front of the N-terminal of Ex-4. A flexible CGGGGS linker (SEQ ID NO: 316) was added immediately before the Factor Xa protease cleavage site and a GGGGSC linker (SEQ ID NO: 317) was added at the end of C-terminal of Ex-4 gene fragment to increase folding and stability of the fusion protein. Then, sequences encoding ETKKYQS (SEQ ID NO: 111) and SYTYNYE (SEQ ID NO: 119) from bovine antibody BLV1H12, which forms antiparallel beta-strands, were added at the ends of the N- and C-terminal of the above designed gene fragments, respectively. Subsequently, PCR fragments encoding genes of interest were grafted into the complementarity determining region 3 of the heavy chain (CDR3H) of trastuzumab IgG antibody by exploiting overlap extension PCR, to replace the Trp99-Met107 loop. The trastuzumab-beta-strand based hEPO fusion protein was further modified to replace the hIgG1 CH1-CH3 constant region of trastuzumab with hIgG4 CH1-CH3 constant region containing triple mutants (S228P, F234A and L235A) to generate trastuzumab-beta hEPO HC (SEQ ID NO: 303). To generate a trastuzumab-beta hGH (CDR2H) fusion protein (SEQ ID NO: 48), a fragment encoding hGH, linkers, and extender peptides was grafted into the complementarity determining region 2 of the heavy chain (CDR2H) of trastuzumab IgG. The expression vectors of trastuzumab-beta-strand based fusion proteins were generated by in-frame ligation of the amplified fusion genes to the pFuse backbone vector (InvivoGen, CA). Similarly, the gene encoding the light chain of trastuzumab IgG antibody was cloned into the pFuse backbone vector. The obtained expression vectors were confirmed by DNA sequencing.

Example 2

Expression and Purification of Trastuzumab-Beta-Strand Based Fusion Proteins

Trastuzumab-beta-strand based fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding trastuzumab-beta-strand fusion protein heavy chain and the trastuzumab light chain. Expressed fusion proteins were secreted into the culture medium and harvested every 48 hours for twice after transfection. The fusion proteins were purified by Protein A/G chromatography (Thermo Fisher Scientific, IL), and analyzed by SDS-PAGE gel. Trastuzumab-beta-strand based Ex-4 fusion protein was further treated with Factor Xa protease (GE Healthcare) following manufacture's protocol to release N-terminal of fused peptide. After treatment, fusion proteins were re-purified by Protein A/G affinity column to remove protease and analyzed by SDS-PAGE gel.

Purified trastuzumab-beta bGCSF IgG (SEQ ID NOs: 77 and 21) are shown in FIG. 5. Lane 1 is a protein molecular weight marker, lane 2 is purified trastuzumab-beta bGCSF IgG, lane 3 is purified trastuzumab-beta bGCSF IgG treated with DTT, lane 4 is purified trastuzumab IgG (SEQ ID NOs: 24 and 21), and lane 5 is purified trastuzumab IgG treated with DTT.

Purified trastuzumab-beta Moka1 IgG (SEQ ID NOs: 79 and 21) and trastuzumab-beta Vm24 (SEQ ID NOs: 80 and 21) are shown in FIG. 10. Lane 1 is a protein molecular weight marker, lane 2 is purified trastuzumab-beta Moka1 IgG, lane 3 is purified trastuzumab-beta Moka1 IgG treated with DTT, lane 4 is purified trastuzumab-beta Vm24 IgG, and lane 5 is purified trastuzumab-beta Vm24 IgG treated with DTT.

Purified trastuzumab-beta hEPO (CDR3H) IgG is shown in FIG. 12. Lane 1 is a protein molecular weight marker, lane 2 is purified trastuzumab-beta hEPO IgG (SEQ ID NOs: 304 and 21), and lane 3 is purified trastuzumab-beta hEPO IgG (SEQ ID NOs: 304 and 21) treated with DTT.

Purified trastuzumab-beta hGH (CDR3H) IgG (SEQ ID NOs: 82 and 21) is shown in FIG. 15. Lane 1 is a protein molecular weight marker, lane 2 is purified trastuzumab-beta hGH (CDR3H) IgG, and lane 3 is purified trastuzumab-beta hGH (CDR3H) IgG treated with DTT.

Purified trastuzumab-beta hGH (CDR2H) IgG (SEQ ID NOs: 298 and 21) is shown in FIG. 16. Lane 1 is a protein molecular weight marker, lane 2 is purified trastuzumab-beta hGH (CDR2H) IgG, and lane 3 is purified trastuzumab-beta hGH (CDR2H) IgG treated with DTT.

Example 3

In Vitro Study of Trastuzumab-Beta-Strand bGCSF Fusion Protein Proliferative Activity on Mouse NFS-60 Cells

Mouse NFS-60 cells were obtained from American Type Culture Collection (ATCC), VA, and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.05 mM 2-mercapoethanol and 62 ng/ml human macrophage colony stimulating factor (M-CSF). For proliferation assays, mouse NFS-60 cells were washed three times with RPMI-1640 medium and re-suspended in RPMI-1640 medium with 10% FBS and 0.05 mM 2-mercapoethanol at a density of 1.5×105 cells/ml. In 96-well plates, 100 μl of cell suspension was added into each well, followed by the addition of varied concentrations of bGCSF (SEQ ID NO: 200), bovine antibody BLV1H12-beta-strand (bAb-beta-strand IgG), bovine antibody BLV1H12-beta-strand-bGCSF fusion protein (bAb-beta-strand-bGCSF L1 IgG), trastuzumab IgG (SEQ ID NOs: 24 and 21) and trastuzumab-beta-strand-bGCSF L1 IgG (SEQ ID NOs: 77 and 21). The plates were incubated at 37° C. in a 5% CO2 incubator for 72 hours. Cells were then treated with AlamarBlue (Invitrogen) ( 1/10 volume of cell suspension) for 4 hours at 37° C. Fluorescence at 595 nm for each well was read to indicate the cell viability. Table 1 and FIG. 6 show the fluorescence of the NFS-60 cells treated with various concentrations of bGCSF, bovine antibody BLV1H12-beta-strand (bAb-beta-strand IgG), bovine antibody BLV1H12-beta-strand-bGCSF fusion protein (bAb-beta-strand-bGCSF L1 IgG), trastuzumab IgG and trastuzumab-beta-strand-bGCSF L1 IgG. As shown in FIG. 6, Line 1 represents bovine antibody BLV1H12-beta-strand (bAb-beta-strand IgG), Line 2 represents bovine antibody BLV1H12-beta-strand-bGCSF fusion protein (bAb-beta-strand-bGCSF L1 IgG), Line 3 represents trastuzumab IgG, Line 4 represents trastuzumab-beta-strand-bGCSF L1 IgG and Line 5 represents bGCSF. The EC50 of bAb-beta-strand-bGCSF L1 was 2.41±0.20 ng/mL. The EC50 of trastuzumab-beta-strand-bGCSF L1 was 2.55±0.19 ng/mL. The EC50 of bGCSF was 4.87±0.29 ng/mL.

TABLE 1
bAb-beta-
bAb-beta-strand bGCSF
strand IgGFluorescenceL1 IgGFluorescenceTrastuzumabFluorescence
(ng/mL)Intensity(ng/mL)IntensityIgG (ng/mL)Intensity
10001536.300510007347.85610001465.7345
333.333331531.6825333.333337767.7615333.333331464.256
111.111111603.698111.111117854.0555111.111111497.443
37.037041595.26137.037047960.98237.037041533.4505
12.345681566.748512.345687724.0212.345681546.9655
4.115231734.5444.115236141.99054.115231613.3125
1.371741629.5751.371743506.50151.371741909.983
0.457251772.2010.457252544.66850.457251751.1505
0.152421684.4850.152422056.65350.152421596.733
0.050811661.19550.050811892.19550.050811674.4565
0.016941764.130.016942062.88350.016941729.6545
0.005651906.98250.005651977.53250.005651929.9635
Trastuzumab-
beta-strand
bGCSF L1FluorescencebGCSFFluorescence
IgG (ng/mL)Intensity(ng/mL)Intensity
10007667.93510007432.54
 333.333337880.162333.333337270.466
 111.111118011.944111.111117464.5905
37.037047745.673537.037046922.3095
12.345687171.862512.345686116.986
 4.115236003.36754.115234445.3315
 1.371743451.57451.371742734.641
 0.457252624.5370.457252178.667
 0.152421919.3860.152421880.8455
 0.050811853.410.050811864.7945
 0.016941974.5660.016942066.8105
 0.005651855.44250.005652012.753

Example 4

Binding of Trastuzumab-Beta-Strand-bGCSF L1 to her2 Receptor

The binding affinity of trastuzumab-beta-strand-bGCSF L1 (SEQ ID NOs: 77 and 21) to Her2 receptor was examined by ELISA. Human Her2-Fc chimera (5 ug/mL) (R&D Systems) was coated on 96-well ELISA plate overnight at 4° C., followed by blocking with 1% BSA in PBS (pH7.4) for 2 hours at 37° C. After washing with 0.05% Tween-20 in PBS (pH7.4), varied concentrations of trastuzumab IgG and trastuzumab-beta-strand-bGCSF L1 were added and incubated for 2 hours at 37° C. Subsequently, goat polyclonal anti-human kappa light chain antibody with HRP conjugate (Sigma) was added and incubated for 2 hours at 37° C. Wells were subsequently washed and binding affinities were examined on the basis of fluorescence intensity at 425 nm by adding fluorogenic peroxidase substrate to each well. Table 2 displays the fluorescence intensity at 425 nm of the trastuzumab IgG and trastuzumab-beta-bGCSF IgG (SEQ ID NOs: 77 and 21). FIG. 7 shows a graphical representation of the data in Table 2. The EC50 of trastuzumab IgG was 110±14 pM.

TABLE 2
Trastuzumab IgGFluorescenceTrastuzumab-beta-strandFluorescence
(pM)IntensitybGCSF L1 IgG (pM)Intensity
4074.0740713113.54754074.074071913.388
1358.0246911544.12751358.02469599.727
452.674910776.7925452.6749336.4235
150.891637846.828150.89163253.0485
50.297214164.89250.29721211.2645
16.765741994.774516.76574198.0155
5.588581023.49855.58858196.9245
1.86286566.87951.86286188.7095

Example 5

In Vitro Study of Trastuzumab-Beta-Strand Moka1 Fusion Protein Inhibitory Activities on Human Peripheral Blood Mononuclear Cells (PBMCs)/T Cells Activation

Human PBMCs were isolated from fresh venous blood of healthy donors through ficoll gradient centrifugation, followed by re-suspension in RPMI1640 medium with 10% FBS and plating in 96-well plates at a density of 1×106 cells/mL. Human T cells were purified from the isolated PBMCs using T cell enrichment kit. Purified PBMCs and T cells were pretreated for 1 h at 37° C. and 5% CO2 with various concentrations of purified trastuzumab-beta-strand Moka1 fusion protein (SEQ ID NOs: 79 and 21), and then activated by anti-CD3 and CD28 antibodies. After 24 h treatment, supernatants were collected and the levels of secreted TNF-α measured using an ELISA kit. A graphical representation of the data is shown in FIG. 11.

Example 6

Binding of Trastuzumab-Beta-Strand-Moka1 to her2 Receptor

The binding affinity of trastuzumab-beta-strand-Moka1 fusion proteins to Her2 receptor is examined by ELISA. Human Her2-Fc chimera (5 ug/mL) (R&D Systems) is coated on 96-well ELISA plate overnight at 4° C., followed by blocking with 1% BSA in PBS (pH7.4) for 2 hours at 37° C. After washing with 0.05% Tween-20 in PBS (pH7.4), varied concentrations of trastuzumab IgG and trastuzumab-beta-strand-Moka1 fusion proteins are added and incubated for 2 hours at 37° C. Subsequently, goat polyclonal anti-human kappa light chain antibody with HRP conjugate (Sigma) is added and incubated for 2 hours at 37° C. Wells are subsequently washed and binding affinities are examined on the basis of fluorescence intensity at 425 nm by adding fluorogenic peroxidase substrate to each well.

Example 7

In Vitro Study of Trastuzumab-Beta-Strand-VM24 Fusion Protein Inhibitory Activities on Human Peripheral Blood Mononuclear Cells (PBMCs) T Cells Activation

Human T cells are purified from isolated PBMCs using a T cell enrichment kit. Purified T cells were pretreated for 1 h at 37° C. and 5% CO2 with various concentrations of purified trastuzumab-beta-strand Vm24 fusion protein (SEQ ID NOs: 80 and 21), and then activated by anti-CD3 and CD28 antibodies. After 24 h treatment, supernatants were collected and the levels of secreted TNF-α were measured using an ELISA kit. A graphical representation of the data is shown in FIG. 11.

Example 8

Binding of Trastuzumab-Beta-Strand-VM24 to her2 Receptor

The binding affinity of trastuzumab-beta-strand-VM24 fusion proteins to Her2 receptor is examined by ELISA. Human Her2-Fc chimera (5 ug/mL) (R&D Systems) is coated on 96-well ELISA plate overnight at 4° C., followed by blocking with 1% BSA in PBS (pH7.4) for 2 hours at 37° C. After washing with 0.05% Tween-20 in PBS (pH7.4), varied concentrations of trastuzumab IgG and trastuzumab-beta-strand-VM24 fusion proteins are added and incubated for 2 hours at 37° C. Subsequently, goat polyclonal anti-human kappa light chain antibody with HRP conjugate (Sigma) is added and incubated for 2 hours at 37° C. Wells are subsequently washed and binding affinities are examined on the basis of fluorescence intensity at 425 nm by adding fluorogenic peroxidase substrate to each well.

Example 9

Expression and Purification of Trastuzumab-Beta-Strand Exendin-4 Based Fusion Proteins

Trastuzumab-beta-strand Exendin-4 based fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding trastuzumab-beta-strand Exendin-4 fusion protein heavy chain (SEQ ID NO: 78) and the trastuzumab light chain (SEQ ID NO: 21). Expressed fusion proteins were secreted into the culture medium and harvested at 48 and 96 hours after transfection. The fusion proteins were purified by Protein A/G chromatography (Thermo Fisher Scientific, IL), and analyzed by SDS-PAGE gel. Trastuzumab-beta-strand based Ex-4 fusion protein was further treated with Factor Xa protease (GE Healthcare) following manufacture's protocol to release N-terminal of fused peptide. After treatment, fusion proteins were re-purified by Protein A/G affinity column to remove protease and analyzed by SDS-PAGE gel. As shown in FIG. 8, Lane 1 and 4 contain the protein ladder, Lane 2 contains trastuzumab-beta strand-Exendin-4 fusion protein (SEQ ID NOs: 78 and 21), Lane 3 contains trastuzumab-beta strand-Exendin-4 fusion protein treated with DTT (SEQ ID NOs: 78 and 21), Lane 5 contains trastuzumab-beta strand-Exendin-4 fusion protein (SEQ ID NOs: 78 and 21) cleaved with Factor Xa, and Lane 6 contains trastuzumab-beta strand-Exendin-4 fusion protein (SEQ ID NOs: 78 and 21) cleaved with Factor Xa and treated with DTT.

Example 10

In Vitro Trastuzumab-Beta Exendin-4 Fusion Protein GLP-1 Receptor Activation Assay

HEK 293 cells overexpressing GLP-1 receptor (GLP-1R) and cAMP responsive element (CRE)-luciferase (Luc) reporter were grown in DMEM with 10% FBS at 37° C. with 5% CO2. Cells were seeded in 384-well plates at a density of 5000 cells per well and treated with various concentrations of Ex-4 peptide (SEQ ID NO: 201), trastuzumab-CDR3H-beta-Ex-4 (SEQ ID NO: 78 and 21) and trastuzumab-CDR3H-beta-Ex-4 RN (SEQ ID NOs: 78 and 21, after cleavage with Factor Xa) fusion proteins for 24 hours at 37° C. with 5% CO2. An immunoglobulin fusion protein which may be cleaved to release the amino-terminus of a therapeutic agent is referred to as RN, for released N-terminus Luminescence intensities were then measured using One-Glo (Promega, WI) luciferase reagent by following manufacturer's instruction. A graphical representation of the data is shown in FIG. 9.

Example 11

Binding of Trastuzumab-Beta-Strand-Exendin-4 to her2 Receptor

The binding affinity of trastuzumab-beta-strand-Exendin-4 fusion proteins to Her2 receptor is examined by ELISA. Human Her2-Fc chimera (5 ug/mL) (R&D Systems) is coated on 96-well ELISA plate overnight at 4° C., followed by blocking with 1% BSA in PBS (pH7.4) for 2 hours at 37° C. After washing with 0.05% Tween-20 in PBS (pH7.4), varied concentrations of trastuzumab IgG and trastuzumab-beta-strand-Exendin-4 fusion proteins are added and incubated for 2 hours at 37° C. Subsequently, goat polyclonal anti-human kappa light chain antibody with HRP conjugate (Sigma) is added and incubated for 2 hours at 37° C. Wells are subsequently washed and binding affinities are examined on the basis of fluorescence intensity at 425 nm by adding fluorogenic peroxidase substrate to each well.

Example 12

In Vitro Study of Trastuzumab-Beta-Strand hGCSF Fusion Protein Proliferative Activity on Mouse NFS-60 Cells

Mouse NFS-60 cells are obtained from American Type Culture Collection (ATCC), VA, and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.05 mM 2-mercapoethanol and 62 ng/ml human macrophage colony stimulating factor (M-CSF). For proliferation assays, mouse NFS-60 cells are washed three times with RPMI-1640 medium and re-suspended in RPMI-1640 medium with 10% FBS and 0.05 mM 2-mercapoethanol at a density of 1.5×105 cells/ml. In 96-well plates, 100 μl of cell suspension is added into each well, followed by the addition of varied concentrations of trastuzumab IgG and trastuzumab-beta-strand-hGCSF fusion proteins. The plates are incubated at 37° C. in a 5% CO2 incubator for 72 hours. Cells are then treated with AlamarBlue (Invitrogen) ( 1/10 volume of cell suspension) for 4 hours at 37° C. Fluorescence at 595 nm for each well was read to indicate the cell viability.

Example 13

Binding of Trastuzumab-Beta-Strand-hGCSF to her2 Receptor

The binding affinity of trastuzumab-beta-strand-hGCSF fusion proteins to Her2 receptor is examined by ELISA. Human Her2-Fc chimera (5 ug/mL) (R&D Systems) is coated on 96-well ELISA plate overnight at 4° C., followed by blocking with 1% BSA in PBS (pH7.4) for 2 hours at 37° C. After washing with 0.05% Tween-20 in PBS (pH7.4), varied concentrations of trastuzumab IgG and trastuzumab-beta-strand-hGCSF fusion proteins are added and incubated for 2 hours at 37° C. Subsequently, goat polyclonal anti-human kappa light chain antibody with HRP conjugate (Sigma) is added and incubated for 2 hours at 37° C. Wells are subsequently washed and binding affinities are examined on the basis of fluorescence intensity at 425 nm by adding fluorogenic peroxidase substrate to each well.

Example 14

Trastuzumab-Beta-Strand-hGH hGHR-Ba/F3 Proliferation Assay

Murine Ba/F3 cells cell lines were stably transduced with hGHR under EF1α promoter. Clonal selected hGHR-Ba/F3 were maintained in 10% FBS in RPMI1640 with 50 ng/mL of hGH. Proliferation assay was performed in 96 well culture plate with 20,000 cells in 200 uL assay medium (10% FBS in RPMI1640) per well. Increasing concentrations of fusion-antibodies were incubated with cells for 72 hours. At the end of incubation period, 20 ul of Prestoblue was added to each well, and fluorescent signal was recorded on a Spectramax fluorescence plate reader at 590 nm with 550 nm excitation. See Table 3 for results of trastuzumab-beta-strand-hGH activity assays.

Example 15

Trastuzumab-Beta-Strand-hGH NB2 Proliferation Assay

Rat Nb2-11 cell lines (Sigma) were maintained in 10% FBS, 10% horse serum (HS) in RPMI with 55 uM β-ME. A proliferation assay was performed in a 96 well culture plate with 50,000 cells in 200 uL assay medium (10% HS in RPMI with 55 uM β-ME) per well. Increasing concentrations of fusion-antibodies were incubated with cells for 72 hours. At the end of incubation period, 20 ul of Prestoblue was added to each well, and fluorescent signal was recorded on a Spectramax fluorescence plate reader at 590 nm with 550 nm excitation.

Example 16

Trastuzumab-Beta-Strand-hGH Stat5 Phosphorylation Assay

Human IM9 cells from ATCC were maintained in 10% FBS in RPMI1640. The night before assay, 2×10e5 IM9 cells were seeded into V bottom 96 well plate in 200 uL assay medium (1% charcoal stripped FBS in RPMI) and starved overnight. On the day of experiment, starved cells were stimulated with hGH and fusion-antibody at various concentration for 10 min at 37° C. After stimulation, cells were fixed by 4% formaldehyde at 37° C. for 10 min, and permeabalized with 90% methanol. Cells were then blocked with 5% BSA at room temperature for 10 min and stained with Alexa Fluor® 488 conjugated anti-pStat5 (Tyr694) (C71E5) Rabbit mAb (Cell Signaling Technology, Inc.) following manufacture suggested protocol. Cells were then washed with PBS and analyzed by a flow cytometer. See Table 3 for results of trastuzumab-beta-strand-hGH activity assays.

TABLE 3
IM9 STAT5
NB2Ba/F3-hGHRphos-
EC50 (nM)proliferationproliferationphorylation
hGH0.084 ± 0.0110.926 ± 0.0590.3525 ± 0.090
hGH-hAb-β (CDR3)0.406 ± 0.0592.851 ± 0.362 2.326 ± 0.441
hGH-hAb-β (CDR2)0.4667 ± 0.038

Example 17

In Vitro Proliferative Activity Assay of Trastuzumab-hEPO Fusion Protein on TF-1 Cells

Human TF-1 cells were cultured at 37° C. with 5% CO2 in RPMI-1640 medium containing 10% fetal bovine serum (FBS), penicillin and streptomycin (50 U/mL), and 2 ng/ml human granulocyte macrophage colony stimulating factor (GM-CSF). To examine the proliferative activity of trastuzumab-hEPO fusion proteins, cells were washed three times with RPMI-1640 medium with 10% FBS, resuspended in RPMI-1640 medium with 10% FBS at a density of 1.5×105 cells/ml, plated in 96-well plates (1.5×104 cells per well) with various concentrations of hEPO (206), trastuzumab, and trastuzumab-beta hEPO fusion protein (SEQ ID NOs: 304 and 21), and then incubated for 72 hours at 37° C. with 5% CO2. Cells were then treated with Alamar Blue (Life Technologies, CA) for 4 hours at 37° C. Fluorescence intensity measured at 595 nm is proportional to cell viability. The EC50 values were determined by fitting data into a logistic sigmoidal function: y=A2+(A1−A2)/(1 (x/x0)p), where A1 is the initial value, A2 is the final value, x0 is the inflection point of the curve, and p is the power. A graphical representation of the data is shown in FIG. 14.

Example 18

Rational Design, Expression and Purification of Beta Fusion Anti-CXCR4 Antibody

CVX15 is a 16 residue cyclic peptide which is an analogue of the horseshoe crab peptide polyphemusin and an antagonist of the chemokine receptor CXCR4. The x-ray crystal structure of a CVX15-CXCR4 complex reveals that the peptide is bound in a β-hairpin conformation with its N- and C-termini inserted into the transmembrane cavity of CXCR4 and its hairpin loop exposed to solvent (FIG. 45A). Optimized variations of modified CVX15 peptides (may be referred to as BCCX2) were used to generate novel CXCR4 antagonist antibodies using antibody BLV1H12. This bovine antibody has an ultralong (61 residues) CDRH3 with an anti-parallel β-sheet 20 Å in length, terminating in a disulfide cross-linked knob domain (FIG. 45B). Replacement of this knob domain afforded an antibody with an extended CDR that can bind the ligand binding cavity of CXCR4. Schematic representations of three candidate antibody fusion proteins are shown in FIG. 45C. Briefly, the unnatural amino acids naphthylamine and citrulline of CVX15 were replaced by tryptophan and lysine based on sequence alignment with the peptide T22 from which CVX15 was derived. Next the N- and C-termini of the peptide were fused to sequences that promote β-turns: Gly-Arg (YRKCRGGRRWCYQK in bAb-AC1 (SEQ ID NO: 231)), Pro-Arg (bAb-AC2, YRKCRGPRRWCYQK (SEQ ID NO: 232)), or Gly-Asn-Gly-Arg (SEQ ID NO: 318) (bAb-AC3, YRKCRGGNGRRWCYQK (SEQ ID NO: 233)). Based on the CVX15-CXCR4 complex structure, it was expected that such a β-turn linker would not affect the interaction of the peptide with CXCR4 Finally, the loop region of CVX15 that resides outside the binding pocket of CXCR4 was removed and the resulting inverse hairpin sequence was substituted for the knob domain of BLV1H12. The final designs of the antibody-CVX fusion proteins are illustrated in FIG. 45D.

Genes containing engineered antibodies were assembled by overlapping PCR and inserted into the pFuse backbone vector (InvivoGen, CA). The antibodies were expressed by transient transfection of FreeStyle 293F cells (Life Technologies, CA). Briefly, 293-F cells at a density of 106 cells/ml were transfected with heavy chain plasmid, light chain plasmid and 293fectin at a ratio of 2:1:6 as suggested by Life Technologies. Expression medium containing secreted proteins was harvested every 48 h twice after transfection. The three engineered antibodies were transiently expressed in FreeStyle 293 cells as a bovine-human chimera in which the Fc domain from human IgG1 was substituted for the bovine Fc. The antibodies were secreted into culture medium and purified by protein G column with yields of more than 5 mg/L (FIG. 46). The antibodies were purified by standard Protein A/G chromatography (Thermo Fisher Scientific, IL) and were analyzed by SDS-PAGE (FIGS. 46 and 47).

Example 19

Competition Assay of Beta Fusion Anti-CXCR4 Antibody by Flow Cytometry Analysis

Binding of the engineered antibodies to CXCR4 was measured by flow cytometry using human Jurkat cells, which highly express CXCR4. As shown in FIGS. 48A and 48B, all three antibodies (1 μg/ml) bind Jurkat cells, while the control antibody (BLV1H12) showed no detectable binding. To confirm that the observed binding is indeed mediated by CXCR4, flow cytometry experiments were performed using Chinese hamster ovary (CHO) cells (which have no detectable CXCR4 expression based on flow cytometry staining with a FITC labeled anti-CXCR4 antibody (clone 12G5)), with and without CXCR4 transfection (FIG. 48C). Incubation of CXCR4 transfected CHO cells with 1 μg/ml of the fusion antibodies resulted in a flow cytometry peak shift of 73.8%, 67.9% and 67.4% for bAb-AC1, bAb-AC2 and bAb-AC3, respectively. No peak shift was observed with non-transfected parental cells. In all cases, the control antibody showed no detectable binding. These results indicate that these engineered antibodies indeed bind specifically to CXCR4.

Cells were first blocked with blocking buffer (PBS supplemented with 3% BSA) at 4° C. for 10 min and then incubated with various concentrations of antibodies in blocking buffer for 1 h. Cells were then washed with PBS and incubated with Alexa Fluor 647 conjugated goat anti-human IgG (Life Technologies, CA) in blocking buffer following manufacturer's instruction. After incubation, cells were washed and analyzed by LSR II flow cytometer (Becton Dickinson). In a competition experiment, cells were pre-incubated with various concentrations of antibodies in blocking buffer at 4° C. for 30 min Fluorescein conjugated mouse anti-human CXCR4 monoclonal antibody (Clone 12G5, R&D system) was added in blocking buffer to a final concentration of 10 μg/mL for an additional 30 min Cells were then washed with PBS and analyzed by a flow cytometer.

Example 20

Tag-Lite HTRF Binding Assay of Beta Fusion Anti-CXCR4 Antibody

To accurately determine the binding affinity between the engineered antibodies and CXCR4, we applied Tag-lite homogeneous time resolved fluorescence (HTRF) (Cisbio Bioassays). Specific binding of fluorescently labeled SDF-1 to labeled SNAP-tag-CXCR4 results in a HTRF signal. The binding constant (Kd) between fluorescently labeled SDF-1 and the Tag-lite CXCR4 receptor was determined to be 14.2±1.2 nM (FIG. 49). A dose dependent competition was observed between the engineered antibodies and 50 nM of labeled SDF-1 (FIG. 50A). Assuming a competitive binding mode, the Kis of bAb-AC1, bAb-AC2 and bAb-AC3 to CXCR4 were calculated to be 2.1 nM, 5.4 nM and 19.8 nM, respectively. These results indicate that bAb-AC1 with a more flexible glycine at i+1 position of the hairpin turn binds the best to CXCR4, which is consistent with the flow cytometry analysis results. On the other hand, bAb-AC3 which has a β-turn promoting sequence (Asn-Gly) added at the end of the β-hairpin, has a decreased affinity compared to bAb-AC1 and bAb-AC2 that is probably due to spatial constraints within the CXCR4 ligand binding pocket.

Monoclonal antibody 12G5 is commonly used to assess CXCR4 expression as well as functionally inhibit the SDF1-CXCR4 interaction. The binding epitope of 12G5 includes extracellular loop (ECL) 2, as well as the N-terminus and ECL3. Because bAb-ACs were designed to bind the CXCR4 pocket, they should compete with binding of 12G5 to the receptor. To confirm this notion, a competition assay was performed between 12G5 and bAb-AC1 by flow cytometry. A dose dependent inhibition was observed for 12G5 binding to Jurkat cells by increasing concentrations of bAb-AC1 (FIG. 51). Flow cytometry analysis (FIG. 50B) indicated that a three-fold excess of bAb-AC1 is sufficient to completely block the binding of 12G5 to CXCR4 on Jurkat cells. FIGS. 59A and 59B show additional Tag-lite HTRF binding assays similarly performed for both bovine and human fusions.

The Tag-lite HTRF binding assay was performed by following manufacturer's suggested procedure. Briefly, 106 Tag-lite labeled CXCR4 cells were thawed at 37° C., centrifuged for 5 min at 1200 g, and re-suspended in 2.7 ml 1×Tag-lite buffer after removal of supernatant. The cells were incubated with increasing concentrations of antibodies and 50 nM of fluorescent ligand (Chemokine CXCR4 receptor red agonist) for 3 h at room temperature. The signal was recorded by an EnVision multi-label plate reader (PerkinElmer) at 620 nm and 665 nm with 340 nm excitation. The binding between CXCR4 and SDF-1 was represented by ratio of signal 665/620*10000. The Kis between antibodies and CXCR4 were calculated based on the Cheng-Prusoff equation: Ki=IC50/([A]/EC50+1), where [A] is the fixed concentration of SDF-1 and EC50 is the concentration of SDF-1 that results in half maximal activation of the CXCR4 receptor. Binding assay results for bovine and human summarized in Table 4 and FIGS. 59A-B. Ki=IC50/(([A]/EC50)+1) is the Cheng-Prusoff equation: where [A] is the fixed concentration of agonist and EC50 is the concentration of agonist that results in half maximal activation of the receptor.

TABLE 4
bAb-bAb-bAb-bAb-
AC1AC2AC3AC4HSCXHMCXHLCX12G5
IC509.5824.3789.334.16>300113.309.364.40
(nM)
Ki (nM)2.125.3919.760.92ND25.062.070.97

Example 21

BVL1H12-Beta BCCX2 HC 4-Antibody with Peptide Fused to CDRH2

BVL1H12-beta BCCX2 HC 4 fusion was designed by grafting the CDRH3 sequence from BVLH12-beta BCCX2 HC 1 into the CDRH2 of the BLV1H12 scaffold. The truncated CDRH3 of the resulting antibody was capped with a GGGGS linker (SEQ ID NO: 164) to give a new CDRH3 sequence, TSVHQGGGGSWHVDV (SEQ ID NO: 234). BLV1H12-beta BCCX2 HC 4 IgG was expressed in 293 cells with a much higher yield (17 mg/L) compared to BLV1H12-beta BCCX2 HC 1 IgG. This may be due to the fact that CDRH2 makes no direct contact with the rest of the antibody, and therefore has less effect on heavy chain and light chain packing compared to the CDRH3 fusion. Binding between BLV1H12-beta BCCX2 HC 4 IgG and CXCR4 was confirmed by both flow cytometry (FIG. 52) and a Tag-lite HTRF assay as described above (FIG. 53) to give a Ki value of 0.92 nM against the receptor. This result indicates that the CDRH2 is indeed a viable alternative to CDRH3 for functional peptide grafting and suggests that it may be possible to simultaneously graft two polypeptide agonists or antagonists into two distinct CDRs of a single antibody fusion protein.

Example 22

Beta Fusion CXCR4 Antibody Inhibits SDF-1 Induced Chemotaxis

The physiological function of SDF-1 is to trigger the migration and recruitment of CXCR4 expressing cells. A chemotaxis assay was used to test if bAb-ACs can block SDF-1 dependent cell migration (FIG. 54). An HTS transwell plate with 5.0 μm pore polycarbonate membrane (Corning Incorporated) was coated with human fibronectin (20 μg/ml) in PBS for 2 h at 37° C. Ramos cells were washed with PBS and re-suspended in assay medium (1% BSA in HEPES buffered RPMI) at a concentration of 106 cells/ml. Cells were starved in assay medium for 4 h at 37° C. and then incubated with various concentrations of antibodies for 1 h. After pre-incubation, 5×105 cells were loaded onto the top chamber of the transwell plate in a volume of 100 μL. The bottom wells were filled with 80 μl of SDF-1 (10 ng/ml) and antibodies at the same concentration as the corresponding top wells. Cell migration was allowed to proceed for 3 h at 37° C. Migrated cells were quantified by the addition of 10 μl prestoblue (Life Technologies, CA) and 10 μl FBS and fluorescent signal was recorded on a Spectramax fluorescence plate reader at 590 nm with 550 nm excitation.

Pre-incubation with the antibodies potently inhibited the migration of Ramos cells in a dose dependent manner (FIG. 55B) with EC50 values of 2.1 nM, 8.5 nM and 3.2 nM for 12G5, bAb-AC1 and bAb-AC4, respectively. 30 nM of bAb-AC4 completely neutralized SDF-1 induced migration of Ramos cells; while 12G5, even at its saturating concentration, could not 100% block the migration (FIGS. 55B and 55C). FIG. 58 shows a migration assay similarly performed for the human fusions.

Example 23

Beta Fusion CXCR4 Antibody Calcium Flux Assay

The engineered antibodies were tested for their ability to block CXCR4 dependent intracellular signaling. Activation of CXCR4 by SDF1 can be measured by intracellular calcium flux, a secondary messenger involved in GPCR signaling. Ramos cells, a non-Hodgkin lymphoma cell line, that highly express CXCR4, were washed with HBSS/HEPES (30 mM), and re-suspended in Fluo-4 direct labeling solution (Life Technologies, CA). The labeling reaction proceeded for 30 min at 37° C. and then room temperature for an additional 30 min Fluo-4 loaded cells were washed with HBSS/HEPES twice and re-suspended in the assay buffer (HBSS with 30 mM HEPES and 2.5 mM probenecid) at a density of 106 cells/ml. Antibodies were added and incubated with loaded cells for 1 h before reading the plate. Calcium flux signals were recorded on a fluorescence laser-imaging plate reader (FLIPR; Molecular Devices) immediately upon addition of SDF-1 at a final concentration of 50 nM. Cells loaded with Fluo-4 calcium indicators were incubated with 300 nM bAb-AC1, bAb-AC4 and the control antibody; SDF-1 mediated release of intracellular calcium was monitored by a fluorescence increase. bAb-AC1 significantly reduced calcium flux induced by 50 nM of SDF-1, whereas the same concentration of bAb-AC4 completely blocked the calcium signaling post SDF-1 activation (FIG. 55A and FIG. 56). These results indicate that these engineered antibodies are CXCR4 antagonists.

Example 24

Beta Fusion Anti-CXCR4 Antibody: Cell Culture and Maintenance

Jurkat and Ramos cells were maintained in RPMI-1640 (Life Technologies, CA) containing 10% (vol/vol) FBS (Life Technologies, CA) in 37° C. incubator with 5% CO2. CHO-S and 293-F cells were maintained between 0.2×106 and 2×106 cells/ml in FreeStyle medium (Life Technologies, CA) in Minitron shakers at 37° C.

Example 25

Expression and Purification of Beta Fusion Elastase Inhibitor Antibodies

BLV1H12 heavy chain or human antibody BVK heavy chain genes containing different CDR3s were cloned as C-terminal His6-tag (SEQ ID NO: 319) into the pFuse backbone vector (InvivoGen, CA). CDRs contained sequences encoding trypsin (control BLV1H12-tryspin inhibitor fusion antibody (BTI)) or neutrophil elastase inhibitor (BLV1H12-elastase inhibitor fusion antibody (BEI) and BVK elastase inhibitor fusion antibody (HEI). The antibodies were expressed through transient transfection of FreeStyle 293F cells using FreeStyle™ 293 Expression System (Life technologies Co., CA). Briefly, 293-F cells were maintained between 0.2×106 to 2×106/ml in 8% CO2 with 125 rpm shaking in Minitron Incubation Shakers at 37° C. Cells were transfected with heavy chain plasmid, light chain plasmid and 293fectin ratio 2:1:6 as suggested by Life technologies. Medium (FreeStyle 293 expression medium) containing secreted proteins was harvested every 48 hours twice after transfection. His6-tag (SEQ ID NO: 319) Fab antibodies were purified by Ni-NTA affinity chromatography (Qiagen, CA) according to the manufacturer's instructions and buffer exchanged four times with 10 KDa Amicon Ultra-15 (1).

Example 26

Beta Fusion Elastase Inhibitor Antibodies: Trypsin Inhibition Assay

BLV1H12 heavy chain genes containing different CDR3 were cloned as C-terminal His6-tag (SEQ ID NO: 319) into the pFuse backbone vector (InvivoGen, CA). The antibodies were expressed through transient transfection of FreeStyle 293F cells using FreeStyle™ 293 Expression System (Life technologies Co., CA). Briefly, 293-F cells were maintained between 0.2×106 to 2×106/ml in 8% CO2 with 125 rpm shaking in Minitron Incubation Shakers at 37° C. Cells were transfected with heavy chain plasmid, light chain plasmid and 293fectin ratio 2:1:6 as suggested by Life technologies. Medium (FreeStyle 293 expression medium) containing secreted proteins was harvested every 48 hours twice after transfection. His6-tag (SEQ ID NO: 319) Fab antibodies were purified by Ni-NTA affinity chromatography (Qiagen, CA) according to the manufacturer's instructions and buffer exchanged four times with 10 KDa Amicon Ultra-15 (Millipore) and were analyzed by SDS-PAGE (see FIGS. 60A, 61 and 63).

Example 27

Beta Fusion Elastase Inhibitor Antibodies: Biolayer Interferometry

Biolayer interferometry experiment was performed using an Octet RED instrument (ForteBio, Inc.). Briefly, his-tag labeled BEI Fab at 50 μg/ml in kinetics buffer (PBS, 0.01% BSA and 0.002% Tween 20) was immobilized onto Ni-NTA coated biosensor. Each biosensor was incubated with a different concentration of bovine trypsin ranging from (12.5 nM to 200 nM). The binding kinetics was monitored in real time with 4 min association and 4 min dissociation time. Kinetics parameters Kon, Koff and Kd was obtained by fitting the data into 1:1 binding mode using Octet system software. The Kon and Koff values were measured in real time at room temperature by Octet RED instrument (ForteBio, Inc.), and were determined to be 9.68×104±4.70×102 M−1 s−1 and 3.26×10−4±6.84×10−6 s−1 respectively. The calculated dissociation constant between BTI and trypsin is 3.37 nM (see FIG. 60B for plotted data).

Example 28

Elastase Inhibition Assay

Human neutrophil elastase was purchased from Elastin Products Company, Inc. Increasing concentration of BEI1 and BEI2 were incubated with 10 nM human NE for 20 min at room temperature, the residue activity of NE was analyzed by the addition of fluorogenic elastase substrate MeOSuc-AAPV-AMC (EMD Millipore) at a final concentration of 100 μM. The slope of reaction was obtained by monitoring at 420 nm wavelength with 325 nm excitation on Spectramax fluorescence plate reader. Each data point was triplicated and fit into morrison equation using Et=10 nM, S=100 μM, Km=130 μM as constant value: Q=(Ki*(1+(S/Km))). Y=Vo*(1−((((Et+X+Q)−(((Et+X+Q)̂2)−4*Et*X)̂0.5))/(2*Et))). FIGS. 62 and 64 show the inhibition of elastase by the bovine and human elastase inhibitor fusion antibodies.

Example 29

Construction of Trastuzumab GCSF/EPO Dual Fusion Protein

Bovine CDR3H fusion proteins were humanized using trastuzumab-based scaffold. Therapeutic polypeptides (EPO) fused to the bovine ultralong CDR3H region was grafted onto the CDR3H of trastuzumab and paired with GCSF fused to the CDR3L region of trastuzumab along with the engineered coiled coil and beta sheet “stalks.” The generated humanized biologically active fusion proteins potentially improve pharmacological propertied for treatment of relevant diseases.

Example 30

In Vitro Proliferative Activity of Trastuzumab GCSF/EPO Dual Fusion Protein on NFS-60 Cells

Mouse NFS-60 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.05 mM 2-mercapoethanol and 62 ng/ml human macrophage colony stimulating factor (M-CSF). For proliferation assay, mouse NFS-60 cells were washed three times with RPMI-1640 medium and resuspended in RPMI-1640 medium with 10% FBS and 0.05 mM 2-mercapoethanol at a density of 1.5×105 cells/ml. In 96-well plates, 100 μl of cell suspension was added into each well, followed by the addition of varied concentrations of hGCSF, His-tagged hGCSF, trastuzumab-H3-beta/hEPO-L3-coil/hGCSF dual fusion. The plates were incubated at 37° C. in a 5% CO2 incubator for 72 hours. Cells were then treated with AlamarBlue (Invitrogen) ( 1/10 volume of cell suspension) for 4 hours at 37° C. Fluorescence at 595 nm for each well was read to indicate the cell viability. See FIG. 66A for plotted data.

Example 31

In Vitro Proliferative Activity Assay of Trastuzumab GCSF/EPO Dual Fusion Protein on TF-1 Cells

Human TF-1 cells were cultured at 37° C. with 5% CO2 in RPMI-1640 medium containing 10% fetal bovine serum (FBS), penicillin and streptomycin (50 U/mL), and 2 ng/ml human granulocyte macrophage colony stimulating factor (GM-CSF). To examine the proliferative activity of trastuzumab-hEPO fusion proteins, cells were washed three times with RPMI-1640 medium with 10% FBS, resuspended in RPMI-1640 medium with 10% FBS at a density of 1.5×105 cells/ml, plated in 96-well plates (1.5×104 cells per well) with various concentrations of hEPO, trastuzumab, and trastuzumab-hEPO fusion proteins (SEQ ID NOs: 304 and 21), and then incubated for 72 hours at 37° C. with 5% CO2. Cells were then treated with Alamar Blue (Life Technologies, CA) for 4 hours at 37° C. Fluorescence intensity measured at 595 nm is proportional to cell viability. The EC50 values were determined by fitting data into a logistic sigmoidal function: y=A2+(A1−A2)/(1 (x/x0p), where A1 is the initial value, A2 is the final value, x0 is the inflection point of the curve, and p is the power. See Table 5 for EC50 values of the dual fusion and related controls, and FIG. 66B for plotted data.

TABLE 5
Trastuzumab-H3-
beta/hEPO-L3-His-tagged
coil/hGCSFhGCSFhGCSF
EC50 (ng/mL)36.1 +/− 8.01.0 +/− 0.010.7 +/− 0.5

Example 32

Electrospray Ionization Mass Spectrometry (ESI-MS) of Immunoglobulin Fusion Proteins

Purified immunoglobulin fusion proteins were treated overnight at 37° C. with Peptide-N-Glycosidase (NEB), followed by the addition of DTT. The fusion proteins were analyzed by ESI-MS using a 6520 Q-TOF LC/MS from Agilent Technology. A chromatograph for the ESI-MS of trastuzumab-CDR3H-beta-hEPO fusion protein (SEQ ID NOs: 304 and 21) is shown in FIG. 13.

Example 33

Construction and Purification of Bovine-Beta Fusion Proteins

To generate BLV1H12-beta Moka fusion proteins, the gene encoding Moka was synthesized by Genscript or IDT, and amplified by PCR. To generate a Moka1 L1 fusion protein, flexible linkers of GGGGS (SEQ ID NO: 164) were added on both ends of the Moka1 gene. Extender peptide sequences having beta strand secondary structure were added to both sides of the Moka1 L1 gene and Moka1 L0 (no linkers). The fragments were grafted into the BLV1H12 heavy chain to generate BLV1H12-beta Moka1 L0 HC and BLV1H12-beta Moka1 L1 HC. The BLV1H12 expression vectors were generated by in-frame ligation of the amplified fusion genes to the pFuse backbone vector (InvivoGen, CA). Similarly, the gene encoding the light chain of BLV1H12 was cloned to the pFuse backbone vector. The obtained expression vectors were confirmed by DNA sequencing.

BLV1H12-beta Moka1 L0 IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta Moka1 L0 protein heavy chain (SEQ ID NO: 261) and the BLV1H12 light chain (SEQ ID NO: 40). BLV1H12-beta Moka1 L1 IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta Moka1 L1 protein heavy chain (SEQ ID NO: 262) and the BLV1H12 light chain (SEQ ID NO: 40).

Purified BLV1H12-beta Moka1 L0 IgG (SEQ ID NOs: 261 and 40) and BLV1H12-beta Moka1 L1 (SEQ ID NOs: 262 and 40) are shown in FIG. 18. Lane 1 is a protein molecular weight marker, lane 2 is purified BLV1H12-beta Moka1 L0 IgG, lane 3 is purified BLV1H12-beta Moka1 L0 IgG treated with DTT, lane 4 is purified BLV1H12-beta Moka1 L1 IgG, and lane 5 is purified BLV1H12-beta Moka1 L1 IgG treated with DTT.

To generate BLV1H12-beta VM24 fusion proteins, the gene encoding VM24 was synthesized by Genscript or IDT, and amplified by PCR. To generate a VM24 L1 fusion protein, flexible linkers of GGGGS (SEQ ID NO: 164) were added on both ends of the VM24 gene. To generate a VM24 L2 fusion protein, flexible linkers of GGGGSGGGGS (SEQ ID NO: 320) were added on both ends of the VM24 gene. Extender peptide sequences having beta strand secondary structure were added to both sides of the VM24 L1 and VM24 L2 genes. The fragments were grafted into the BLV1H12 heavy chain to generate BLV1H12-beta VM24 L1 HC and BLV1H12-beta VM24 L2 HC. The BLV1H12 expression vectors were generated by in-frame ligation of the amplified fusion genes to the pFuse backbone vector (InvivoGen, CA). Similarly, the gene encoding the light chain of BLV1H12 was cloned to the pFuse backbone vector. The obtained expression vectors were confirmed by DNA sequencing.

BLV1H12-beta VM24 L1 IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta VM24 L1 protein heavy chain (SEQ ID NO: 263) and the BLV1H12 light chain (SEQ ID NO: 40). BLV1H12-beta VM24 L2 IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta VM24 L2 protein heavy chain (SEQ ID NO: 264) and the BLV1H12 light chain (SEQ ID NO: 40).

Purified BLV1H12-beta VM24 L1 IgG (SEQ ID NOs: 263 and 40) and BLV1H12-beta VM24 L2 (SEQ ID NOs: 264 and 40) are shown in FIG. 21. Lane 1 is a protein molecular weight marker, lane 2 is purified BLV1H12-beta VM24 L1 IgG, lane 3 is purified BLV1H12-beta VM24 L1 IgG treated with DTT, lane 4 is purified BLV1H12-beta VM24 L2 IgG, and lane 5 is purified BLV1H12-beta VM24 L2 IgG treated with DTT.

To generate BLV1H12-beta hEPO fusion proteins, the gene encoding hEPO was synthesized by Genscript or IDT, and amplified by PCR. To generate a hEPO fusion protein, flexible linkers of GGGGS (SEQ ID NO: 164) were added on both ends of the hEPO gene. Extender peptide sequences having beta strand secondary structure were added to both sides of the hEPO gene. The fragment was grafted into the BLV1H12 heavy chain to generate BLV1H12-beta hEPO HC. The BLV1H12 expression vector was generated by in-frame ligation of the amplified fusion genes to the pFuse backbone vector (InvivoGen, CA). Similarly, the gene encoding the light chain of BLV1H12 was cloned to the pFuse backbone vector. The obtained expression vectors were confirmed by DNA sequencing.

BLV1H12-beta hEPO IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta hEPO protein heavy chain (SEQ ID NO: 267) and the BLV1H12 light chain (SEQ ID NO: 40).

Purified BLV1H12-beta hEPO IgG (SEQ ID NOs: 267 and 40) are shown in FIG. 24. Lane 1 is a protein molecular weight marker, lane 2 is purified BLV1H12-beta hEPO IgG, and lane 3 is purified BLV1H12-beta hEPO IgG treated with DTT.

To generate BLV1H12-beta GLP-1 fusion proteins, the gene encoding GLP-1 was synthesized by Genscript or IDT, and amplified by PCR. To generate a GLP-1 RN (released N-terminus) heavy chain fusion protein, a Factor Xa cleavage site of IEGR was added to the N-terminus of the GLP-1 gene to generate a GLP-1 RN fragment. Flexible linkers of GGGGS (SEQ ID NO: 164) were added on both ends of the GLP-1 RN fragment. Extender peptide sequences having beta strand secondary structure were added to both sides of the GLP-1 RN fragment having linker peptide sequences. The GLP-1 RN, linker, extender peptide fragment was grafted into the BLV1H12 heavy chain to generate BLV1H12-beta GLP-1 RN HC. The BLV1H12 expression vector was generated by in-frame ligation of the amplified fusion genes to the pFuse backbone vector (InvivoGen, CA). Similarly, the gene encoding the light chain of BLV1H12 was cloned to the pFuse backbone vector. The obtained expression vectors were confirmed by DNA sequencing.

BLV1H12-beta GLP-1 RN IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta GLP-1 RN protein heavy chain (SEQ ID NO: 265) and the BLV1H12 light chain (SEQ ID NO: 40). The fusion proteins were purified and cleaved with Factor Xa to generated a clipped fusion protein.

Purified BLV1H12-beta GLP-1 RN IgG (SEQ ID NOs: 265 and 40) are shown in FIG. 29. Lane 1 is a protein molecular weight marker, lane 2 is purified BLV1H12-beta GLP-1 RN IgG, lane 3 is purified BLV1H12-beta GLP-1 RN IgG treated with DTT, lane 6 is purified BLV1H12-beta GLP-1 RN IgG cleaved with Factor Xa, and lane 7 is purified BLV1H12-beta GLP-1 RN IgG cleaved with Factor Xa and treated with DTT.

To generate BLV1H12-beta Ex-4 fusion proteins, the gene encoding Ex-4 was synthesized by Genscript or IDT, and amplified by PCR. To generate an Ex-4 RN (released N-terminus) heavy chain fusion protein, a Factor Xa cleavage site of IEGR was added to the N-terminus of the Ex-4 gene to generate an Ex-4 RN fragment. Flexible linkers of GGGGS (SEQ ID NO: 164) were added on both ends of the Ex-4 RN fragment. Extender peptide sequences having beta strand secondary structure were added to both sides of the Ex-4 RN fragment having linker peptide sequences. The Ex-4 RN, linker, extender peptide fragment was grafted into the BLV1H12 heavy chain to generate BLV1H12-beta Ex-4 RN HC. The BLV1H12 expression vector was generated by in-frame ligation of the amplified fusion genes to the pFuse backbone vector (InvivoGen, CA). Similarly, the gene encoding the light chain of BLV1H12 was cloned to the pFuse backbone vector. The obtained expression vectors were confirmed by DNA sequencing.

BLV1H12-beta Ex-4 RN IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta Ex-4 RN protein heavy chain (SEQ ID NO: 266) and the BLV1H12 light chain (SEQ ID NO: 40). The fusion proteins were purified and cleaved with Factor Xa to generate a clipped fusion protein.

Purified BLV1H12-beta Ex-4 RN IgG (SEQ ID NOs: 266 and 40) are shown in FIG. 29. Lane 1 is a protein molecular weight marker, lane 4 is purified BLV1H12-beta Ex-4 RN IgG, lane 5 is purified BLV1H12-beta Ex-4 RN IgG treated with DTT, lane 8 is purified BLV1H12-beta Ex-4 RN IgG cleaved with Factor Xa, and lane 9 is purified BLV1H12-beta Ex-4 RN IgG cleaved with Factor Xa and treated with DTT.

To generate BLV1H12-beta hLeptin fusion proteins, the gene encoding hLeptin was synthesized by Genscript or IDT, and amplified by PCR. Flexible linkers of GGGGS (SEQ ID NO: 164) were added on both ends of the hLeptin fragment. Extender peptide sequences having beta strand secondary structure were added to both sides of the hLeptin fragment having linker peptide sequences. The hLeptin, linker, extender peptide fragment was grafted into the BLV1H12 heavy chain to generate BLV1H12-beta hLeptin HC. The BLV1H12 expression vector was generated by in-frame ligation of the amplified fusion genes to the pFuse backbone vector (InvivoGen, CA). Similarly, the gene encoding the light chain of BLV1H12 was cloned to the pFuse backbone vector. The obtained expression vectors were confirmed by DNA sequencing.

BLV1H12-beta hLeptin IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta hLeptin protein heavy chain (SEQ ID NO: 230) and the BLV1H12 light chain (SEQ ID NO: 40).

Purified BLV1H12-beta hLeptin IgG (SEQ ID NOs: 230 and 40) are shown in FIG. 43A. Lane 1 is a protein molecular weight marker, lane 2 is purified BLV1H12-beta hLeptin IgG, and lane 3 is purified BLV1H12-beta hLeptin IgG treated with DTT.

To generate BLV1H12-beta relaxin fusion proteins, fragments encoding relaxin2, relaxin2 (GGSIEGR (SEQ ID NO: 307)), and relaxin2 (IEGRCpeptideIEGR (SEQ ID NO: 321)) were synthesized by Genscript or IDT, and amplified by PCR. Linker peptides encoding GGGGS (SEQ ID NO: 164) were added to the N-terminus and C-terminus of the relaxin fragments. Extender peptide ETKKYQS (SEQ ID NO: 111) was then added to the N-terminus of relaxin-linker fragments and extender peptide SYTYNYE (SEQ ID NO: 119) was added to the C-terminus of the relaxin-linker fragments. The relaxin, linker, extender peptide fragments were grafted into the BLV1H12 heavy chain to generate BLV1H12-beta relaxin HC fusions. The BLV1H12 expression vectors were generated by in-frame ligation of the amplified fusion genes to the pFuse backbone vector (InvivoGen, CA). Similarly, the gene encoding the light chain of BLV1H12 was cloned to the pFuse backbone vector. The obtained expression vectors were confirmed by DNA sequencing.

BLV1H12-beta relaxin2 IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta relaxin2 heavy chain (SEQ ID NO: 274) and the BLV1H12 light chain (SEQ ID NO: 40). BLV1H12-beta relaxin2 (GGSIEGR (SEQ ID NO: 307)) IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta relaxin2 (GGSIEGR (SEQ ID NO: 307)) heavy chain (SEQ ID NO: 275) and the BLV1H12 light chain (SEQ ID NO: 40). BLV1H12-beta relaxin2 (IEGRCpepIEGR (SEQ ID NO: 321)) IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta relaxin2 (IEGRCpepIEGR (SEQ ID NO: 321)) heavy chain (SEQ ID NO: 276) and the BLV1H12 light chain (SEQ ID NO: 40). The fusion proteins were purified and some were cleaved with Factor Xa to generated a clipped fusion protein.

Purified BLV1H12-beta relaxin IgGs are shown in FIGS. 44 A-C: (A) BLV1H12-CDR3H-beta human relaxin2 clip fusion protein (SEQ ID NOs: 274 and 40), with and without reducing agent; (B) BLV1H12-CDR3H-beta human relaxin clip fusion protein with engineered connector peptide (SEQ ID NOs: 276 and 40), with and without reducing agent; and (C) BLV1H12-CDR3H-beta human relaxin clip fusion protein with GGSIEGR linker (SEQ ID NO: 307) (SEQ ID NOs: 275 and 40), with and without reducing agent.

To generate BLV1H12-beta BCCX fusion proteins, fragments encoding BCCX2 were synthesized by Genscript or IDT, and amplified by PCR. Peptides encoding ETKKYQS (SEQ ID NO: 111) were added to the N-terminus and peptides encoding SYTYNYE (SEQ ID NO: 119) were added to the C-terminus of the BCCX2 fragments. The fragments were grafted into the BLV1H12 heavy chain to generate BLV1H12-beta BCCX2 HC fusions. The BLV1H12 expression vectors were generated by in-frame ligation of the amplified fusion genes to the pFuse backbone vector (InvivoGen, CA). Similarly, the gene encoding the light chain of BLV1H12 was cloned to the pFuse backbone vector. The obtained expression vectors were confirmed by DNA sequencing.

BLV1H12-beta BCCX2 HC 1 IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta BCCX2 HC 1 (SEQ ID NO: 92) and the BLV1H12 light chain (SEQ ID NO: 40). BLV1H12-beta BCCX2 HC 4 fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta HC4 (SEQ ID NO: 95) and the BLV1H12 light chain (SEQ ID NO: 40).

Purified BLV1H12-beta BCCX2 IgGs are shown in FIG. 46. Lane 1 is a protein molecular weight marker, lane 2 is BLV1H12-beta BCCX2 HC4 (bAb-AC4) IgG (SEQ ID NOs: 94 and 40), lane 3 is BLV1H12-beta BCCX2 HC4 (bAb-AC4) IgG (SEQ ID NOs: 94 and 40) treated with DTT, lane 4 is BLV1H12-beta BCCX2 HCl (bAb-AC1) IgG (SEQ ID NOs: 92 and 40), and lane 5 is BLV1H12-beta BCCX2 HCl (bAb-AC1) IgG (SEQ ID NOs: 92 and 40) treated with DTT.

Example 34

In Vitro Study of BLV1H12-Beta-Strand bGCSF Fusion Protein Proliferative Activity on Mouse NFS-60 Cells

Mouse NFS-60 cells were obtained from American Type Culture Collection (ATCC), VA, and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.05 mM 2-mercapoethanol and 62 ng/ml human macrophage colony stimulating factor (M-CSF). For proliferation assays, mouse NFS-60 cells were washed three times with RPMI-1640 medium and re-suspended in RPMI-1640 medium with 10% FBS and 0.05 mM 2-mercapoethanol at a density of 1.5×105 cells/ml. In 96-well plates, 100 μl of cell suspension was added into each well, followed by the addition of varied concentrations of bGCSF (SEQ ID NO: 200), hGCSF, BLV1H12 IgG, BLV1H12-beta bGCSF L0 fusion protein (Ab-bGCSF L0), and BLV1H12-beta bGCSF L1 fusion protein (Ab-bGCSF L1). The plates were incubated at 37° C. in a 5% CO2 incubator for 72 hours. Cells were then treated with AlamarBlue (Invitrogen) ( 1/10 volume of cell suspension) for 4 hours at 37° C. Fluorescence at 595 nm for each well was read to indicate the cell viability. FIGS. 37 A-E shows the fluorescence of the NFS-60 cells treated with various concentrations of bGCSF (SEQ ID NO: 200), hGCSF, BLV1H12 IgG, BLV1H12-beta bGCSF L0 fusion protein (Ab-bGCSF L0), and BLV1H12-beta bGCSF L1 fusion protein (Ab-bGCSF L1).

Example 35

In Vitro Study of BLV1H12-Beta-Strand Moka1 Fusion Protein Inhibitory Activities on Human Peripheral Blood Mononuclear Cells (PBMCs)/T Cells Activation

Human PBMCs were isolated from fresh venous blood of healthy donors through ficoll gradient centrifugation, followed by re-suspension in RPMI1640 medium with 10% FBS and plating in 96-well plates at a density of 1×106 cells/mL. PBMCs and T cells were pretreated for 1 h at 37° C. and 5% CO2 with various concentrations of BLV1H12 IgG, purified BLV1H12-beta Moka1L0 fusion protein (SEQ ID NOs: 261 and 40) and purified BLV1H12-beta Moka L1 fusion protein (SEQ ID NOs:262 and 40), and then activated by anti-CD3 and CD28 antibodies. After 24 h treatment, supernatants were collected and the levels of secreted TNF-α measured using an ELISA kit. A tabular representation of the data is shown in FIG. 19.

Example 36

In Vitro Study of BLV1H12-Beta-Strand Moka1 Fusion Protein Inhibitory Activities on Human Peripheral Blood Mononuclear Cells (PBMCs)/T Cells Activation

Human PBMCs were isolated from fresh venous blood of healthy donors through ficoll gradient centrifugation, followed by re-suspension in RPMI1640 medium with 10% FBS and plating in 96-well plates at a density of 1×106 cells/mL. Human T cells were purified from the isolated PBMCs using T cell enrichment kit. Purified PBMCs and T cells were pretreated for 1 h at 37° C. and 5% CO2 with various concentrations of purified BLV1H12-beta Moka L1 fusion protein (SEQ ID NOs:262 and 40), and then activated by anti-CD3 and CD28 antibodies. After 24 h treatment, supernatants were collected and the levels of secreted TNF-α measured using an ELISA kit. A graphical representation of the data is shown in FIG. 20.

Example 37

In Vitro Study of BLV1H12-Beta-Strand VM24 Fusion Protein Inhibitory Activities on Human Peripheral Blood Mononuclear Cells (PBMCs)/T Cells Activation

Human PBMCs were isolated from fresh venous blood of healthy donors through ficoll gradient centrifugation, followed by re-suspension in RPMI1640 medium with 10% FBS and plating in 96-well plates at a density of 1×106 cells/mL. Human T cells were purified from the isolated PBMCs using T cell enrichment kit. Purified PBMCs and T cells were pretreated for 1 h at 37° C. and 5% CO2 with various concentrations of purified BLV1H12-beta VM24 L1 fusion protein (SEQ ID NOs:263 and 40) and BLV1H12-beta VM24 L2 fusion protein (SEQ ID NOs: 264 and 40), and then activated by anti-CD3 and CD28 antibodies. After 24 h treatment, supernatants were collected and the levels of secreted TNF-α measured using an ELISA kit. A graphical representation of the data is shown in FIGS. 22A and 22B.

Example 38

In Vitro Proliferative Activity Assay of BLV1H12-Beta hEPO Fusion Protein on TF-1 Cells

Human TF-1 cells were cultured at 37° C. with 5% CO2 in RPMI-1640 medium containing 10% fetal bovine serum (FBS), penicillin and streptomycin (50 U/mL), and 2 ng/ml human granulocyte macrophage colony stimulating factor (GM-CSF). To examine the proliferative activity of BLV1H12-hEPO fusion proteins, cells were washed three times with RPMI-1640 medium with 10% FBS, resuspended in RPMI-1640 medium with 10% FBS at a density of 1.5×105 cells/ml, plated in 96-well plates (1.5×104 cells per well) with various concentrations of hEPO (SEQ ID NO: 206), BLV1H12 IgG, and BLV1H12-beta hEPO fusion protein (SEQ ID NOs: 267 and 40), and then incubated for 72 hours at 37° C. with 5% CO2. Cells were then treated with Alamar Blue (Life Technologies, CA) for 4 hours at 37° C. A graphical representation of the data is shown in FIG. 25.

Example 39

Pharmacokinetics of BLV1H12-Beta hEPO Fusion Proteins in Mice

hEPO (0.18 mg/kg) and BLV1H12-beta hEPO IgG (SEQ ID NOs: 267 and 40) (1.5 mg/kg) in PBS (pH 7.4) were administrated by intravenous (i.v.) injection into three CD1 mice per group. Blood was collected from day 0 to day 14 and analyzed by ELISA using anti-human IgG Fc (Abcam) and anti-hEPO (R&D systems) antibodies. Data were normalized by taking the maximal concentration at the first time point (30 minutes). The percentage of maximal concentration was plotted versus time, and the half-lives were determined by fitting data into the first-order equation, A=A0e-kt, where A0 is the initial concentration, t is the time, and k is the first order rate constant. A graphical representation of the data is shown in FIG. 26.

Example 40

Pharmacodynamics of BLV1H12-hEPO Fusion Proteins in Mice

Vehicle (PBS, pH 7.4), BLV1H12 IgG (810 μg/kg), hEPO (90 μg/kg) and BLV1H12-beta hEPO IgG (SEQ ID NOs: 267 and 40) (810 μg/kg) were administrated by subcutaneous (s.c.) injection into CD1 mice (three per group) at day 0 and day 2. Blood was collected at different time points and the hematocrit levels were measured by centrifugation in micro-hematocrit capillary tubes. A graphical representation of the data is shown in FIG. 27.

Example 41

In Vitro BLV1H12-Beta GLP-1 and BLV1H12-Beta Exendin-4 Based Fusion Proteins Activation Activities on GLP-1 Receptor

HEK 293 cells overexpressing GLP-1 receptor (GLP-1R) and cAMP responsive element (CRE)-luciferase (Luc) reporter were grown in DMEM with 10% FBS at 37° C. with 5% CO2. Cells were seeded in 384-well plates at a density of 5000 cells per well and treated with various concentrations of Ex-4 peptide (SEQ ID NO: 201), BLV1H12-beta Ex-4 (SEQ ID NOs: 266 and 40), BLV1H12-beta Ex-4 RN (SEQ ID NOs: 266 and 40, treated with Factor Xa), BLV1H12-beta GLP-1 (SEQ ID NOs: 265 and 40), and BLV1H12-beta GLP RN (SEQ ID NOs: 265 and 40, treated with Factor Xa) for 24 hours at 37° C. with 5% CO2. An immunoglobulin fusion protein which may be cleaved to release the amino-terminus of a therapeutic agent is referred to as RN, for released N-terminus. Luminescence intensities were then measured using One-Glo (Promega, WI) luciferase reagent by following manufacturer's instruction. A graphical representation of the data is shown in FIG. 30.

Example 42

In Vitro Stability Assay of BLV1H12-Beta Exendin-4 Fusion Proteins

Exendin-4 (200 nM), BLV1H12-beta Ex-4 (SEQ ID NOs: 266 and 40) (1 uM) and BLV1H12-beta Ex-4 RN (SEQ ID NOs: 266 and 40, treated with Factor Xa) (200 nM) were incubated in fresh mouse and human plasma at 37° C. The mixtures were collected at times between 0 and 96 hours, and the remaining activities on GLP-1R activation were measured using HEK293-GLP-1R-CRE-Luc cells. A graphical depiction of the data is shown in FIGS. 31A and 31B.

Example 43

Pharmacokinetics of BLV1H12-Beta Ex-4 RN Fusion Proteins in Mice

Exendin-4 (1.6 mg/kg) and BLV1H12-beta Ex-4 RN (SEQ ID NOs: 266 and 40, cleaved with Factor Xa) fusion protein (2.8 mg/kg) were administrated by injection into CD1 mice (N=3). Blood samples were collected from day 0 to day 8. The remaining activities were analyzed using HEK 293-GLP-1R-CRE-Luc cells. Data were normalized by taking the maximal concentration at the first time point (30 minutes). Percentages of the maximal concentration were plotted versus time points of blood sample collection, and half-lives were determined by fitting data into the first-order equation, A=A0e-kt, where A0 is the initial concentration, t is the time, and k is the first-order rate constant. FIG. 32 depicts a graphical representation of the data. The t1/2 of Exendin-4 was 1.5±0.2 hours. The t1/2 of BLV1H12-beta Ex-4 RN was 2.2±1.1 days.

Example 44

Pharmacodynamics of BLV1H12-Beta Ex-4 RN Fusion Proteins in Mice

Doses of Exendin-4 (0.5 μg), BLV1H12, PBS, and BLV1H12-beta Ex-4 RN (SEQ ID NOs: 266 and 40, cleaved with Factor Xa) (100 μg) were administrated by intravenous injection into CD1 mice (N=5). Glucose (3 g/kg, p.o.) was given post treatment, followed by blood glucose measurements. FIGS. 33A and 33B depicts a graphical representation of the data.

Example 45

Pharmacodynamics of BLV1H12-Beta Ex-4 RN Fusion Proteins in Mice

Doses of Exendin-4 (0.5 μg), BLV1H12 (100, 200 μg), PBS, and BLV1H12-beta Ex-4 RN (SEQ ID NOs: 266 and 40, cleaved with Factor Xa) (100, 200 μg) were administrated by subcutaneous injection into CD1 mice (N=5). Glucose (3 g/kg, p.o.) was given 24 hr post treatment, followed by blood glucose measurements. FIGS. 34A and 34B depicts a graphical representation of the data.

Example 46

Pharmacodynamics of BLV1H12-Beta Ex-4 RN Fusion Proteins in Mice

Doses of Exendin-4 (0.5 μg), BLV1H12 (100, 200 μg), PBS, and BLV1H12-beta Ex-4 RN (SEQ ID NOs: 266 and 40, cleaved with Factor Xa) (100, 200 μg) were administrated by subcutaneous injection into CD1 mice (N=5). Glucose (3 g/kg, p.o.) was given 48 hr post treatment, followed by blood glucose measurements. FIGS. 35A and 35B depicts a graphical representation of the data.

Example 47

In Vitro Study of BLV1H12-Beta-Strand bGCSF Fusion Protein Proliferative Activity on Human Granulocyte Progenitors

Human mobilized peripheral blood CD34+ cells were purchased from AllCells (Emeryville, Calif.). Cells were resuspended in HSC expansion medium (StemSpan SFEM, StemCell Technologies), and supplemented with 1× antibiotics and the following recombinant human cytokines: thrombopoietin, IL6, Flt3 ligand, and stem cell factor (100 ng/mL, R&D Systems). The cells were then plated in 96-well plates (1000 cells per well) with various concentrations of bGCSF (SEQ ID NO: 200), hGCSF, BLV1H12 full-length IgG, BLV1H12-beta bGCSF L0 fusion protein (Ab-bGCSF L0), and BLV1H12-beta bGCSF L1 fusion protein (Ab-bGCSF L1). Cells were cultured for 7 days at 37° C. with 5% CO2, and then analyzed by flow cytometry to measure cell number and expression of CD45ra and CD41 using PE-Cy7 anti-CD45ra and eFluor 450 anti-CD41 (eBiosciences) antibodies. FIGS. 38 A-E show the fluorescence of the cells treated with various concentrations of bGCSF (SEQ ID NO: 200), hGCSF, BLV1H12 full-length IgG, BLV1H12-beta bGCSF L0 fusion protein (Ab-bGCSF L0), and BLV1H12-beta bGCSF L1 fusion protein (Ab-bGCSF L1).

Example 48

Pharmacokinetics of BLV1H12-Beta bGCSF Fusion Proteins in Mice

BLV1H12 full-length IgG (2.8 mg/kg), BLV1H12-beta bGCSF L0 fusion protein (Ab-bGCSF L0) (2.8 mg/kg), BLV1H12-beta bGCSF L1 fusion protein (Ab-bGCSF L1) (2.8 mg/kg) and bGCSF (8 mg/kg) were administrated by intravenous (i.v.) injection into 3 BALB/c mice per group. Blood was collected from day 0 to day 14 and analyzed by ELISA using anti-human IgG Fc antibody (KPL) for BLV1H12 full-length IgG and anti-bGCSF antibody (Abbiotec) for Ab-bGCSF fusion proteins and bGCSF. Data were normalized by taking maximal concentration at the first time point (30 minutes). FIGS. 39A and 39B depicts a graphical representation of the data. The t1/2 of bGCSF was 4.8 hours. The t1/2 of BLV1H12 full-length IgG was 12.6 days. The t1/2 of BLV1H12-beta bGCSF L0 IgG was 8.1 days. The t1/2 of BLV1H12-beta bGCSF L1 IgG was 9.2 days.

Example 49

Pharmacodynamics of BLV1H12-Beta bGCSF Fusion Proteins in Mice

Single doses of bGCSF (10 μg/kg), BLV1H12 IgG, BLV1H12-beta bGCSF L0 fusion protein (Ab-bGCSF L0) (50 μg/kg), and BLV1H12-beta bGCSF L1 fusion protein (Ab-bGCSF L1) (50 μg/kg) were administrated by subcutaneous (s.c.) injection into 3 BALB/c mice per group. Blood was collected from day 0 to day 21 and analyzed by flow cytometry to measure percentages of neutrophil populations in white blood cells using FITC anti-CD45 (Miltenyi Biotec), PE anti-CD11b (Miltenyi Biotec), and APC anti-Ly-6G antibodies (BD Biosciences). FIG. 40A depicts a graphical representation of the data. FIG. 40B depicts stained monocytes and neutrophils.

Example 50

Construction and Purification of Bovine-Beta hGH Fusion Proteins

To generate BLV1H12-beta hGH fusion proteins, the gene encoding hGH was synthesized by Genscript or IDT, and amplified by PCR. Flexible linkers of GGGGS (SEQ ID NO: 164) were added on both ends of the hGH gene. Extender peptide sequences having beta strand secondary structure were added to both sides of the hGH-linker fragment. The fragment was grafted into the BLV1H12 Fab heavy chain CDR3H to generate BLV1H12-beta Fab hGH (CDR3H). The fragment was grafted into the BLV1H12 hFc (IgG) heavy chain CDR3H to generate BLV1H12-beta hFc (IgG) hGH (CDR3H). The BLV1H12 expression vectors were generated by in-frame ligation of the amplified fusion genes to the pFuse backbone vector (InvivoGen, CA). Similarly, the gene encoding the light chain of BLV1H12 was cloned to the pFuse backbone vector. The obtained expression vectors were confirmed by DNA sequencing.

BLV1H12-beta hGH IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta Fab hGH protein heavy chain (SEQ ID NO: 300) and the BLV1H12 light chain (SEQ ID NO: 40). BLV1H12-beta hGH IgG fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding BLV1H12-beta hFc (IgG) hGH protein heavy chain (SEQ ID NO: 302) and the BLV1H12 light chain (SEQ ID NO: 40).

Purified BLV1H12-beta Fab hGH IgG (SEQ ID NOs: 300 and 40) and BLV1H12-beta hFc (IgG) (SEQ ID NOs: 302 and 40) are shown in FIGS. 41A and 41B. FIG. 41A depicts purified BLV1H12-beta Fab hGH IgG (SEQ ID NOs: 300 and 40), with and without DTT. FIG. 41B depicts purified BLV1H12-beta hFc (IgG) hGH IgG (SEQ ID NOs: 302 and 40), with and without DTT.

Example 51

BLV1H12-Beta hGH hGHR-Ba/F3 Proliferation Assay

Murine Ba/F3 cells cell lines were stably transduced with hGHR under EF1α promoter. Clonal selected hGHR-Ba/F3 were maintained in 10% FBS in RPMI1640 with 50 ng/mL of hGH. The proliferation assay was performed in 96 well culture plate with 20,000 cells in 200 uL assay medium (10% FBS in RPMI1640) per well. Increasing concentrations of BLV1H12-beta Fab hGH IgG (SEQ ID NOs: 300 and 40) and BLV1H12-beta hFc (IgG) (SEQ ID NOs: 302 and 40) were incubated with cells for 72 hours. At the end of the incubation period, 20 ul of Prestoblue was added to each well, and the fluorescent signal recorded on a Spectramax fluorescence plate reader at 590 nm with 550 nm excitation. FIG. 42 B depicts a graphical representation of the data.

Example 52

BLV1H12-Beta hGH NB2 Proliferation Assay

Rat Nb2-11 cell lines (Sigma) were maintained in 10% FBS, 10% horse serum (HS) in RPMI with 55 uM β-ME. A proliferation assay was performed in a 96 well culture plate with 50,000 cells in 200 uL assay medium (10% HS in RPMI with 55 uM β-ME) per well. Increasing concentrations of BLV1H12-beta Fab hGH IgG (SEQ ID NOs: 300 and 40) and BLV1H12-beta hFc (IgG) (SEQ ID NOs: 302 and 40) were incubated with cells for 72 hours. At the end of the incubation period, 20 ul of Prestoblue was added to each well, and the fluorescent signal recorded on a Spectramax fluorescence plate reader at 590 nm with 550 nm excitation. FIG. 42 A depicts a graphical representation of the data.

Example 53

BLV1H12-Beta hGH Stat5 Phosphorylation Assay

Human IM9 cells from ATCC were maintained in 10% FBS in RPMI1640. The night before the assay, 2×10e5 IM9 cells were seeded into V bottom 96 well plate in 200 uL assay medium (1% charcoal stripped FBS in RPMI) and starved overnight. On the day of the experiment, starved cells were stimulated with hGH, BLV1H12-beta Fab hGH IgG (SEQ ID NOs: 300 and 40) and BLV1H12-beta hFc (IgG) (SEQ ID NOs: 302 and 40) at various concentrations for 10 min at 37° C. After stimulation, cells were fixed by 4% formaldehyde at 37° C. for 10 min, and permeabalized with 90% methanol. Cells were then blocked with 5% BSA at room temperature for 10 min and stained with Alexa Fluor® 488 conjugated anti-pStat5 (Tyr694) (C71E5) Rabbit mAb (Cell Signaling Technology, Inc.) following the manufacturer's suggested protocol. Cells were then washed with PBS and analyzed by a flow cytometer. FIG. 42 C depicts a graphical representation of the data.

Example 54

BLV1H12-Beta hLeptin IgG Leptin Receptor Activity

Baf3 stable cells overexpressed with Leptin receptor (LepR) were seeded in a 96-well plate and subsequently treated with different doses of hLeptin and BLV1H12-beta hLeptin IgG (SEQ ID NOs: 230 and 40) for 72 hours. AlamarBlue regent was added as 1/10 volume, the plate was incubated for 2 hrs, and the fluorescent read at 590 nm under excitation at 560 nm. Data were analyzed using GraphPad Prism 6. A graphical depiction of the data is shown in FIG. 43B.

Example 55

Expression and Purification of Trastuzumab-Beta BCCX2 Fusion Proteins

Trastuzumab-beta BCCX2 fusion proteins were expressed through transient transfections of free style HEK293 cells with vectors encoding trastuzumab-beta BCCX2 fusion protein heavy chain (SEQ ID NOs: 96, 97, or 98) and the trastuzumab light chain (SEQ ID NO: 21). Expressed fusion proteins were secreted into the culture medium and harvested every 48 hours for twice after transfection. The fusion proteins were purified by Protein A/G chromatography (Thermo Fisher Scientific, IL), and analyzed by SDS-PAGE gel.

Purified trastuzumab-beta BCCX2 IgGs are shown in FIG. 47: trastuzumab-beta BCCX2 HC long (HLCX) (SEQ ID NOs: 96 and 40), trastuzumab-beta BCCX2 HC medium HMCX (SEQ ID NOs: 97 and 40), and trastuzumab-beta BCCX2 HC short (HSCX) (SEQ ID NOs: 98 and 40), with or without reducing reagent DTT.

Example 56

Flow Cytometry Analysis of CXCR4 Antibodies and BLV1H12-Beta BCCX2 Fusion Proteins

As shown in FIG. 48, engineered antibodies (A) bind to CXCR4 positive Jurkat cells, (B) do not bind to CXCR4 negative CHO cells (C) and bind to CXCR4 transfected CHO cells. In all cases, the control antibody (BLV1H12) showed no peak shift by flow cytometry analysis. The shaded peaks are cells without antibody treatment. The engineered antibodies include BLV1H12-beta BCCX2 HC 1 (bAb-AC1) (SEQ ID NOs: 92 and 40), BLV1H12-beta BCCX2 HC 2 (bAb-AC2) (SEQ ID NOs: 93 and 40), and BLV1H12-beta BCCX2 HC 3 (bAb-AC3) (SEQ ID NOs: 94 and 40).

Example 57

BLV12H12-Beta BCCX2IgG Binding to CXCR4

A Tag-lite HTFR binding assay was performed to determine binding between BLV1H12-beta BCCX2 fusion proteins and CXCR4. The binding affinities were calculated based on the Cheng-Prusoff equation to give Ki values of 2.1 nM, 5.4 nM and 19.8 nM for BLV1H12-beta BCCX2 HC 1, BLV1H12-beta BCCX2 HC 2 and BLV1H12-beta BCCX2 HC 3, respectively. FIG. 50B depicts a flow cytometry histogram demonstrating nearly complete inhibition of 12G5 binding to CXCR4 by a three-fold excess of BLV1H12-beta BCCX2 HC 1.

Example 58

BLV12H12-Beta BCCX2IgG FACS Competition

Jurkat cells were incubated with increasing concentrations of BLVH12-beta BCCX2 HC 1 (SEQ ID NOs: 92 and 40) in blocking buffer (PBS supplemented with 3% BSA) at 4° C. for 30 min followed by fluorescein conjugated 12G5 treatment at a final concentration of 10 μg/mL for an additional 30 min. Cells were then washed with PBS and analyzed by a flow cytometer. A graph representing the data is shown in FIG. 51.

The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

TABLE 6
Immunoglobulin Light Chain (LC) and Heavy Chain (HC)-
Nucleotide Sequence
SEQ ID
NAMENOSEQUENCE
BVK heavy1CAGGTCCAGCTCCAGGAAAGCGGTCCCGGCCTCGTGCGTCCCAGCC
chain (HC)AGACTCTCTCCCTCACTTGTACTGTGTCAGGTTTTAGCCTCACTGGC
TACGGAGTGAACTGGGTCCGCCAGCCACCTGGTAGGGGACTGGAG
TGGATCGGCATGATTTGGGGAGACGGTAACACCGATTATAATTCTG
CTCTGAAGTCAAGAGTGACAATGCTCAAGGACACCTCCAAAAATC
AGTTCTCTCTGCGTCTCTCCAGCGTGACCGCCGCTGATACTGCAGTC
TACTATTGCGCCCGCGAAAGAGATTATCGTCTGGATTATTGGGGTC
AGGGTAGTCTGGTCACAGTGTCCTCAGCCTCCACCAAGGGCCCATC
GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACA
GCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGA
CGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTT
CCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTG
GTGACTGTGCCCTCTAGCAGCTTGGGCACCCAGACCTACATCTGCA
ACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTT
Trastuzumab LC2GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG
GAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGATGTGAATA
CCGCGGTCGCATGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGC
TCCTGATCTATTCTGCATCCTTCTTGTATAGTGGGGTCCCATCAAGG
TTCAGTGGCAGTAGATCTGGGACAGATTTCACTCTCACCATCAGCA
GTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCATTA
CACTACCCCTCCGACGTTCGGCCAAGGTACCAAGCTTGAGATCAAA
CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTCGTGTGCCTGCTGAATAAC
TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC
CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC
AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA
GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT
CAGGGCCTGTCCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG
TGT
Trastuzumab N-3GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG
terminal LCGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAG
Trastuzumab C-4ACCGCGGTCGCATGGTATCAGCAGAAACCAGGGAAAGCCCCTAAG
terminal LCCTCCTGATCTATTCTGCATCCTTCTTGTATAGTGGGGTCCCATCAAG
GTTCAGTGGCAGTAGATCTGGGACAGATTTCACTCTCACCATCAGC
AGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCATT
ACACTACCCCTCCGACGTTCGGCCAAGGTACCAAGCTTGAGATCAA
ACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATG
AGCAGTTGAAATCTGGAACTGCCTCTGTCGTGTGCCTGCTGAATAA
CTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGC
CCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA
AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCA
TCAGGGCCTGTCCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA
GTGT
Trastuzumab HC5GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGC
GGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAG
ATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGA
ATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCG
GATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAA
AACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACC
GCGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGA
TGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCGA
GCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGAGCAGCAAAA
GCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGTGAAAGATT
ATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGCGCGCTGAC
CAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCAGCGGCCTG
TATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAGCCTGGGC
ACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGCAACACCA
AAGTGGATAAAAAAGTGGAACCGCCGAAAAGCTGCGATAAAACCC
ATACCTGCCCGCCGTGCCCGGCGCCGGAACTGCTGGGCGGCCCGAG
CGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGATGATTAGC
CGCACCCCGGAAGTGACCTGCGTGGTGGTGGATGTGAGCCATGAA
GATCCGGAAGTGAAATTTAACTGGTATGTGGATGGCGTGGAAGTGC
ATAACGCGAAAACCAAACCGCGCGAAGAACAGTATAACAGCACCT
ATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAA
CGGCAAAGAATATAAATGCAAAGTGAGCAACAAAGCGCTGCCGGC
GCCGATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGA
ACCGCAGGTGTATACCCTGCCGCCGAGCCGCGATGAACTGACCAA
AAACCAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCCGAGC
GATATTGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACAAC
TATAAAACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTTTTC
TGTATAGCAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCA
ACGTGTTTAGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTA
TACCCAGAAAAGCCTGAGCCTGAGCCCGGGCAAA
Trastuzumab wt6GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCAGCCTGGG
hIgG1 HCGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCAATATTAAGG
ACACTTACATCCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG
AGTGGGTCGCACGTATTTATCCTACCAATGGTTACACACGCTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCGCAGACACTTCCAAG
AACACGGCGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACG
GCCGTGTATTACTGTTCGAGATGGGGCGGTGACGGCTTCTATGCCA
TGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGCCTC
CACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC
ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACT
TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAG
CGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC
TCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCC
AGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG
TGGACAAGAAAGTTGAACCCAAATCTTGCGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT
CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC
CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT
GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA
AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCAT
CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCG
CCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA
CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC
AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC
TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA
AGAGCCTCTCCCTGTCTCCGGGTAAA
Trastuzumab7GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCAGCCTGGG
heptad mutationGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCAATATTAAGG
in hIgG1 HCACACTTACATCCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG
AGTGGGTCGCACGTATTTATCCTACCAATGGTTACACACGCTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCGCAGACACTTCCAAG
AACACGGCGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACG
GCCGTGTATTACTGTTCGAGATGGGGCGGTGACGGCTTCTATGCCA
TGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGCCTC
CACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC
ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACT
TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAG
CGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC
TCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCC
AGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG
TGGACAAGAAAGTTGAACCCAAATCTTGCGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTCCAGTCGCCGGACCGTCAGTCTTCCT
CTTCCCTCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT
GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGG
TCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA
GTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAAGCTCCATCGAG
AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TACACCCTGCCTCCATCCCGGGATGAGCTGACCAAGAACCAGGTCA
GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT
GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC
GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGC
TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT
GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGA
GCCTCTCCCTGTCTCCGGGTAAA
Trastuzumab8GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCAGCCTGGG
triple mutationsGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCAATATTAAGG
in hIgG4 HCACACTTACATCCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG
AGTGGGTCGCACGTATTTATCCTACCAATGGTTACACACGCTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCGCAGACACTTCCAAG
AACACGGCGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACG
GCCGTGTATTACTGTTCGAGATGGGGCGGTGACGGCTTCTATGCCA
TGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGCCAG
CACTAAAGGTCCATCTGTGTTCCCTCTGGCTCCTTGCAGCCGGAGC
ACCTCCGAGTCCACAGCCGCTCTGGGATGTCTGGTGAAAGATTACT
TCCCCGAGCCCGTCACCGTGAGCTGGAATAGCGGAGCACTGACCTC
CGGCGTCCACACATTCCCCGCCGTGCTCCAAAGCTCCGGCCTGTAC
AGCCTCTCCTCCGTGGTCACCGTGCCCAGCAGCTCTCTGGGCACAA
AGACCTATACCTGTAACGTGGATCACAAGCCTAGCAACACCAAAGT
GGATAAGCGGGTGGAGAGCAAGTACGGCCCTCCCTGTCCCCCTTGC
CCCGCTCCTGAGGCCGCTGGCGGACCTTCCGTGTTCCTGTTTCCCCC
TAAGCCCAAGGACACCCTCATGATTAGCCGGACACCCGAAGTGAC
CTGCGTGGTCGTGGATGTGTCCCAGGAGGACCCTGAAGTGCAATTT
AACTGGTACGTGGACGGCGTCGAGGTGCACAACGCCAAGACCAAG
CCTCGGGAAGAGCAGTTCAACAGCACCTACCGGGTGGTCAGCGTG
CTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAG
TGCAAGGTGAGCAACAAGGGCCTGCCCAGCTCCATCGAGAAGACC
ATCAGCAAGGCCAAGGGCCAGCCCAGGGAACCCCAGGTGTATACC
CTGCCCCCTAGCCAGGAGGAAATGACCAAAAACCAGGTGAGCCTG
ACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGT
GGGAGAGCAACGGCCAGCCCGAGAACAATTACAAGACCACCCCTC
CTGTGCTGGACAGCGACGGCTCCTTCTTTCTGTATAGCCGGCTGAC
CGTGGACAAGAGCAGGTGGCAGGAGGGCAACGTGTTCTCCTGTAG
CGTGATGCACGAGGCCCTGCACAACCATTACACCCAGAAGAGCTTG
AGCCTGAGCCTGGGCAAA
Trastuzumab N-9GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGC
terminal HCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAG
ATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGA
ATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCG
GATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAA
AACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACC
GCGGTGTATTATTGCAGCCGC
Trastuzumab C-10GATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCGAGC
terminal HCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGAGCAGCAAAAGC
ACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGTGAAAGATTATT
TTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGCGCGCTGACCA
GCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCAGCGGCCTGTA
TAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAGCCTGGGCAC
CCAGACCTATATTTGCAACGTGAACCATAAACCGAGCAACACCAA
AGTGGATAAAAAAGTGGAACCGCCGAAAAGCTGCGATAAAACCCA
TACCTGCCCGCCGTGCCCGGCGCCGGAACTGCTGGGCGGCCCGAGC
GTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGATGATTAGCC
GCACCCCGGAAGTGACCTGCGTGGTGGTGGATGTGAGCCATGAAG
ATCCGGAAGTGAAATTTAACTGGTATGTGGATGGCGTGGAAGTGCA
TAACGCGAAAACCAAACCGCGCGAAGAACAGTATAACAGCACCTA
TCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAAC
GGCAAAGAATATAAATGCAAAGTGAGCAACAAAGCGCTGCCGGCG
CCGATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAA
CCGCAGGTGTATACCCTGCCGCCGAGCCGCGATGAACTGACCAAA
AACCAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCCGAGCG
ATATTGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACAACT
ATAAAACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTTTTCT
GTATAGCAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAA
CGTGTTTAGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTAT
ACCCAGAAAAGCCTGAGCCTGAGCCCGGGCAAA
Palivizumab wt11CAGGTGACCCTGCGCGAGTCCGGCCCCGCCCTGGTGAAGCCCACCC
hIgG1 HCAGACCCTGACCCTGACCTGCACCTTCTCCGGCTTCTCCCTGTCCACC
TCCGGCATGTCCGTGGGCTGGATCCGCCAGCCCCCCGGCAAGGCCC
TGGAGTGGCTGGCCGACATCTGGTGGGACGACAAGAAGGACTACA
ACCCCTCCCTGAAGTCCCGCCTGACCATCTCCAAGGACACCTCCAA
GAACCAGGTGGTGCTGAAGGTGACCAACATGGACCCCGCCGACAC
CGCCACCTACTACTGCGCCCGCTCCATGATCACCAACTGGTACTTC
GACGTGTGGGGCGCCGGCACCACCGTGACCGTGTCCTCCGCCTCCA
CCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC
CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT
CCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCA
GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGT
GGACAAGAAAGTTGAACCCAAATCTTGCGACAAAACTCACACATG
CCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTC
CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTG
AGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTG
TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA
GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC
CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC
CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCA
AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT
CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA
GAGCCTCTCCCTGTCTCCGGGTAAA
Palivizumab12CAGGTGACCCTGCGCGAGTCCGGCCCCGCCCTGGTGAAGCCCACCC
heptad mutationAGACCCTGACCCTGACCTGCACCTTCTCCGGCTTCTCCCTGTCCACC
in hIgG1 HCTCCGGCATGTCCGTGGGCTGGATCCGCCAGCCCCCCGGCAAGGCCC
TGGAGTGGCTGGCCGACATCTGGTGGGACGACAAGAAGGACTACA
ACCCCTCCCTGAAGTCCCGCCTGACCATCTCCAAGGACACCTCCAA
GAACCAGGTGGTGCTGAAGGTGACCAACATGGACCCCGCCGACAC
CGCCACCTACTACTGCGCCCGCTCCATGATCACCAACTGGTACTTC
GACGTGTGGGGCGCCGGCACCACCGTGACCGTGTCCTCCGCCTCCA
CCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC
CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT
CCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCA
GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGT
GGACAAGAAAGTTGAACCCAAATCTTGCGACAAAACTCACACATG
CCCACCGTGCCCAGCACCTCCAGTCGCCGGACCGTCAGTCTTCCTC
TTCCCTCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG
AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA
GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGT
CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAAGCTCCATCGAGA
AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCTCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG
CCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG
CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC
CTCTCCCTGTCTCCGGGTAAA
Palivizumab13CAGGTGACCCTGCGCGAGTCCGGCCCCGCCCTGGTGAAGCCCACCC
triple mutationsAGACCCTGACCCTGACCTGCACCTTCTCCGGCTTCTCCCTGTCCACC
in hIgG4 HCTCCGGCATGTCCGTGGGCTGGATCCGCCAGCCCCCCGGCAAGGCCC
TGGAGTGGCTGGCCGACATCTGGTGGGACGACAAGAAGGACTACA
ACCCCTCCCTGAAGTCCCGCCTGACCATCTCCAAGGACACCTCCAA
GAACCAGGTGGTGCTGAAGGTGACCAACATGGACCCCGCCGACAC
CGCCACCTACTACTGCGCCCGCTCCATGATCACCAACTGGTACTTC
GACGTGTGGGGCGCCGGCACCACCGTGACCGTGTCCTCCGCCAGCA
CTAAAGGTCCATCTGTGTTCCCTCTGGCTCCTTGCAGCCGGAGCAC
CTCCGAGTCCACAGCCGCTCTGGGATGTCTGGTGAAAGATTACTTC
CCCGAGCCCGTCACCGTGAGCTGGAATAGCGGAGCACTGACCTCCG
GCGTCCACACATTCCCCGCCGTGCTCCAAAGCTCCGGCCTGTACAG
CCTCTCCTCCGTGGTCACCGTGCCCAGCAGCTCTCTGGGCACAAAG
ACCTATACCTGTAACGTGGATCACAAGCCTAGCAACACCAAAGTGG
ATAAGCGGGTGGAGAGCAAGTACGGCCCTCCCTGTCCCCCTTGCCC
CGCTCCTGAGGCCGCTGGCGGACCTTCCGTGTTCCTGTTTCCCCCTA
AGCCCAAGGACACCCTCATGATTAGCCGGACACCCGAAGTGACCT
GCGTGGTCGTGGATGTGTCCCAGGAGGACCCTGAAGTGCAATTTAA
CTGGTACGTGGACGGCGTCGAGGTGCACAACGCCAAGACCAAGCC
TCGGGAAGAGCAGTTCAACAGCACCTACCGGGTGGTCAGCGTGCT
GACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTG
CAAGGTGAGCAACAAGGGCCTGCCCAGCTCCATCGAGAAGACCAT
CAGCAAGGCCAAGGGCCAGCCCAGGGAACCCCAGGTGTATACCCT
GCCCCCTAGCCAGGAGGAAATGACCAAAAACCAGGTGAGCCTGAC
CTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG
GAGAGCAACGGCCAGCCCGAGAACAATTACAAGACCACCCCTCCT
GTGCTGGACAGCGACGGCTCCTTCTTTCTGTATAGCCGGCTGACCG
TGGACAAGAGCAGGTGGCAGGAGGGCAACGTGTTCTCCTGTAGCG
TGATGCACGAGGCCCTGCACAACCATTACACCCAGAAGAGCTTGA
GCCTGAGCCTGGGCAAA
BVK light chain14GACATTCAGATGACACAGAGCCCCAGCAGCCTCAGTGCCTCAGTCG
(LC)GTGACAGAGTGACCATTACTTGCCGTGCCAGCGGAAACATTCACAA
CTACCTGGCCTGGTATCAGCAGAAGCCCGGCAAAGCTCCTAAGCTG
CTCATCTACTATACCACTACACTCGCAGACGGCGTGCCATCTCGCTT
CTCTGGCTCAGGATCCGGTACAGACTACACCTTTACTATCTCCAGC
CTGCAGCCCGAGGATATTGCTACCTACTATTGCCAGCATTTTTGGTC
AACCCCCCGCACATTCGGTCAGGGCACTAAGGTGGAGATTAAGAG
AACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGC
AGTTGAAATCTGGAACTGCCTCTGTCGTGTGCCTGCTGAATAACTT
CTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA
GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGC
AGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCA
GGGCCTGTCCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT
Palivizumab LC15GACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCCGTGG
GCGACCGCGTGACCATCACCTGCAAGTGCCAGCTGTCCGTGGGCTA
CATGCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCT
GATCTACGACACCTCCAAGCTGGCCTCCGGCGTGCCCTCCCGCTTC
TCCGGCTCCGGCTCCGGCACCGAGTTCACCCTGACCATCTCCTCCCT
GCAGCCCGACGACTTCGCCACCTACTACTGCTTCCAGGGCTCCGGC
TACCCCTTCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAACGAA
CTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAG
TTGAAATCTGGAACTGCCTCTGTCGTGTGCCTGCTGAATAACTTCTA
TCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCA
ATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGA
CAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGA
CTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGG
CCTGTCCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT
Palivizumab N-16CAGGTGACCCTGCGCGAGTCCGGCCCTGCACTGGTGAAGCCCACCC
terminal HCAGACCCTGACCCTGACCTGCACCTTCTCCGGCTTCTCCCTGTCCACC
TCCGGCATGTCCGTGGGCTGGATCCGGCAGCCTCCCGGCAAGGCCC
TGGAGTGGCTGGCTGACATCTGGTGGGACGACAAGAAGGACTACA
ACCCCTCCCTGAAGTCCCGCCTGACCATCTCCAAGGACACCTCCAA
GAACCAGGTGGTGCTGAAGGTGACCAACATGGACCCCGCCGACAC
CGCCACCTACTACTGCGCCCGC
Palivizumab C-17GACGTGTGGGGAGCCGGTACCACCGTGACCGTGTCTTCCGCCTCCA
terminal HCCCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC
CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT
CCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCA
GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGT
GGACAAGAAAGTTGAACCCAAATCTTGCGACAAAACTCACACATG
CCCACCGTGCCCAGCACCTCCAGTCGCCGGACCGTCAGTCTTCCTC
TTCCCTCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG
AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA
GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGT
CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAAGCTCCATCGAGA
AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCTCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG
CCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG
CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC
CTCTCCCTGTCTCCGGGTAAATGATAAGTGCTAGCTGGCCAGA
BLV1H12 N-18CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCC
terminal HCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCG
ACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGG
AATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATC
CCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAA
GTCAGGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGC
AACTTACTATTGCACCTCTGTGCACCAG
BLV1H12 C-19TGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTA
terminal HCGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTG
TGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCA
AGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCC
TGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGG
CCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCA
GGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCA
AAGTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAAACTCACA
CATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT
CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC
CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATA
ATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACC
GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG
CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAC
CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA
TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC
AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC
TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC
AGAAGAGCCTCTCCCTGTCTCCGGGTAAA
BLV1H12 LC20CAGGCCGTCCTGAACCAGCCAAGCAGCGTCTCCGGGTCTCTGGGGC
AGCGGGTCTCAATCACCTGTAGCGGGTCTTCCTCCAATGTCGGCAA
CGGCTACGTGTCTTGGTATCAGCTGATCCCTGGCAGTGCCCCACGA
ACCCTGATCTACGGCGACACATCCAGAGCTTCTGGGGTCCCCGATC
GGTTCTCAGGGAGCAGATCCGGAAACACAGCTACTCTGACCATCAG
CTCCCTGCAGGCTGAGGACGAAGCAGATTATTTCTGCGCATCTGCC
GAGGACTCTAGTTCAAATGCCGTGTTTGGAAGCGGCACCACACTGA
CAGTCCTGGGGCAGCCCAAGAGTCCCCCTTCAGTGACTCTGTTCCC
ACCCTCTACCGAGGAACTGAACGGAAACAAGGCCACACTGGTGTG
TCTGATCAGCGACTTTTACCCTGGATCCGTCACTGTGGTCTGGAAG
GCAGATGGCAGCACAATTACTAGGAACGTGGAAACTACCCGCGCC
TCCAAGCAGTCTAATAGTAAATACGCCGCCAGCTCCTATCTGAGCC
TGACCTCTAGTGATTGGAAGTCCAAAGGGTCATATAGCTGCGAAGT
GACCCATGAAGGCTCAACCGTGACTAAGACTGTGAAACCATCCGA
GTGCTCC

TABLE 7
Immunoglobulin Light Chain (LC) and Heavy Chain (HC)-
Amino Acid Sequence
SEQ ID
NameNOSequence
Trastuzumab LC21DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI
YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF
GQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC
Trastuzumab N-22DIQMTQSPSSLSASVGDRVTITCRASQ
terminal LC
Trastuzumab C-23TAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQ
terminal LCPEDFATYYCQQHYTTPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Trastuzumab HC24EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW
VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY
YCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK
Trastuzumab wt25EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW
hIgG1 HCVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY
YCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK
Trastuzumab26EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW
heptad mutationVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY
in hIgG1 HCYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPV
AGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP
SSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK
Trastuzumab27EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW
triple mutationsVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY
in hIgG4 HCYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSES
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
MHEALHNHYTQKSLSLSLGK
Light chain28DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI
paired withYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF
Trastuzumab AbGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC
Trastuzumab N-29EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW
terminal HCVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY
YCSR
Trastuzumab C-30DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
terminal HCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK
Palivizumab wt31QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALE
hIgG1 HCWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTAT
YYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK
Palivizumab32QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALE
heptad mutationWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTAT
in hIgG1 HCYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVA
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
SIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK
Palivizumab33QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALE
triple mutationsWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTAT
in hIgG4 HCYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSEST
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
MHEALHNHYTQKSLSLSLGK
Light chain34DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLI
paired withYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTF
Palivizumab AbGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC
Palivizumab LC35DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLI
YDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTF
GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC
Palivizumab N-36QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALE
terminal HCWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTAT
YYCAR
Palivizumab C-37DVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
terminal HCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK
SLSLSPGK
BLV1H12 N-38QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
terminal HCLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
SVHQ
BLV1H12 C-39WHVDVWGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSS
terminal HCYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQ
TFTCNVAHPASSTKVDKAVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK
BLV1H12 LC40QAVLNQPSSVSGSLGQRVSITCSGSSSNVGNGYVSWYQLIPGSAPRTLI
YGDTSRASGVPDRFSGSRSGNTATLTISSLQAEDEADYFCASAEDSSSN
AVFGSGTTLTVLGQPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSV
TVVWKADGSTITRNVETTRASKQSNSKYAASSYLSLTSSDWKSKGSYS
CEVTHEGSTVTKTVKPSECS

TABLE 8
Immunoglobulin fusion protein-Nucleotide Sequence
SEQ ID
NAMENOSEQUENCE
Trastuzumab- beta hEPO LC41embedded image
embedded image
embedded image
Trastuzumab-42GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCAGCCTGGG
beta bGCSF HCGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCAATATTAAGG
ACACTTACATCCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG
AGTGGGTCGCACGTATTTATCCTACCAATGGTTACACACGCTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCGCAGACACTTCCAAG
AACACGGCGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACG
GCCGTGTATTACTGTTCGAGAGAAACTAAGAAATACCAGAGCGGT
GGCGGAGGATCTACCCCCCTTGGCCCTGCCCGATCCCTGCCCCAGA
GCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAAATCCAGGCTGA
TGGCGCCGAGCTGCAGGAGAGGCTGTGTGCCGCCCACAAGCTGTG
CCACCCGGAGGAGCTGATGCTGCTCAGGCACTCTCTGGGCATCCCC
CAGGCTCCCCTAAGCAGCTGCTCCAGCCAGTCCCTGCAGCTGACGA
GCTGCCTGAACCAACTACACGGCGGCCTCTTTCTCTACCAGGGCCT
CCTGCAGGCCCTGGCGGGCATCTCCCCAGAGCTGGCCCCCACCTTG
GACACACTGCAGCTGGACGTCACTGACTTTGCCACGAACATCTGGC
TGCAGATGGAGGACCTGGGGGCGGCCCCCGCTGTGCAGCCCACCC
AGGGCGCCATGCCGACCTTCACTTCAGCCTTCCAACGCAGAGCAGG
AGGGGTCCTGGTTGCTTCCCAGCTGCATCGTTTCCTGGAGCTGGCA
TACCGTGGCCTGCGCTACCTTGCTGAGCCCGGTGGCGGAGGATCTT
CTTATACCTACAATTATGAAGACTACTGGGGCCAAGGAACCCTGGT
CACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTG
GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAA
CTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTA
CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCT
CTAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAA
GCCCAGCAACACCAAGGTGGACAAGAAAGTTGAACCCAAATCTTG
CGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG
GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC
TCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGT
GAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGG
CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG
GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA
GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG
CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG
AGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC
GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG
CTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG
CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC
ACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
Trastuzumab-43GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCAGCCTGGG
beta Exendin-4GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCAATATTAAGG
HCACACTTACATCCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG
AGTGGGTCGCACGTATTTATCCTACCAATGGTTACACACGCTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCGCAGACACTTCCAAG
AACACGGCGTATCTTCQAAATGAACAGCCTGAGAGCCGAGGACACG
GCCGTGTATTACTGTTCGAGAGAAACTAAGAAATACCAGAGCTGCG
GGGGTGGCGGAAGCATCGAAGGTCGTCACGGAGAAGGAACATTTA
CCAGCGACCTCAGCAAGCAGATGGAGGAAGAGGCCGTGAGGCTGT
TCATCGAGTGGCTGAAGAACGGCGGACCCTCCTCTGGCGCTCCACC
CCCTAGCGGCGGAGGTGGGAGTTGCTCTTATACCTACAATTATGAA
GACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGCCTCCA
CCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC
CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT
CCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCA
GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGT
GGACAAGAAAGTTGAACCCAAATCTTGCGACAAAACTCACACATG
CCCACCGTGCCCAGCACCTCCAGTCGCCGGACCGTCAGTCTTCCTC
TTCCCTCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG
AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA
GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGT
CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAAGCTCCATCGAGA
AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCTCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG
CCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG
CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC
CTCTCCCTGTCTCCGGGTAAA
Trastuzumab- beta Moka1 HC44embedded image
embedded image
embedded image
Trastuzumab- beta VM24 HC45embedded image
embedded image
embedded image
Trastuzumab-46GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCAGCCTGGG
beta hGCSF HCGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCAATATTAAGG
ACACTTACATCCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG
AGTGGGTCGCACGTATTTATCCTACCAATGGTTACACACGCTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCGCAGACACTTCCAAG
AACACGGCGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACG
GCCGTGTATTACTGTTCGAGAGAAACTAAGAAATACCAGAGCGCC
ACACCTCTGGGCCCCGCCTCCTCCCTGCCTCAGAGCTTTCTGCTCAA
ATGTCTGGAGCAGGTGCGGAAGATCCAGGGCGACGGCGCCGCTCT
GCAAGAGAAACTGGTCAGCGAATGCGCCACATATAAGCTGTGTCA
CCCCGAGGAACTGGTCCTCTTGGGCCACAGCCTGGGCATCCCCTGG
GCCCCTCTCAGCTCCTGCCCCTCCCAAGCTCTCCAACTCGGCTGGATG
TCTGTCCCAACTGCACTCCGGCCTCTTCCTGTACCAGGGACTCCTCC
AGGCTCTCGAAGGGATCAGCCCCGAACTGGGCCCCACACTGGACA
CCTTGCAACTCGATGTGGCCGATTTCGCCACAACCATCTGGCAGCA
GATGGAAGAACTCGGAATGGCTCCTGCTCTCCAGCCCACACAGGG
AGCTATGCCTGCTTTCGCCTCTGCTTTCCAGCGGAGAGCTGGTGGT
GTGCTCGTCGCATCCCACCTCCAGAGCTTCTTGGAGGTGTCCTATCG
GGTGCTCCGGCATCTGGCCCAACCCTCTTATACCTACAATTATGAA
GACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGCCAGCA
CTAAAGGTCCATCTGTGTTCCCTCTGGCTCCTTGCAGCCGGAGCAC
CTCCGAGTCCACAGCCGCTCTGGGATGTCTGGTGAAAGATTACTTC
CCCGAGCCCGTCACCGTGAGCTGGAATAGCGGAGCACTGACCTCCG
GCGTCCACACATTCCCCGCCGTGCTCCAAAGCTCCGGCCTGTACAG
CCTCTCCTCCGTGGTCACCGTGCCCAGCAGCTCTCTGGGCACAAAG
ACCTATACCTGTAACGTGGATCACAAGCCTAGCAACACCAAAGTGG
ATAAGCGGGTGGAGAGCAAGTACGGCCCTCCCTGTCCCCCTTGCCC
CGCTCCTGAGGCCGCTGGCGGACCTTCCGTGTTCCTGTTTCCCCCTA
AGCCCAAGGACACCCTCATGATTAGCCGGACACCCGAAGTGACCT
GCGTGGTCGTGGATGTGTCCCAGGAGGACCCTGAAGTGCAATTTAA
CTGGTACGTGGACGGCGTCGAGGTGCACAACGCCAAGACCAAGCC
TCGGGAAGAGCAGTTCAACAGCACCTACCGGGTGGTCAGCGTGCT
GACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTG
CAAGGTGAGCAACAAGGGCCTGCCCAGCTCCATCGAGAAGACCAT
CAGCAAGGCCAAGGGCCAGCCCAGGGAACCCCAGGTGTATACCCT
GCCCCCTAGCCAGGAGGAAATGACCAAAAACCAGGTGAGCCTGAC
CTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG
GAGAGCAACGGCCAGCCCGAGAACAATTACAAGACCACCCCTCCT
GTGCTGGACAGCGACGGCTCCTTCTTTCTGTATAGCCGGCTGACCG
TGGACAAGAGCAGGTGGCAGGAGGGCAACGTGTTCTCCTGTAGCG
TGATGCACGAGGCCCTGCACAACCATTACACCCAGAAGAGCTTGA
GCCTGAGCCTGGGCAAA
Trastuzumab- beta hGH CDRH3 HC47embedded image
embedded image
embedded image
embedded image
Trastuzumab- beta hGH CDRH2 HC48embedded image
embedded image
embedded image
embedded image
Trastuzumab- beta hLeptin HC49embedded image
embedded image
embedded image
embedded image
Trastuzumab- beta hIFNalpha HC50embedded image
GAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATCTCTCTTTTCTCC
TGCTTGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCA
ACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCA
GCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGAT
GAGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATG
ACCTGGAAGCCTGTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCC
TGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAATACTTCCAAAGAAT
CACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTT
GTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGA
embedded image
embedded image
embedded image
Trastuzumab- beta GLP1 HC51embedded image
embedded image
embedded image
embedded image
Trastuzumab- beta Elafin HC52embedded image
GCCTGGCTCCTGCCCCATTATCTTGATCCGGTGCGCCATGTTGAATCCC
CCTAACCGCTGCTTGAAAGATACTGACTGCCCAGGAATCAAGAAGTGCT
embedded image
embedded image
embedded image
Trastuzumab- beta Mambalgin HC53embedded image
GAGATATGAAGTTTTGCTATCATAACACTGGCATGCCTTTTCGAAATCTC
AAGCTCATCCTACAGGGATGTTCTTCTTCGTGCAGTGAAACAGAAAACAA
embedded image
embedded image
Trastuzumab- beta betatrophin HC54embedded image
embedded image
embedded image
embedded image
Trastuzumab- beta hGH fusion HC55embedded image
CTATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCA
GGAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCTG
CAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACCCT
CCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCAT
CTCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTCCTCAG
GAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTGACAGCAACGT
CTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGG
GAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAAGCAGAC
CTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAG
AACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAG
ACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGC
embedded image
embedded image
Trastuzumab- beta hIFNB1 HC56embedded image
AGCAATTTTCAGTGTCAGAAGCTCCTGTGGCAATTGAATGGGAGGCTTG
AATACTGCCTCAAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAA
GCAGCTGCAGCAGTTCCAGAAGGAGGACGCCGCATTGACCATCTATGA
GATGCTCCAGAACATCTTTGCTATTTTCAGACAAGATTCATCTAGCACTG
GCTGGAATGAGACTATTGTTGAGAACCTCCTGGCTAATGTCTATCATCAG
ATAAACCATCTGAAGACAGTCCTGGAAGAAAAACTGGAGAAAGAAGATT
TCACCAGGGGAAAACTCATGAGCAGTCTGCACCTGAAAAGATATTATGG
GAGGATTCTGCATTACCTGAAGGCCAAGGAGTACAGTCACTGTGCCTGG
ACCATAGTCAGAGTGGAAATCCTAAGGAACTTTTACTTCATTAACAGACT
embedded image
embedded image
Trastuzumab- beta Mambalgin HC57embedded image
embedded image
embedded image
Palivizumab- beta Mambalgin HC58embedded image
embedded image
embedded image
BLV1H12-beta- HEI 159embedded image
embedded image
BLV1H12-beta- HEI 260embedded image
embedded image
Human BVK antibody -beta- HEI LC61embedded image
embedded image
Human BVK antibody -beta- HEI HC 462 embedded image
embedded image
Human BVK antibody -beta- HEI HC 563embedded image
embedded image
Human BVK- beta-HEI HC 664embedded image
embedded image
Human BVK antibody -beta- HEI HC 765embedded image
embedded image
Human BVK antibody -beta- HEI HC 866embedded image
embedded image
Human BVK antibody -beta- HEI HC 967embedded image
embedded image
BLV1H12-beta68CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCC
trypsin inhibitorAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCG
ACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGG
AATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATC
CCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAA
GTCAGGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGC
AACTTACTATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAG
AGCAGATGTACCAAGAGCATACCACCCATCTGCTTCTCTTATACCTAC
AATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGA
CAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTC
AAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGC
CTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACT
CAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCA
GTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGC
AGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCA
GCTCCACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTGC
BLV1H12-beta69CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCC
BCCX2 HC 1AGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCG
(bAB-AC1)ACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGG
AATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATC
CCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAA
GTCAGGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGC
AACTTACTATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAG
AGCTATCGCAAATGTAGAGGAGGCAACGGACGAAGGTGGTGCTACCAA
AAGTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGGAC
AGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAA
GGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACC
GTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGA
CTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTT
CCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATG
GTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATG
TGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAAC
CCAAATCTTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACC
TGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC
AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG
TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT
ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGG
AGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT
CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA
GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA
TCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGG
TCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG
ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAA
GAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC
CGGGTAAATGATAA
BLV1H12-beta70CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCC
BCCX2 HC 2AGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCG
(bAb-AC2)ACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGG
AATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATC
CCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAA
GTCAGGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGC
AACTTACTATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAG
AGCTATCGCAAATGTAGAGGACCTCGAAGGTGGTGCTACCAAAAGTCTT
ATACCTACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCT
GCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTAC
CCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACAC
TGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCAC
CTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCT
GTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAG
TCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCA
TCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAAATCT
TGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC
TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC
CCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGAC
GTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG
TACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC
AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG
GGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG
ATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG
GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG
ACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAG
GTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTA
AATGATAA
BLV1H12-beta71CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCC
BCCX2 HC 3AGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCG
(bAb-AC3)ACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGG
AATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATC
CCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAA
GTCAGGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGC
AACTTACTATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAG
AGCTATCGCAAATGTAGAGGAGGCAACGGACGAAGGTGGTGCTACCAA
AAGTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGGAC
AGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAA
GGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACC
GTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGA
CTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTT
CCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATG
GTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATG
TGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAAC
CCAAATCTTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACC
TGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC
AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG
TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT
ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGG
AGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT
CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA
GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA
TCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGG
TCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG
ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAA
GAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC
CGGGTAAATGATAA
BLV1H12-beta72CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCC
BCCX2 HC 4AGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCG
(bAB-AC4)ACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGG
AATGGCTGGGCAGCATCGATGAAACTAAGAAATACCAGAGCTATCG
CAAATGTAGAGGAGGCCGAAGGTGGTGCTACCAAAAGTCTTATACCTA
CAATTATGAAACAGGGTACAATCCCGGACTGAAGAGCAGACTGTC
CATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGC
TCCGTCACCACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC
ACCAGGGAGGTGGCGGAAGCTGGCATGTGGATGTCTGGGGACAGG
GCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGT
GTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTG
ACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTG
TCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCC
AGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTG
ACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGG
CCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCA
AATCTTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA
ACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGG
TGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA
GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG
CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCC
AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC
CGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC
AAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC
TCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGA
GCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGA
GGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG
GGTAAATGATAA
Trastuzumab-73CAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCAGCCTGGG
beta BCCX2 HCGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCAATATTAAGG
long (HLCX)ACACTTACATCCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG
AGTGGGTCGCACGTATTTATCCTACCAATGGTTACACACGCTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCGCAGACACTTCCAAG
AACACGGCGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACG
GCCGTGTATTACTGTTCGAGAGAAACTAAGAAATACCAGAGCTATC
GCAAATGTAGAGGAGGCCGAAGGTGGTGCTACCAAAAGTCTTATACCT
ACAATTATGAAGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTC
CTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT
CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA
AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGC
CCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA
GGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCT
TGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCA
ACACCAAGGTGGACAAGAAAGTTGAACCCAAATCTTGCGACAAAA
CTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACC
GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT
CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG
AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGG
TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA
CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCC
AGCCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG
AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACC
AAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA
GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA
ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT
CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGG
GAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGATAA
Trastuzumab-74CAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCAGCCTGGG
beta BCCX2 HCGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCAATATTAAGG
mediumACACTTACATCCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG
(HMCX)AGTGGGTCGCACGTATTTATCCTACCAATGGTTACACACGCTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCGCAGACACTTCCAAG
AACACGGCGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACG
GCCGTGTATTACTGTTCGAGAGAAACTAAGAAATATCGCAAATGTAG
AGGAGGCCGAAGGTGGTGCTACCAAAAGTACAATTATGAAGACTACT
GGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGG
CCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGG
GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAAC
CGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC
ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGC
AGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCAGACCTACA
TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGA
AAGTTGAACCCAAATCTTGCGACAAAACTCACACATGCCCACCGTG
CCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC
CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCA
CATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAA
AGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG
TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA
GTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC
ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC
CTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT
CCCTGTCTCCGGGTAAATGATAA
Trastuzumab-75CAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCAGCCTGGG
beta BCCX2 HCGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCAATATTAAGG
short (HSCX)ACACTTACATCCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG
AGTGGGTCGCACGTATTTATCCTACCAATGGTTACACACGCTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCGCAGACACTTCCAAG
AACACGGCGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACG
GCCGTGTATTACTGTTCGAGATATCGCAAATGTAGAGGAGGCCGAAG
GTGGTGCTACCAAAAGGACTACTGGGGCCAAGGAACCCTGGTCACC
GTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC
CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCT
GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA
GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGT
CCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAG
CAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCC
AGCAACACCAAGGTGGACAAGAAAGTTGAACCCAAATCTTGCGAC
AAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT
GATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG
GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC
AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC
TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC
CCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCT
GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT
CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA
GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC
CACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGATAA
embedded image

TABLE 9
Immunoglobulin fusion protein - Amino Acid Sequence
NameSEQ ID NOSequence
Trastuzumat-76embedded image
beta hEPOVLERYLLEAKEAENITTGCAEHCSLNENITBPDTKVNFYAWKRMEVGQQAV
LCEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRAL
GAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTG
embedded image
embedded image
embedded image
embedded image
Trastuzumab-77embedded image
betaembedded image
bGCSF HCembedded image
RLCAAHKLCHPEELMLLRHSLGIPQAPLSSCSSQSLQLTSCLNQLHGGLFL
YQGLLQALAGISPELAPTLDTLQLDVTDFATNIWLQMEDLGAAPAVQPTQ
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-78embedded image
betaembedded image
Exendin-4embedded image
HCembedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-79embedded image
betaembedded image
MokalHCembedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-80embedded image
beta VM24embedded image
HCembedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-81embedded image
betaembedded image
hGCSF HCembedded image
embedded image
GLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPAL
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-82embedded image
beta hGHembedded image
HCembedded image
AYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWL
EPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQI
FKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGS
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-83embedded image
betaembedded image
hLeptin HCembedded image
VTGLDFIPGLHPILTISKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLL
HVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLW
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-84embedded image
betaembedded image
hlFNalphaembedded image
HCHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDK
FYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKY
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-85embedded image
beta GLP1embedded image
HCembedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-86embedded image
beta Elafinembedded image
HCembedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-87embedded image
betaembedded image
Mambalginembedded image
HCembedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-88embedded image
betaembedded image
betatrophinembedded image
HCRATEARLTEAGHSLGLYDRALEFLGTEVRQGQDATQELRTSLSEIQVEEDS
LHLRAEATARSLGEVARAQQALRDTVRRLQVQRGAWLGQAIIGEFETLKA
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-89embedded image
beta hGHembedded image
HCembedded image
AYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWL
EPVQFLRSVFANSLVYGASDSNYVDLLKDLEEGIQTLMGRLEDGSPRTGQI
FKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGS
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-90embedded image
betaembedded image
hIFNB1 HCembedded image
KDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIV
ENLLANVYHQIHLKTVLEEKLELEDFTRGLMSSLHLKRYYGRILHYLKA
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Palivizumab-91embedded image
betaembedded image
Mambalginembedded image
HCembedded image
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BLV1H12-92embedded image
beta BCCX2embedded image
HC 1 (bAb-embedded image
AC1)embedded image
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BLV1H12-93embedded image
beta BCCX2embedded image
HC 2 (bAb-embedded image
AC2)embedded image
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BLV1H12-94embedded image
beta BCCX2embedded image
HC 3 (bAb-embedded image
AC3)embedded image
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BLV1H12-95embedded image
beta BCCX2embedded image
HC 4 (bAb-embedded image
AC4)embedded image
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Trastuzumab-96embedded image
betaembedded image
BCCX2 HCembedded image
longembedded image
(HLCX)embedded image
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Trastuzumab-97embedded image
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BCCX2 HCembedded image
mediumembedded image
(HMCX)embedded image
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Trastuzumab-98embedded image
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BCCX2 HCembedded image
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(HSCX)embedded image
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BLV1H12-99embedded image
beta trypsinembedded image
inhibitorembedded image
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BLV1H12-100 embedded image
beta-EI 1embedded image
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BLV1H12-101 embedded image
beta-EI 2embedded image
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Human102 embedded image
BVK- betaembedded image
HEI LCembedded image
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Human103 embedded image
BVK- betaembedded image
HEI HC 4embedded image
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Human104 embedded image
BVK- betaembedded image
HEI HC 5embedded image
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Human105 embedded image
BVK- betaembedded image
HEI HC 6embedded image
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Human106 embedded image
BVK- betaembedded image
HEI HC 7embedded image
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Human107 embedded image
BVK- betaembedded image
HEI HC 8embedded image
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Human108 embedded image
BVK- betaembedded image
HEI HC 9embedded image
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TABLE 10
Extender Peptide Sequences
SEQ ID
NameNOSequence
Beta strand 1A109X1X2X3X4X5X6X7, wherein X1 may be a negatively charged amino acid;
X2 may be a polar, uncharged amino acid; X3 may be a positively charged
amino acid; X4 may be a positively charged amino acid; X5 may be a
hydrophobic amino acid; X6 may be a polar, uncharged amino acid; and
X7 may be a polar, uncharged amino acid
Beta strand 1B110ETKKYQXnS
Beta strand 1C111ETKKYQS
Beta Strand 1D112ETKKYQKHRHS
Beta Strand 1E113ETKKYQKHKNS
Beta Strand 1F114ETKKYQKHRHTTERS
Beta strand 2A115X1TX2NX3
Beta strand 2B116SX1TX2NX3E
Beta strand 2C117SX1TX2NX3X4
Beta strand 2D118SXnX1TX2NX3X4
Beta strand 2E119SYTYNYE
Beta strand 2F120SATYNYE
Beta strand 2G121SATANAE
Beta strand 2H122SYTANYE
Beta strand 2I123SYTYNAE
Beta strand 2J124SYTYNYA
Beta strand 2K125SYTYDYTYNYE
Beta strand 2L126SYTYDYTYNYE
Beta strand 2M127SITYNYTYDYTYNYE
Beta Strand 2N128YX1YX2Y; wherein X1 and X2 may be independently selected from a
polar amino acid
Alpha Helix 1A129X1X2X3X4X5X6X7X8X9X10X11X12X13X14; wherein in X1-X14 may be
independently selected from a positively charged amino acid or a
hydrophobic amino acid; wherein X1-X14 may be independently selected
from the group comprising A, L and K; wherein A may comprise at least
about 30% of the total amino acid composition; wherein A may comprise
less than about 70% of the total amino acid composition; wherein L may
comprise at least about 20% of the total amino acid composition; wherein
L may comprise less than about 50% of the total amino acid composition;
wherein K may comprise at least about 20% of the total amino acid
composition; wherein K may comprise less than about 50% of the total
amino acid composition; wherein the hydrophobic amino acids may
comprise at least about 50% of the total amino acid composition; wherein
the hydrophobic amino acids may comprise at least about 60% of the
total amino acid composition; wherein the hydrophobic amino acids may
comprise at least about 70% of the total amino acid composition; wherein
the hydrophobic amino acids may comprise less than about 90% of the
total amino acid composition
Alpha Helix 1B130(X1X2X3X4X5X6X7)n
Alpha Helix 1C131XaXbXcXd(X1X2X3X4X5X6X7)n; wherein n may be 1-3; wherein Xa, Xb
and Xd may be independently selected from a hydrophobic amino acid;
wherein Xc may be a polar, uncharged amino acid; wherein Xa, Xb and Xd
may be the same amino acid; wherein Xa, Xb and Xd may be different
amino acids.
Alpha Helix 1D132XaXbXcXd (AKLAALK)n; wherein n may be 1-3; wherein Xa, Xb and Xd
may be independently selected from a hydrophobic amino acid; wherein
Xc may be a polar, uncharged amino acid; wherein Xa, Xb and Xd may be
the same amino acid; wherein Xa, Xb and Xd may be different amino
acids.
Alpha Helix 1E133(AKLAALK)n
Alpha Helix 1F134GGSG(AKLAALK)n
Alpha Helix 1G135AKLAALKAKLAALK
Alpha Helix 1H136GGSGAKLAALKAKLAALK
Alpha Helix 1I137CAALKSKVSALKSKVASLKSKVAAL
Alpha Helix 1J138ALKKELQANKKELAQLKKELQALKKELAQ
Alpha Helix 2A139X1X2X3X4X5X6X7X8X9X10X11X12X13X14; wherein in X1-X14 may be
independently selected from a negatively charged amino acid or a
hydrophobic amino acid; wherein X1-X14 may be independently selected
from the group comprising A, L and E; wherein A may comprise at least
about 30% of the total amino acid composition; wherein A may comprise
less than about 70% of the total amino acid composition; wherein L may
comprise at least about 20% of the total amino acid composition; wherein
L may comprise less than about 50% of the total amino acid composition;
wherein E may comprise at least about 20% of the total amino acid
composition; wherein E may comprise less than about 50% of the total
amino acid composition; wherein the hydrophobic amino acids may
comprise at least about 50% of the total amino acid composition; wherein
the hydrophobic amino acids may comprise at least about 60% of the
total amino acid composition; wherein the hydrophobic amino acids may
comprise at least about 70% of the total amino acid composition; wherein
the hydrophobic amino acids may comprise less than about 90% of the
total amino acid composition
Alpha Helix 2B140(X1X2X3X4X5X6X7)n
Alpha Helix 2C141(X1X2X3X4X5X6X7)n XaXbXcXd; wherein n may be 1-3; wherein Xa, Xb
and Xd may be independently selected from a hydrophobic amino acid;
wherein Xc may be a polar, uncharged amino acid; wherein Xa, Xb and Xd
may be the same amino acid; wherein Xa, Xb and Xd may be different
amino acids.
Alpha Helix 2D142(ELAALEA)n XaXbXcXd; wherein n may be 1-3; wherein Xa, Xb and Xd
may be independently selected from a hydrophobic amino acid; wherein
Xc may be a polar, uncharged amino acid; wherein Xa, Xb and Xd may be
the same amino acid; wherein Xa, Xb and Xd may be different amino
acids.
Alpha Helix 2E143(ELAALEA)n
Alpha Helix 2F144(ELAALEA)nGGSG
Alpha Helix 2G145ELAALEA ELAALEA
Alpha Helix 2H146ELAALEA ELAALEAGGSG
Alpha Helix 2I147LAAVESELSAVESELASVESELAAC
Alpha Helix 2J148QLEKKLQALEKKLAQLEKKNQALEKKLAQ
Alpha Helix 3149CAALKSKVSALKSKVASLKSKVAAL
Alpha Helix 4150LAAVESELSAVESELASVESELAAC
Alpha Helix 5151ALKKELQANKKELAQLKKELQALKKELAQ
Alpha Helix 6152QLEKKLQALEKKLAQLEKKNQALEKKLAQ
Alpha Helix 7153LKLELQLIKQYREAL
Alpha Helix 8154LAKILEDEEKHIEWL
Alpha Helix 9155LSDLHRQVSRLV
Alpha Helix 10156LQDAKVLLEAAL
Alpha Helix 11157LQQKIHELEGLIAQH
Alpha Helix 12158AAQIRDQLHQLRELF
Alpha Helix 13159ELARLIRLYFAL
Alpha Helix 14160QESLYVDLFDKF

TABLE 11
Linker sequences
SEQ ID
NameNOSequence
Linker 1161XeXfXgXh; wherein Xe, Xf and Xg may be independently selected from a
hydrophobic amino acid; wherein Xh may be a polar, uncharged amino acid;
wherein Xe, Xf and Xg may be the same amino acid; wherein Xe, Xf and Xg may be
different amino acids.
Linker 2162CXeXfXgXh; wherein Xe, Xf and Xg may be independently selected from a
hydrophobic amino acid; wherein Xh may be a polar, uncharged amino acid;
wherein Xe, Xf and Xg may be the same amino acid; wherein Xe, Xf and Xg may be
different amino acids.
Linker 3163XeXfXgXhC; wherein Xe, Xf and Xg may be independently selected from a
hydrophobic amino acid; wherein Xh may be a polar, uncharged amino acid;
wherein Xe, Xf and Xg may be the same amino acid; wherein Xe, Xf and Xg may be
different amino acids.
Linker 4164(GGGGS)n
Linker 5165GGGSGGGGS
Linker 6166GGGGSGGGS

TABLE 12
Therapeutic agents-Nucleic acid sequence
SEQ ID
NameNOSequence
bGCSF167ACCCCCCTTGGCCCTGCCCGATCCCTGCCCCAGAGCTTCCTGCTCAAG
TGCTTAGAGCAAGTGAGGAAAATCCAGGCTGATGGCGCCGAGCTGCA
GGAGAGGCTGTGTGCCGCCCACAAGCTGTGCCACCCGGAGGAGCTGA
TGCTGCTCAGGCACTCTCTGGGCATCCCCCAGGCTCCCCTAAGCAGCT
GCTCCAGCCAGTCCCTGCAGCTGACGAGCTGCCTGAACCAACTACACG
GCGGCCTCTTTCTCTACCAGGGCCTCCTGCAGGCCCTGGCGGGCATCT
CCCCAGAGCTGGCCCCCACCTTGGACACACTGCAGCTGGACGTCACTG
ACTTTGCCACGAACATCTGGCTGCAGATGGAGGACCTGGGGGCGGCC
CCCGCTGTGCAGCCCACCCAGGGCGCCATGCCGACCTTCACTTCAGCC
TTCCAACGCAGAGCAGGAGGGGTCCTGGTTGCTTCCCAGCTGCATCGT
TTCCTGGAGCTGGCATACCGTGGCCTGCGCTACCTTGCTGAGCCC
hGCSF168GCCACACCTCTGGGCCCCGCCTCCTCCCTGCCTCAGAGCTTTCTGCTCA
AATGTCTGGAGCAGGTGCGGAAGATCCAGGGCGACGGCGCCGCTCTG
CAAGAGAAACTGGTCAGCGAATGCGCCACATATAAGCTGTGTCACCC
CGAGGAACTGGTCCTCTTGGGCCACAGCCTGGGCATCCCCTGGGCCCC
TCTCAGCTCCTGCCCCTCCCAAGCTCTCCAACTGGCTGGATGTCTGTCC
CAACTGCACTCCGGCCTCTTCCTGTACCAGGGACTCCTCCAGGCTCTC
GAAGGGATCAGCCCCGAACTGGGCCCCACACTGGACACCTTGCAACT
CGATGTGGCCGATTTCGCCACAACCATCTGGCAGCAGATGGAAGAAC
TCGGAATGGCTCCTGCTCTCCAGCCCACACAGGGAGCTATGCCTGCTT
TCGCCTCTGCTTTCCAGCGGAGAGCTGGTGGTGTGCTCGTCGCATCCC
ACCTCCAGAGCTTCTTGGAGGTGTCCTATCGGGTGCTCCGGCATCTGG
CCCAACCC
Exendin-4169CACGGAGAAGGAACATTTACCAGCGACCTCAGCAAGCAGATGGAGGA
AGAGGCCGTGAGGCTGTTCATCGAGTGGCTGAAGAACGGCGGACCCT
CCTCTGGCGCTCCACCCCCTAGC
Moka1170ATCAACGTGAAGTGCAGCCTGCCCCAGCAGTGCATCAAGCCCTGCAA
GGACGCCGGCATGCGGTTCGGCAAGTGCATGAACAAGAAGTGCAGGT
GCTACAGC
VM24171GCCGCTGCAATCTCCTGCGTCGGCAGCCCCGAATGTCCTCCCAAGTGC
CGGGCTCAGGGATGCAAGAACGGCAAGTGTATGAACCGGAAGTGCAA
GTGCTACTATTGC
hGLP-1172CATGCGGAAGGCACCTTTACCAGCGATGTGAGCAGCTATCTGGAAGG
CCAGGCGGCGAAAGAATTTATTGCGTGGCTGGTGAAAGGCCGC
hEPO173GCCCCACCACGCCTCATCTGTGACAGCCGAGTCCTGGAGAGGTACCTC
TTGGAGGCCAAGGAGGCCGAGAATATCACGACGGGCTGTGCTGAACA
CTGCAGCTTGAATGAGAATATCACTGTCCCAGACACCAAAGTTAATTT
CTATGCCTGGAAGAGGATGGAGGTCGGGCAGCAGGCCGTAGAAGTCT
GGCAGGGCCTGGCCCTGCTGTCGGAAGCTGTCCTGCGGGGCCAGGCC
CTGTTGGTCAACTCTTCCCAGCCGTGGGAGCCCCTGCAGCTGCATGTG
GATAAAGCCGTCAGTGGCCTTCGCAGCCTCACCACTCTGCTTCGGGCT
CTGGGAGCCCAGAAGGAAGCCATCTCCCCTCCAGATGCGGCCTCAGC
TGCTCCACTCCGAACAATCACTGCTGACACTTTCCGCAAACTCTTCCG
AGTCTACTCCAATTTCCTCCGGGGAAAGCTGAAGCTGTACACAGGGG
AGGCCTGCAGGACAGGGGACAGA
GMCSF174GCGCCGGCGCGCAGCCCGAGCCCGAGCACCCAGCCGTGGGAACATGT
GAACGCGATTCAGGAAGCGCGCCGCCTGCTGAACCTGAGCCGCGATA
CCGCGGCGGAAATGAACGAAACCGTGGAAGTGATTAGCGAAATGTTT
GATCTGCAGGAACCGACCTGCCTGCAGACCCGCCTGGAACTGTATAA
ACAGGGCCTGCGCGGCAGCCTGACCAAACTGAAAGGCCCGCTGACCA
TGATGGCGAGCCATTATAAACAGCATTGCCCGCCGACCCCGGAAACC
AGCTGCGCGACCCAGATTATTACCTTTGAAAGCTTTAAAGAAAACCTG
AAAGATTTTCTGCTGGTGATTCCGTTTGATTGCTGGGAACCGGTGCAG
GAA
IFN-beta175ATGAGCTATAACCTGCTGGGCTTTCTGCAGCGCAGCAGCAACTTTCAG
TGCCAGAAACTGCTGTGGCAGCTGAACGGCCGCCTGGAATATTGCCTG
AAAGATCGCATGAACTTTGATATTCCGGAAGAAATTAAACAGCTGCA
GCAGTTTCAGAAAGAAGATGCGGCGCTGACCATTTATGAAATGCTGC
AGAACATTTTTGCGATTTTTCGCCAGGATAGCAGCAGCACCGGCTGGA
ACGAAACCATTGTGGAAAACCTGCTGGCGAACGTGTATCATCAGATT
AACCATCTGAAAACCGTGCTGGAAGAAAAACTGGAAAAAGAAGATTT
TACCCGCGGCAAACTGATGAGCAGCCTGCATCTGAAACGCTATTATGG
CCGCATTCTGCATTATCTGAAAGCGAAAGAATATAGCCATTGCGCGTG
GACCATTGTGCGCGTGGAAATTCTGCGCAACTTTTATTTTATTAACCG
CCTGACCGGCTATCTGCGCAAC
Oxyntomodulin176CACTCTCAGGGTACCTTCACCTCTGACTACTCTAAATACCTGGACTCTC
GTCGTGCTCAGGACTTCGTTCAGTGGCTGATGAACACCAAACGTAACC
GTAACAACATCGCT
Leptin177GTTCCAATTCAAAAGGTTCAAGATGATACCAAAACTCTGATTAAAACT
ATTGTCACGCGTATAAACGACATCAGCCATACCCAGTCGGTTAGCTCA
AAGCAAAAAGTTACCGGTTTGGACTTTATTCCGGGACTGCACCCGATC
CTGACCCTTAGTAAAATGGACCAGACACTGGCCGTCTACCAGCAAATC
CTGACATCGATGCCATCCAGAAATGTGATACAAATTAGCAACGATTTG
GAAAACCTTCGCGATCTGCTGCACGTGCTGGCCTTCAGTAAGTCCTGT
CATCTGCCGTGGGCGTCGGGACTGGAGACTCTTGACTCGCTGGGTGGA
GTGTTAGAGGCCTCTGGCTATTCTACTGAAGTCGTTGCGCTGTCACGC
CTCCAGGGGAGCCTGCAGGACATGCTGTGGCAGCTGGACCTGTCACCT
GGCTGC
Betatrophin178GCTCCTCTGGGCGGTCCTGAACCAGCACAGTACGAGGAACTGACACT
GTTGTTCCATGGAGCCTTGCAGCTGGGCCAGGCCCTCAACGGCGTGTA
CCGCGCCACAGAGGCACGTTTGACCGAGGCCGGACACAGCCTGGGTT
TGTACGACAGAGCCCTGGAGTTTCTGGGTACCGAAGTGCGTCAGGGC
CAGGACGCAACTCAGGAGCTGAGAACCTCCCTCTCTGAGATCCAGGT
GGAGGAGGACGCCCTGCACCTGCGCGCCGAGGCGACAGCACGCTCTT
TGGGAGAAGTTGCTCGCGCTCAGCAGGCCCTGCGTGATACCGTGCGG
AGACTCCAAGTTCAGCTCAGAGGCGCTTGGCTCGGACAGGCGCATCA
GGAGTTCGAGACCCTGAAAGCTCGTGCCGACAAACAGTCCCACCTGC
TGTGGGCGCTCACCGGTCACGTCCAGCGCCAGCAACGCGAAATGGCC
GAGCAGCAGCAATGGCTGCGCCAAATCCAGCAGCGCCTGCATACCGC
GGCCCTGCCAGCGTAA
GDF11179AACCTGGGTCTGGACTGCGACGAACACTCTTCTGAATCTCGTTGCTGC
CGTTACCCGCTGACCGTTGACTTCGAGGCGTTCGGTTGGGACTGGATC
ATCGCTCCGAAACGTTACAAAGCTAACTACTGCTCTGGTCAGTGCGAA
TACATGTTCATGCAGAAATACCCGCACACCCACCTGGTTCAGCAGGCT
AACCCGCGTGGTTCTGCTGGTCCGTGCTGCACCCCGACCAAAATGTCT
CCGATCAACATGCTGTACTTCAACGACAAACAGCAGATCATCTACGGT
AAAATCCCGGGTATGGTTGTTGACCGTTGCGGTTGCTCTTAA
ANGPTL3180GGATCCGGTGGTTTCACCATCAAACTGCTGCTGTTCATCGTTCCGCTG
GTTATCTCTTCTCGTATCGACCAGGACAACTCTTCTTTCGACTCTCTGT
CTCCGGAACCGAAATCTCGTTTCGCTATGCTGGACGACGTTAAAATCC
TGGCTAACGGTCTGCTGCAGCTGGGTCACGGTCTGAAAGACTTCGTTC
ACAAAACCAAAGGTCAGATCAACGACATCTTCCAGAAACTGAACATC
TTCGACCAGTCTTTCTACGACCTGTCTCTGCAGACCTCTGAAATCAAA
GAAGAAGAAAAAGAACTGCGTCGTACCACCTACAAACTGCAGGTTAA
AAACGAAGAAGTTAAAAACATGTCTCTGGAACTGAACTCTAAACTGG
AATCTCTGCTGGAAGAAAAAATCCTGCTGCAGCAGAAAGTTAAATAC
CTGGAAGAACAGCTGACCAACCTGATCCAGAACCAGCCGGAAACCCC
GGAACACCCGGAAGTTACCTCTCTGAAAACCTTCGTTGAAAAACAGG
ACAACTCTATCAAAGACCTGCTGCAGACCGTTGAAGACCAGTACAAA
CAGCTGAACCAGCAGCACTCTCAGATCAAAGAAATCGAAAACCAGCT
GCGTCGTACCTCTATCCAGGAACCGACCGAAATCTCTCTGTCTTCTAA
ACCGCGTGCTCCGCGTACCACCCCGTTCCTGCAGCTGAACGAAATCCG
TAACGTTAAACACGACGGTATCCCGGCTGAATGCACCACCATCTACAA
CCGTGGTGAACACACCTCTGGTATGTACGCTATCCGTCCGTCTAACTC
TCAGGTTTTCCACGTTTACTGCGACGTTATCTCTGGTTCTCCGTGGACC
CTGATCCAGCACCGTATCGACGGTTCTCAGAACTTCAACGAAACCTGG
GAAAACTACAAATACGGTTTCGGTCGTCTGGACGGTGAATTCTGGCTG
GGTCTGGAAAAAATCTACTCTATCGTTAAACAGTCTAACTACGTTCTG
CGTATCGAACTGGAAGACTGGAAAGACAACAAACACTACATCGAATA
CTCTTTCTACCTGGGTAACCACGAAACCAACTACACCCTGCACCTGGT
TGCTATCACCGGTAACGTTCCGAACGCTATCCCGAAGAAGAAGAAGA
AAAAAAAGAAGAAGAAAT
hGH181TTCCCAACCATTCCCTTATCCAGGCTTTTTGACAACGCTATGCTCCGCG
CCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAGGAGTTTGAAG
AAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCTGCAGAACCCCC
AGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACCCTCCAACAGGG
AGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATCTCCCTG
CTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTCCTCAGGAGTGTC
TTCGCCAACAGCCTGGTGTACGGCGCCTCTGACAGCAACGTCTATGAC
CTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGGAGGCT
GGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAAGCAGACCTACA
GCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAAC
TACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGAC
ATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTC
hIFN-alpha182TGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATG
CTCCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGAC
AGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAA
AAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTC
AATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTC
CTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAA
GCCTGTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAA
GGAGGACTCCATTCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCT
CTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCA
GAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGAAA
GTTTAAGAAGTAAGGAA
Mamba183CTGAAATGTTACCAACATGGTAAAGTTGTGACTTGTCATCGAGATATG
AAGTTTTGCTATCATAACACTGGCATGCCTTTTCGAAATCTCAAGCTC
ATCCTACAGGGATGTTCTTCTTCGTGCAGTGAAACAGAAAACAATAAG
TGTTGCTCAACAGACAGATGCAACAA
Parathyroid184TCTGTGAGTGAAATACAGCTTATGCATAACCTGGGAAAACATCTGAAC
hormoneTCGATGGAGAGAGTAGAATGGCTGCGTAAGAAGCTGCAGGATGTGCA
CAATTTTGTTGCCCTTGGAGCTCCTCTAGCTCCCAGAGATGCTGGTTCC
CAGAGGCCCCGAAAAAAGGAAGACAATGTCTTGGTTGAGAGCCATGA
AAAAAGTCTTGGAGAGGCAGACAAAGCTGATGTGAATGTATTAACTA
AAGCTAAATCCCAG
IL-11185ATGAACTGCGTGTGCCGCCTGGTGCTGGTGGTGCTGAGCCTGTGGCCG
GATACCGCGGTGGCGCCGGGCCCGCCGCCGGGCCCGCCGCGCGTGAG
CCCGGATCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCA
GCCTGCTGGCGGATACCCGCCAGCTGGCGGCGCAGCTGCGCGATAAA
TTTCCGGCGGATGGCGATCATAACCTGGATAGCCTGCCGACCCTGGCG
ATGAGCGCGGGCGCGCTGGGCGCGCTGCAGCTGCCGGGCGTGCTGAC
CCGCCTGCGCGCGGATCTGCTGAGCTATCTGCGCCATGTGCAGTGGCT
GCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGG
GCACCCTGCAGGCGCGCCTGGATCGCCTGCTGCGCCGCCTGCAGCTGC
TGATGAGCCGCCTGGCGCTGCCGCAGCCGCCGCCGGATCCGCCGGCG
CCGCCGCTGGCGCCGCCGAGCAGCGCGTGGGGCGGCATTCGCGCGGC
GCTGGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCG
CGGCCTGCTGCTGCTGAAAACCCGCCTG
Relaxin186GACTCTTGGATGGAAGAAGTTATCAAACTGTGCGGTCGTGAACTGGTT
CGTGCTCAGATCGCTATCTGCGGTATGTCTACCTGGTCTGGTGGCGGT
CGTGGCGGTCGTCAGCTGTACTCTGCTCTGGCTAACAAATGCTGCCAC
GTTGGTTGCACCAAACGTTCTCTGGCTCGTTTCTGCTAA
Relaxin-187GATAGCTGGATGGAAGAAGTGATTAAACTGTGCGGCCGCGAACTGGT
FactorXaGCGCGCGCAGATTGCGATTTGCGGCATGAGCACCTGGAGCATTGAAG
GCCGCAGCCTGAGCCAGGAAGATGCGCCGCAGACCCCGCGCCCGGTG
GCGGAAATTGTGCCGAGCTTTATTAACAAAGATACCGAAACCATTAA
CATGATGAGCGAATTTGTGGCGAACCTGCCGCAGGAACTGAAACTGA
CCCTGAGCGAAATGCAGCCGGCGCTGCCGCAGCTGCAGCAGCATGTG
CCGGTGCTGAAAGATAGCAGCCTGCTGTTTGAAGAATTTAAAAAACT
GATTCGCAACCGCCAGAGCGAAGCGGCGGATAGCAGCCCGAGCGAAC
TGAAATATCTGGGCCTGGATACCCATAGCATTGAAGGCCGCCAGCTGT
ATAGCGCGCTGGCGAACAAATGCTGCCATGTGGGCTGCACCAAACGC
AGCCTGGCGCGCTTTTGC
Relaxin188AGCCTGAGCCAGGAAGATGCGCCGCAGACCCCGCGCCCGGTGGCGGA
fragmentAATTGTGCCGAGCTTTATTAACAAAGATACCGAAACCATTAACATGAT
GAGCGAATTTGTGGCGAACCTGCCGCAGGAACTGAAACTGACCCTGA
GCGAAATGCAGCCGGCGCTGCCGCAGCTGCAGCAGCATGTGCCGGTG
CTGAAAGATAGCAGCCTGCTGTTTGAAGAATTTAAAAAACTGATTCGC
AACCGCCAGAGCGAAGCGGCGGATAGCAGCCCGAGCGAACTGAAAT
ATCTGGGCCTGGATACCCATAGC
Relaxin2 A189GACTCTTGGATGGAAGAAGTTATCAAACTGTGCGGTCGTGAACTGGTT
chainCGTGCTCAGATCGCTATCTGCGGTATGTCTACCTGGTCTAAACGTTCTC
TGTCTCAGGAAGACGCTCCGCAGACCCCGCGTCCGGTT
Relaxin2 B190CAGCTGTACTCTGCTCTGGCTAACAAATGCTGCCACGTTGGTTGCACC
chainAAACGTTCTCTGGCTCGTTTCTGC
IL8191CCGCGCAGCGCGAAAGAACTGCGCTGCCAGTGCATTAAAACCTATAG
CAAACCGTTTCATCCGAAATTTATTAAAGAACTGCGCGTGATTGAAAG
CGGCCCGCATTGCGCGAACACCGAAATTATTGTGAAACTGAGCGATG
GCCGCGAACTGTGCCTGGATCCGAAAGAAAACTGGGTGCAGCGCGTG
GTGGAAAAATTTCTGAAACGCGCGGAAAACAGC
ziconotide192TGCAAAGGCAAAGGCGCGAAATGCAGCCGCCTGATGTATGATTGCTG
CACCGGCAGCTGCCGCAGCGGCAAATGC
somatostatin193GCGGGCTGCAAAAACTTTTTTTGGAAAACCTTTACCAGCTGCGGC
chlorotoxin194ATGTGCATGCCGTGCTTTACCACCGATCATCAGATGGCGCGCAAATGC
GATGATTGCTGCGGCGGCAAAGGCCGCGGCAAATGCTATGGCCCGCA
GTGCCTG
SDF1(alpha)195AAACCGGTGAGCCTGAGCTATCGCTGCCCGTGCCGCTTTTTTGAAAGC
CATGTGGCGCGCGCGAACGTGAAACATCTGAAAATTCTGAACACCCC
GAACTGCGCGCTGCAGATTGTGGCGCGCCTGAAAAACAACAACCGCC
AGGTGTGCATTGATCCGAAACTGAAATGGATTCAGGAATATCTGGAA
AAAGCGCTGAACAAA
IL21196CAGGGCCAGGATCGCCATATGATTCGCATGCGCCAGCTGATTGATATT
GTGGATCAGCTGAAAAACTATGTGAACGATCTGGTGCCGGAATTTCTG
CCGGCGCCGGAAGATGTGGAAACCAACTGCGAATGGAGCGCGTTTAG
CTGCTTTCAGAAAGCGCAGCTGAAAAGCGCGAACACCGGCAACAACG
AACGCATTATTAACGTGAGCATTAAAAAACTGAAACGCAAACCGCCG
AGCACCAACGCGGGCCGCCGCCAGAAACATCGCCTGACCTGCCCGAG
CTGCGATAGCTATGAAAAAAAACCGCCGAAAGAATTTCTGGAACGCT
TTAAAAGCCTGCTGCAGAAAATGATTCATCAGCATCTGAGCAGCCGC
ACCCATGGCAGCGAAGATAGC
Elafin197GCGCAAGAGCCAGTCAAAGGTCCAGTCTCCACTAAGCCTGGCTCCTGC
CCCATTATCTTGATCCGGTGCGCCATGTTGAATCCCCCTAACCGCTGCT
TGAAAGATACTGACTGCCCAGGAATCAAGAAGTGCTGTGAAGGCTCT
TGCGGGATGGCCTGTTTCGTTCCCCAG
BCCX2 (of198TATCGCAAATGTAGAGGAGGCCGAAGGTGGTGCTACCAAAAG
bAb-AC1)
Elastase199ATGTGTACCGCAAGCATACCACCCCAATGCTAC
inhibitor

TABLE 13
Therapeutic agents - Amino acid sequences
SEQ ID
NameNOSequence
bGCSF200TPLGPARSLPQSFLLKCLEQVRKIQADGAELQERLCAAHKLCHPEELMLL
RHSLGIPQAPLSSCSSQSLQLTSCLNQLHGGLFLYQGLLQALAGISPELAPT
LDTLQLDVTDFATNIWLQMEDLGAAPAVQPTQGAMPTFTSAFQRRAGG
VLVASQLHRFLELAYRGLRYLAEP
Exendin-4201HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS
Moka1202INVKCSLPQQCIKPCKDAGMRFGKCMNKKCRCYS
VM24203AAAISCVGSPECPPKCRAQGCKNGKCMNRKCKCYYC
hGCSF204ATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLVSECATYKLCHPEE
LVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISP
ELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQR
RAGGVLVASHLQSFLEVSYRVLRHLAQP
hGLP-1205HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR
hEPO206APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYA
WKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDK
AVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNF
LRGKLKLYTGEACRTGDR
GMCSF207APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQ
EPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQI
ITFESFKENLKDFLLVIPFDCWEPVQE
IFN-beta208MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQ
FQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKT
VLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEI
LRNFYFINRLTGYLRN
Oxyntomodulin209HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA
Leptin210VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSK
MDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASG
LETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC
Betatrophin211APLGGPEPAQYEELTLLFHGALQLGQALNGVYRATEARLTEAGHSLGLY
DRALEFLGTEVRQGQDATQELRTSLSEIQVEEDALHLRAEATARSLGEVA
RAQQALRDTVRRLQVQLRGAWLGQAHQEFETLKARADKQSHLLWALT
GHVQRQQREMAEQQQWLRQIQQRLHTAALPA
GDF11212NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEY
MFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIP
GMVVDRCGCS
ANGPTL3213GSGGFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGL
LQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRR
TTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQN
QPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQ
LRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEH
TSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYG
FGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHE
TNYTLHLVAITGNVPNAIPKKKKKKKKKK
hGH214FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTS
LCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVY
GASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHND
DALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF
hIFN-alpha215CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKA
ETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQ
GVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSF
SLSTNLQESLRSKE
Mamba216LKCYQHGKVVTCHRDMKFCYHNTGMPFRNLKLILQGCSSSCSETENNKC
CSTDRCN
Parathyroid217SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGS
HormoneQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ
IL-11218MNCVCRLVLVVLSLWPDTAVAPGPPPGPPRVSPDPRAELDSTVLLTRSLL
ADTRQLAAQLRDKFPADGDHNLDSLPTLAMSAGALGALQLPGVLTRLR
ADLLSYLRHVQWLRRAGGSSLKTLEPELGTLQARLDRLLRRLQLLMSRL
ALPQPPPDPPAPPLAPPSSAWGGIRAALAILGGLHLTLDWAVRGLLLLKT
RL
Relaxin219DSWMEEVIKLCGRELVRAQIAICGMSTWSGGGRGGRQLYSALANKCCH
VGCTKRSLARFC
Relaxin-220DSWMEEVIKLCGRELVRAQIAICGMSTWSIEGRSLSQEDAPQTPRPVAEIV
FactorXaPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLKDSS
LLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSIEGRQLYSALANKCCHV
GCTKRSLARFC
Relaxin221SLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQ
fragmentPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHS
Relaxin2 A222DSWMEEVIKLCGRELVRAQIAICGMSTWS
chain
Relaxin2 B223QLYSALANKCCHVGCTKRSLARFC
chain
IL8224PRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELC
LDPKENWVQRVVEKFLKRAENS
ziconotide225CKGKGAKCSRLMYDCCTGSCRSGKC
somatostatin226AGCKNFFWKTFTSCG
chlorotoxin227MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCL
SDF1(alpha)228KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVC
IDPKLKWIQEYLEKALNK
IL21229QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSC
FQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSY
EKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
Elafin230AQEPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGM
ACFVPQ
BCCX2 (of231YRKCRGGRRWCYQK
bAb-AC1)
BCCX2 (of232YRKCRGPRRWCYQK
bAB-AC2)
BCCX2 (of233YRKCRGGNGRRWCYQK
bAB-AC3)
BCCX2 (of234TSVHQGGGGSWHVDV
bAB-AC4)
Elastase235MCTASIPPQCY
inhibitor

TABLE 14
Miscellaneous sequences
SEQ ID
NameNOSequence
Factor Xa nucleotide236ATCGAAGGTCGT
Factor Xa peptide237IEGR
PC2 Cleavage Site - Nucleotide238CGTAAAAAACGT
PC2 Cleavage Site - Amino acid239RKKR

TABLE 15
Immunoglobulin Fusion Proteins for Dual Fusions
SEQ ID
NameNOSequence
Trastuzumab-coil hEPO LC240embedded image
embedded image
NITTGCAEHCSLNENITVPDTKVNFYAWRMEVGQQ
AVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHV
DKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTTT
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-coil bGCSF HC241embedded image
embedded image
embedded image
embedded image
EQVRKLQADGAELQERLCSSHKLCHPEELMLLRHSL
GIPQAPLSSCSSQSLQLTSCLNQLHGGLFLYQGLLQAL
AGISPELAPTLDTLQLDVTDFATNIWLQMEDLGAAPA
VPTQGAMPTFTSAFQRRAGGVLVASQLHRFLELAY
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-coil hGCSF HC242embedded image
embedded image
embedded image
embedded image
LEQVRKIQGDGAALQEKLVSECATVKLCHPEELVLLG
HSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGL
LQALEGISPELGPTLDTLQLDVADFATTIWQQMEELG
MAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFL
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-coil hLeptin HC243embedded image
embedded image
embedded image
embedded image
TRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQ
TLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKS
CHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGS
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-direct-hEPO LC244embedded image
LICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVP
DTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRG
QALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGA
QKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGK
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-direct-bGCSF HC245embedded image
embedded image
embedded image
embedded image
AAHKLCHPEELMLLRHSLGIPQAPLSSCSSQSLQLTSC
LNQLHGGLFLYQGLLQALAGISPELAPTLDTLQLDVT
DFATNIWLQMEDIGAAPAVQPTQGAMPTFTSAFQRR
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-direct hGCSF HC246embedded image
embedded image
embedded image
embedded image
VSECATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQ
LAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQ
LDVADFATTIWQQMEELGMAPALQPTQGAMPAFASA
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
embedded image
Trastuzumab-direct hLeptin HC247embedded image
embedded image
embedded image
embedded image
LDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQIS
NDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVL
embedded image
embedded image
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embedded image
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embedded image
embedded image

TABLE 16
Ultralong CDR3 containing bovine antibody heavy chain and
light chain amino acid sequences
SEQ ID
NameNOSequence
BLV1H1248QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEWLGS
2 HCIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCTSVHQE
TKKYQSCPDGYRERSDCSNRPACGTSDCCRVSVFGNCLTTLPVSYSYTYN
YEWHVDVWGQGLLVTVSS
BLV5B8249QVQLRESGPSLVQPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEWLGS
HCIDTGGSTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCTTVHQET
RKTCSDGYIAVDSCGRGQSDGCVNDCNSCYYGWRNCRRQPAIHSYEFHV
DAWGRGLLVTVSS
BLV5D3250QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRRAPGKALEWLGT
HCTDTGGSAAYNPGLKSRLSITKDNSKSQVSLSVSNVATEDSATYYCSSVTQR
THVSRSCPDGCSDGDGCVDGCCCSAYRCYTPGVRDLSCTSYSITYTYEWN
VDAWGQGLLVTVSS
BLV1H1251QAVLNQPSSVSGSLGQRVSITCSGSSSNVGNGYVSWYQLIPGSAPRTLIYG
2 LCDTSRASGVPDRFSGSRSGNTATLTISSLQAEDEADYFCASAEDSSSNAVFG
SGTTLTVLGQPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSVTVVWK
ADGSTITRNVETTRASKQSNSKYAASSYLSLTSSDWKSKGSYSCEVTHEGS
TVTKTVKPSECS
BLV5B8252QAVLNQPSSVSGSLGQRVSITCSGSSSNVGNGYVSWYQLIPGSAPRTLIYG
LCDTSRASGVPDRFSGSRSGNTATLTISSLQAEDEADYFCASAEDSSSNAVFG
SGTTLTVLGQPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSVTVVWK
ADGSTITRNVETTRASKQSNSKYAASSYLSLTSSDWKSKGSYSCEVTHEGS
TVTKTVKPSECS
BLV5D3253QAVLNQPSSVSGSLGQRVSITCSGSSSNVGNGYVSWYQLIPGSAPRTLIYG
LCDTSRASGVPDRFSGSRSGNTATLTISSLQAEDEADYFCASAEDSSSNAVFG
SGTTLTVLGQPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSVTVVWK
ADGSTITRNVETTRASKQSNSKYAASSYLSLTSSDWKSKGSYSCEVTHEGS
TVTKTVKPSECS
For select SEQ ID NOs, the Ultralong CDR3 Stalks are underlined and knobs are double underlined

TABLE 17
Ultralong CDR3 containing bovine antibody heavy chain and
light chain nucleotide sequences
SEQ ID
NameNOSequence
BLV1H12 Fab254CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
heavy chainGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
(VH + CH1)AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCTGTCCT
GACGGCTATCGGGAGAGATCTGATTGCAGTAATAGGCCAGCTTGTGG
CACATCCGACTGCTGTCGCGTGTCTGTCTTCGGGAACTGCCTGACTAC
CCTGCCTGTGTCCTACTCTTATACCTACAATTATGAATGGCATGTGGA
TGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAA
CTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCC
TCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGA
GCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGC
ACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTT
CAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGT
AATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGA
ACCCAAATCTTGCGACGGCAGCCATCACCATCATCATCAC
BLV5B8 Fab255CAGGTCCAGCTGAGAGAGAGCGGGCCTTCACTGGTCCAGCCTTCACA
heavy chainGACACTGAGCCTGACTTGTACTGCCTCCGGGTTTTCACTGTCTGACAA
(VH + CH1)GGCTGTGGGATGGGTCCGACAGGCACCAGGGAAAGCTCTGGAGTGG
CTGGGAAGTATCGATACCGGCGGGTCAACAGGGTACAACCCTGGACT
GAAGTCCAGACTGTCTATTACTAAGGACAATTCTAAAAGTCAGGTGT
CACTGAGCGTGAGCTCCGTCACCACAGAGGATTCTGCAACATACTAT
TGCACTACCGTGCACCAGGAAACAAGGAAAACTTGTAGTGACGGCTA
TATCGCAGTGGATAGCTGCGGACGAGGACAGTCCGACGGATGCGTG
AACGATTGCAATAGCTGTTACTATGGATGGCGAAACTGCCGGAGACA
GCCAGCAATTCATTCATACGAGTTTCATGTGGATGCTTGGGGGCGGG
GGCTGCTGGTCACCGTCTCCTCAGCTTCCACAACTGCACCAAAGGTG
TACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGAC
ACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCA
CCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCT
GTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTC
CCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCC
TGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTGCG
ACGGCAGCCATCACCATCATCATCAC
BLV5D3 Fab256CAGGTCCAGCTGAGGGAATCCGGCCCATCACTGGTCAAGCCTTCACA
heavy chainGACACTGAGCCTGACATGTACTGCAAGCGGGTTTTCACTGAGTGACA
(VH + CH1)AGGCAGTGGGATGGGTCCGGAGAGCACCAGGAAAAGCCCTGGAGTG
GCTGGGAACCACAGATACTGGAGGATCCGCCGCTTACAACCCTGGCC
TGAAGTCCCGGCTGTCTATCACCAAGGACAACTCTAAAAGTCAGGTG
TCACTGAGCGTGTCCAATGTCGCTACAGAAGATTCTGCAACTTACTAT
TGTAGCTCCGTGACTCAGAGGACCCACGTCTCTCGCAGTTGTCCAGA
CGGGTGCAGTGACGGAGATGGCTGCGTGGATGGATGCTGTTGCTCAG
CTTACCGATGTTATACACCCGGGGTCAGAGACCTGAGCTGCACCTCA
TATAGCATTACATACACTTACGAATGGAATGTGGATGCTTGGGGACA
GGGACTGCTGGTGACCGTCTCTTCAGCTTCCACAACTGCACCAAAGG
TGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTG
ACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGT
CACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAG
CTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAG
TCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCAT
CCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTG
CGACGGCAGCCATCACCATCATCATCAC
BLV1H12 Fab257CAGGCCGTCCTGAACCAGCCAAGCAGCGTCTCCGGGTCTCTGGGGCA
light chainGCGGGTCTCAATCACCTGTAGCGGGTCTTCCTCCAATGTCGGCAACG
(VL + CL)GCTACGTGTCTTGGTATCAGCTGATCCCTGGCAGTGCCCCACGAACC
CTGATCTACGGCGACACATCCAGAGCTTCTGGGGTCCCCGATCGGTT
CTCAGGGAGCAGATCCGGAAACACAGCTACTCTGACCATCAGCTCCC
TGCAGGCTGAGGACGAAGCAGATTATTTCTGCGCATCTGCCGAGGAC
TCTAGTTCAAATGCCGTGTTTGGAAGCGGCACCACACTGACAGTCCT
GGGGCAGCCCAAGAGTCCCCCTTCAGTGACTCTGTTCCCACCCTCTAC
CGAGGAACTGAACGGAAACAAGGCCACACTGGTGTGTCTGATCAGC
GACTTTTACCCTGGATCCGTCACTGTGGTCTGGAAGGCAGATGGCAG
CACAATTACTAGGAACGTGGAAACTACCCGCGCCTCCAAGCAGTCTA
ATAGTAAATACGCCGCCAGCTCCTATCTGAGCCTGACCTCTAGTGATT
GGAAGTCCAAAGGGTCATATAGCTGCGAAGTGACCCATGAAGGCTC
AACCGTGACTAAGACTGTGAAACCATCCGAGTGCTCC
BLV5B8 Fab258CAGGCCGTCCTGAACCAGCCAAGCAGCGTCTCCGGGTCTCTGGGGCA
light chainGCGGGTCTCAATCACCTGTAGCGGGTCTTCCTCCAATGTCGGCAACG
(VL + CL)GCTACGTGTCTTGGTATCAGCTGATCCCTGGCAGTGCCCCACGAACC
CTGATCTACGGCGACACATCCAGAGCTTCTGGGGTCCCCGATCGGTT
CTCAGGGAGCAGATCCGGAAACACAGCTACTCTGACCATCAGCTCCC
TGCAGGCTGAGGACGAAGCAGATTATTTCTGCGCATCTGCCGAGGAC
TCTAGTTCAAATGCCGTGTTTGGAAGCGGCACCACACTGACAGTCCT
GGGGCAGCCCAAGAGTCCCCCTTCAGTGACTCTGTTCCCACCCTCTAC
CGAGGAACTGAACGGAAACAAGGCCACACTGGTGTGTCTGATCAGC
GACTTTTACCCTGGATCCGTCACTGTGGTCTGGAAGGCAGATGGCAG
CACAATTACTAGGAACGTGGAAACTACCCGCGCCTCCAAGCAGTCTA
ATAGTAAATACGCCGCCAGCTCCTATCTGAGCCTGACCTCTAGTGATT
GGAAGTCCAAAGGGTCATATAGCTGCGAAGTGACCCATGAAGGCTC
AACCGTGACTAAGACTGTGAAACCATCCGAGTGCTCC
BLV5D3 Fab259CAGGCCGTCCTGAACCAGCCAAGCAGCGTCTCCGGGTCTCTGGGGCA
light chainGCGGGTCTCAATCACCTGTAGCGGGTCTTCCTCCAATGTCGGCAACG
(VL + CL)GCTACGTGTCTTGGTATCAGCTGATCCCTGGCAGTGCCCCACGAACC
CTGATCTACGGCGACACATCCAGAGCTTCTGGGGTCCCCGATCGGTT
CTCAGGGAGCAGATCCGGAAACACAGCTACTCTGACCATCAGCTCCC
TGCAGGCTGAGGACGAAGCAGATTATTTCTGCGCATCTGCCGAGGAC
TCTAGTTCAAATGCCGTGTTTGGAAGCGGCACCACACTGACAGTCCT
GGGGCAGCCCAAGAGTCCCCCTTCAGTGACTCTGTTCCCACCCTCTAC
CGAGGAACTGAACGGAAACAAGGCCACACTGGTGTGTCTGATCAGC
GACTTTTACCCTGGATCCGTCACTGTGGTCTGGAAGGCAGATGGCAG
CACAATTACTAGGAACGTGGAAACTACCCGCGCCTCCAAGCAGTCTA
ATAGTAAATACGCCGCCAGCTCCTATCTGAGCCTGACCTCTAGTGATT
GGAAGTCCAAAGGGTCATATAGCTGCGAAGTGACCCATGAAGGCTC
AACCGTGACTAAGACTGTGAAACCATCCGAGTGCTCC

TABLE 18
Bovine Immunoglobulin Fusion Protein Amino Acid Sequences
NameSEQ ID NOSequence
BLV1H1260QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
hLeptinSVHQETKKYQSGGGGSVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSK
HCQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLEN
LRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQ
GSLQDMLWQLDLSPGCGGGGSSYTYNYEWHVDVWGQGLLVTVSSA
STTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSG
VHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
BLV1H1261QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
Moka1SVHQETKKYQSINVKCSLPQQCIKPCKDAGMRFGKCMNKKCRCYSSY
L0 HCTYNYEWHVDVWGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLG
CLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGS
TSGQTFTCNVAHPASSTKVDKAVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK
BLV1H1262QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
Moka1SVHQETKKYQSGGGGSINVKCSLPQQCIKPCKDAGMRFGKCMNKKCR
L1 HCCYSGGGGSSYTYNYEWHVDVWGQGLLVTVSSASTTAPKVYPLSSCC
GDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLY
SLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK
BLV1H1263QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
VM24 L1SVHQETKKYQSGGGGSAAAISCVGSPECPPKCRAQGCKNGKCMNRKC
HCKCYYCGGGGSSYTYNYEWHVDVWGQGLLVTVSSASTTAPKVYPLSS
CCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSG
LYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK
BLV1H1264QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
VM24 L2SVHQETKKYQSGGGSGGGGSAAAISCVGSPECPPKCRAQGCKNGKCM
HCNRKCKCYYCGGGGSGGGSSYTYNYEWHVDVWGQGLLVTVSSASTT
APKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHT
FPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPK
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
BLV1H1265QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
GLP-1SVHQETKKYQSCGGGGSIEGRHAEGTFTSDVSSYLEGQAAKEFIAWLV
HCKGRGGGGSCSYTYNYEWHVDVWGQGLLVTVSSASTTAPKVYPLSSC
CGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGL
YSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKSCDKTHTCPP
CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK
BLV1H1266QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
Exendin-SVHQETKKYQSCGGGGSIEGRHGEGTFTSDLSKQMEEEAVRLFIEWLK
4 HCNGGPSSGAPPPSGGGGSCSYTYNYEWHVDVWGQGLLVTVSSASTTAP
KVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFP
AVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
BLV1H1267QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
EPO HCSVHQETKKYQSGGGGSAPPRLICDSRVLERYLLEAKEAENITTGCAEH
CSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQ
ALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAAS
AAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRGGGGSSYT
YNYEWHVDVWGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGC
LVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGST
SGQTFTCNVAHPASSTKVDKAVEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGK
BLV1H1268QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-LGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
betaoxyntomodulinSVHQETKKYQSGGGGSHSQGTFTSDYSKYLDSRRAQDFVQWLMNTK
HCRNRNNIAGGGGSSYTYNYEWHVDVWGQGLLVTVSSASTTAPKVYPL
SSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQS
SGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
BLV1H1269QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
Mamba1SVHQETKKYQSGGGGSLKCYQHGKVVTCHRDMKFCYHNTGMPFRNL
HCKLILQGCSSSCSETENNKCCSTDRCNKGGGGSSYTYNYEWHVDVWGQ
GLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVT
WNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHP
ASSTKVDKAVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
LSPGK
BLV1H1270QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
GMCSFSVHQETKKYQSGGGGSAPARSPSPSTQPWEHVNAIQEARRLLNLSRDT
HCAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTM
MASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQEG
GGGSSYTYNYEWHVDVWGQGLLVTVSSASTTAPKVYPLSSCCGDKSS
STVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSM
VTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK
BLV1H1271QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
IL11 HCSVHQETKKYQSGGGGSPGPPPGPPRVSPDPRAELDSTVLLTRSLLADTR
QLAAQLRDKFPADGDHNLDSLPTLAMSAGALGALQLPGVLTRLRADL
LSYLRHVQWLRRAGGSSLKTLEPELGTLQARLDRLLRRLQLLMSRLA
LPQPPPDPPAPPLAPPSSAWGGIRAAHAILGGLHLTLDWAVRGLLLLKT
RLGGGGSSYTYNYEWHVDVWGQGLLVTVSSASTTAPKVYPLSSCCG
DKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYS
LSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK
BLV1H1272QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
IFN betaSVHQETKKYQSGGGGSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEY
HCCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGW
NETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYG
RILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNGGGGSSYTY
NYEWHVDVWGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCL
VSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTS
GQTFTCNVAHPASSTKVDKAVEPKSCDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGK
BLV1H1273QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
parathyroidSVHQETKKYQSGGGGSSVSEIQLMHNLGKHLNSMERVEWLRKKLQD
hormoneVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVN
HCVLTKAKSQGGGGSSYTYNYEWHVDVWGQGLLVTVSSASTTAPKVYP
LSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQ
SSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKSCDKTH
TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
BLV1H1274QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
Relaxin2SVHQETKKYQSGGGGSDSWMEEVIKLCGRELVRAQIAICGMSTWSKR
HCSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSE
MQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGL
DTHSRKKRQLYSALANKCCHVGCTKRSLARFCGGGGSSYTYNYEWH
VDVWGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMP
EPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTC
NVAHPASSTKVDKAVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK
BLV1H1275QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
Relaxin2SVHQETKKYQSGGGGSDSWMEEVIKLCGRELVRAQIAICGMSTWSGG
(GGSIEGR)SIEGRQLYSALANKCCHVGCTKRSLARFCGGGGSSYTYNYEWHVDV
HCWGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPV
TVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVA
HPASSTKVDKAVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGK
BLV1H1276QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
Relaxin2SVHQETKKYQSGGGGSDSWMEEVIKLCGRELVRAQIAICGMSTWSIE
(IEGRCpepGRSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLS
IEGR)EMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLG
HCLDTHSIEGRQLYSALANKCCHVGCTKRSLARFCGGGGSSYTYNYEWH
VDVWGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMP
EPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTC
NVAHPASSTKVDKAVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK
BLV1H1277QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEW
2-betaLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCT
hGHSVHQETKKYQSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFE
heavyEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLI
chainQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLED
GSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFL
RIVQCRSVEGSCGFGGGGSSYTYNYEWHVDVWGQGLLVTVSSASTTA
PKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTF
PAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
BLV1H12278QAVLNQPSSVSGSLGQRVSITCSGSSSNVGNGYVSWYQLIPGSAPRTLI
immunoglobulinYGDTSRASGVPDRFSGSRSGNTATLTISSLQAEDEADYFCASAEDSSSN
fusionAVFGSGTTLTVLGQPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSV
proteinTVVWKADGSTITRNVETTRASKQSNSKYAASSYLSLTSSDWKSKGSYS
LCCEVTHEGSTVTKTVKPSECS

TABLE 19
Bovine Immunoglobulin Fusion Protein Nucleotide Sequences
NameSEQ ID NOSequence
BLV1H12-beta279CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
hLeptin L1 HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGTGGC
GGAGGATCTGTTCCAATTCAAAAGGTTCAAGATGATACCAAAACTCT
GATTAAAACTATTGTCACGCGTATAAACGACATCAGCCATACCCAGT
CGGTTAGCTCAAAGCAAAAAGTTACCGGTTTGGACTTTATTCCGGGA
CTGCACCCGATCCTGACCCTTAGTAAAATGGACCAGACACTGGCCGT
CTACCAGCAAATCCTGACATCGATGCCATCCAGAAATGTGATACAAA
TTAGCAACGATTTGGAAAACCTTCGCGATCTGCTGCACGTGCTGGCC
TTCAGTAAGTCCTGTCATCTGCCGTGGGCGTCGGGACTGGAGACTCTT
GACTCGCTGGGTGGAGTGTTAGAGGCCTCTGGCTATTCTACTGAAGT
CGTTGCGCTGTCACGCCTCCAGGGGAGCCTGCAGGACATGCTGTGGC
AGCTGGACCTGTCACCTGGCTGCGGAGGTGGTGGTTCATCTTATACCT
ACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTG
ACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTC
AAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCC
TGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCA
GGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTC
CTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTA
CTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCA
CCAAAGTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAAACTCA
CACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAG
TCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT
GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC
CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA
GTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGA
AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCC
TGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG
TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCG
TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG
ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT
GTCTCCGGGTAAA
BLV1H12-beta280CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
Moka1 L0 HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCATCAAC
GTGAAGTGCAGCCTGCCCCAGCAGTGCATCAAGCCCTGCAAGGACGC
CGGCATGCGGTTCGGCAAGTGCATGAACAAGAAGTGCAGGTGCTAC
AGCTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGGACA
GGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGG
TGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTG
ACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGT
CACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAG
CTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAG
TCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCAT
CCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTG
CGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG
GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC
ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG
CCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGG
AGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAG
CACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC
TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG
AACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAG
AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA
CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC
AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCT
TCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG
AAGAGCCTCTCCCTGTCTCCGGGTAAA
BLV1H12-beta281CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
Moka1 L1 HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGTGGC
GGAGGATCTATCAACGTGAAGTGCAGCCTGCCCCAGCAGTGCATCAA
GCCCTGCAAGGACGCCGGCATGCGGTTCGGCAAGTGCATGAACAAG
AAGTGCAGGTGCTACAGCGGAGGTGGTGGTTCATCTTATACCTACAA
TTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAG
TCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCT
GCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTC
TCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGC
CCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTG
GCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCA
GGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAA
AGTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAAACTCACACA
TGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT
CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA
GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTC
AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTA
CAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA
CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC
CCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG
GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCG
TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG
CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC
TCCGGGTAAA
BLV1H12-beta282CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
VM24 L1 HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCGCCGCTGCAATCTCCTGCGTCGGCAGCCCCGAATGTCC
TCCCAAGTGCCGGGCTCAGGGATGCAAGAACGGCAAGTGTATGAAC
CGGAAGTGCAAGTGCTACTATTGCGGCGGAGGTGGGAGTTCTTATAC
CTACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGG
TGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGT
CAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGC
CTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTC
AGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGT
CCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGT
ACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCC
ACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAAACTC
ACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA
GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC
TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGT
GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGG
AGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG
AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGC
CTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC
TGTCTCCGGGTAAA
BLV1H12-beta283CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
VM24 L2 HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGCGGT
GGATCTGGGGGTGGCGGAAGCGCCGCTGCAATCTCCTGCGTCGGCAG
CCCCGAATGTCCTCCCAAGTGCCGGGCTCAGGGATGCAAGAACGGCA
AGTGTATGAACCGGAAGTGCAAGTGCTACTATTGCGGCGGAGGTGGG
AGTGGAGGCGGTAGCTCTTATACCTACAATTATGAATGGCATGTGGA
TGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAA
CTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCC
TCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGA
GCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGC
ACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTT
CAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGT
AATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGA
ACCCAAATCTTGCGACAAAACTCACACATGCCCACCGTGCCCAGCAC
CTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG
GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG
CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG
GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG
AGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC
TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA
GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
BLV1H12-beta284CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
GLP-1 HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCTGCGGG
GGTGGCGGAAGCATCGAAGGTCGTCACGCTGAGGGAACATTCACTTC
CGATGTGTCCTCCTACCTGGAGGGCCAGGCTGCCAAAGAGTTCATCG
CTTGGCTCGTCAAGGGCAGGGGCGGAGGTGGGAGTTGCTCTTATACC
TACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGT
GACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGT
CAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGC
CTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTC
AGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGT
CCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGT
ACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCC
ACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAAACTC
ACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA
GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC
TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGT
GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGG
AGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG
AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGC
CTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC
TGTCTCCGGGTAAA
BLV1H12-beta285CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
Exendin-4 HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCTGCGGG
GGTGGCGGAAGCATCGAAGGTCGTCACGCTGAGGGAACATTCACTTC
CGATGTGTCCTCCTACCTGGAGGGCCAGGCTGCCAAAGAGTTCATCG
CTTGGCTCGTCAAGGGCAGGGGCGGAGGTGGGAGTTGCTCTTATACC
TACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGT
GACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGT
CAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGC
CTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTC
AGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGT
CCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGT
ACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCC
ACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAAACTC
ACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA
GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC
TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGT
GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGG
AGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG
AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGC
CTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC
TGTCTCCGGGTAAA
BLV1H12-beta286CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
EPO HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCGCCCCACCACGCCTCATCTGTGACAGCCGAGTCCTGGA
GAGGTACCTCTTGGAGGCCAAGGAGGCCGAGAATATCACGACGGGC
TGTGCTGAACACTGCAGCTTGAATGAGAATATCACTGTCCCAGACAC
CAAAGTTAATTTCTATGCCTGGAAGAGGATGGAGGTCGGGCAGCAGG
CCGTAGAAGTCTGGCAGGGCCTGGCCCTGCTGTCGGAAGCTGTCCTG
CGGGGCCAGGCCCTGTTGGTCAACTCTTCCCAGCCGTGGGAGCCCCT
GCAGCTGCATGTGGATAAAGCCGTCAGTGGCCTTCGCAGCCTCACCA
CTCTGCTTCGGGCTCTGGGAGCCCAGAAGGAAGCCATCTCCCCTCCA
GATGCGGCCTCAGCTGCTCCACTCCGAACAATCACTGCTGACACTTTC
CGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGGGAAAGCTGAA
GCTGTACACAGGGGAGGCCTGCAGGACAGGGGACAGAGGCGGAGGT
GGGAGTTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGG
ACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAA
AGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACC
GTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGAC
TGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCC
CAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGA
CAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCC
CATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAAATC
TTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC
TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC
CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGT
GAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGC
GTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACA
ACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCT
CCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC
GAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACC
AAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAG
CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC
TACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG
TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC
AGAAGAGCCTCTCCCTGTCTCCGGGTAAA
BLV1H12-287CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
betaoxyntomodulinGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
HCAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCCACTCTCAGGGTACCTTCACCTCTGACTACTCTAAATA
CCTGGACTCTCGTCGTGCTCAGGACTTCGTTCAGTGGCTGATGAACAC
CAAACGTAACCGTAACAACATCGCTGGCGGAGGTGGGAGTTCTTATA
CCTACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTG
GTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCT
GTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGAT
GCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAAC
TCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCA
GTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCA
GTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCT
CCACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAAAC
TCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT
CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGA
CCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATA
ATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCG
TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA
AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGG
TGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTC
AGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT
GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC
ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC
CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT
CCCTGTCTCCGGGTAAA
BLV1H12-beta288CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
Mamba1 HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCCTGAAATGTTACCAACATGGTAAAGTTGTGACTTGTCA
TCGAGATATGAAGTTTTGCTATCATAACACTGGCATGCCTTTTCGAAA
TCTCAAGCTCATCCTACAGGGATGTTCTTCTTCGTGCAGTGAAACAGA
AAACAATAAGTGTTGCTCAACAGACAGATGCAACAAAGGCGGAGGT
GGGAGTTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGG
ACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAA
AGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACC
GTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGAC
TGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCC
CAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGA
CAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCC
CATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAAATC
TTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC
TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC
CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGT
GAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGC
GTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACA
ACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCT
CCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC
GAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACC
AAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAG
CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC
TACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG
TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC
AGAAGAGCCTCTCCCTGTCTCCGGGTAAA
BLV1H12-beta289CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
GMCSF HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTG
GGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGA
GTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCA
GAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGA
GCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGC
CCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAAC
CCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAA
AGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGA
GCCAGTCCAGGAGGGCGGAGGTGGGAGTTCTTATACCTACAATTATG
AATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCT
AGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTG
TGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAA
GCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTG
AAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCT
GTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGC
AGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGTG
GACAAAGCAGTGGAACCCAAATCTTGCGACAAAACTCACACATGCCC
ACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTT
CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG
TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG
TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC
CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG
CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCT
CCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC
CCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCC
TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGC
AATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG
ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAG
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGA
GGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAA
BLV1H12-beta290CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
IL11 HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCCCTGGGCCACCACCTGGCCCCCCTCGAGTTTCCCCAGA
CCCTCGGGCCGAGCTGGACAGCACCGTGCTCCTGACCCGCTCTCTCCT
GGCGGACACGCGGCAGCTGGCTGCACAGCTGAGGGACAAATTCCCA
GCTGACGGGGACCACAACCTGGATTCCCTGCCCACCCTGGCCATGAG
TGCGGGGGCACTGGGAGCTCTACAGCTCCCAGGTGTGCTGACAAGGC
TGCGAGCGGACCTACTGTCCTACCTGCGGCACGTGCAGTGGCTGCGC
CGGGCAGGTGGCTCTTCCCTGAAGACCCTGGAGCCCGAGCTGGGCAC
CCTGCAGGCCCGACTGGACCGGCTGCTGCGCCGGCTGCAGCTCCTGA
TGTCCCGCCTGGCCCTGCCCCAGCCACCCCCGGACCCGCCGGCGCCC
CCGCTGGCGCCCCCCTCCTCAGCCTGGGGGGGCATCAGGGCCGCCCA
CGCCATCCTGGGGGGGCTGCACCTGACACTTGACTGGGCCGTGAGGG
GACTGCTGCTGCTGAAGACTCGGCTGGGCGGAGGTGGGAGTTCTTAT
ACCTACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCT
GGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCC
TGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGA
TGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAA
CTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGC
AGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGC
AGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAG
CTCCACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAA
ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACC
GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTC
CCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAG
ACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACC
GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC
AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCAT
CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT
CAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC
GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT
CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC
TCCCTGTCTCCGGGTAAA
BLV1H12-beta291CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
IFN beta HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAG
CAATTTTCAGTGTCAGAAGCTCCTGTGGCAATTGAATGGGAGGCTTG
AATACTGCCTCAAGGACAGGATGAACTTTGACATCCCTGAGGAGATT
AAGCAGCTGCAGCAGTTCCAGAAGGAGGACGCCGCATTGACCATCTA
TGAGATGCTCCAGAACATCTTTGCTATTTTCAGACAAGATTCATCTAG
CACTGGCTGGAATGAGACTATTGTTGAGAACCTCCTGGCTAATGTCT
ATCATCAGATAAACCATCTGAAGACAGTCCTGGAAGAAAAACTGGA
GAAAGAAGATTTCACCAGGGGAAAACTCATGAGCAGTCTGCACCTG
AAAAGATATTATGGGAGGATTCTGCATTACCTGAAGGCCAAGGAGTA
CAGTCACTGTGCCTGGACCATAGTCAGAGTGGAAATCCTAAGGAACT
TTTACTTCATTAACAGACTTACAGGTTACCTCCGAAACGGCGGAGGT
GGGAGTTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGG
ACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAA
AGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACC
GTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGAC
TGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCC
CAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGA
CAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCC
CATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAAATC
TTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC
TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC
CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGT
GAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGC
GTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACA
ACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCT
CCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC
GAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACC
AAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAG
CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC
TACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG
TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC
AGAAGAGCCTCTCCCTGTCTCCGGGTAAA
BLV1H12-292CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
betaparathyroidGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
hormone HCAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCTCTGTGAGTGAAATACAGCTTATGCATAACCTGGGAAA
ACATCTGAACTCGATGGAGAGAGTAGAATGGCTGCGTAAGAAGCTG
CAGGATGTGCACAATTTTGTTGCCCTTGGAGCTCCTCTAGCTCCCAGA
GATGCTGGTTCCCAGAGGCCCCGAAAAAAGGAAGACAATGTCTTGGT
TGAGAGCCATGAAAAAAGTCTTGGAGAGGCAGACAAAGCTGATGTG
AATGTATTAACTAAAGCTAAATCCCAGGGCGGAGGTGGGAGTTCTTA
TACCTACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGC
TGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCC
CTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGG
ATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGA
ACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTG
CAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGG
CAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCA
GCTCCACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTGCGACAA
AACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGAC
CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT
CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA
GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA
TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG
CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA
CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGC
TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC
TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCT
CTCCCTGTCTCCGGGTAAA
BLV1H12-beta293CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
Relaxin2 HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCGACTCTTGGATGGAAGAAGTTATCAAACTGTGCGGTCG
TGAACTGGTTCGTGCTCAGATCGCTATCTGCGGTATGTCTACCTGGTC
TAAACGTTCTCTGTCTCAGGAAGACGCTCCGCAGACCCCGCGTCCGG
TTGCTGAAATCGTTCCGTCTTTCATCAACAAAGACACCGAAACCATC
AACATGATGTCTGAATTCGTTGCTAACCTGCCGCAGGAACTGAAACT
GACCCTGTCTGAAATGCAGCCGGCTCTGCCGCAGCTGCAGCAGCACG
TTCCGGTTCTGAAAGACTCTTCTCTGCTGTTCGAAGAATTCAAAAAAC
TGATCCGTAACCGTCAGTCTGAAGCTGCTGACTCTTCTCCGTCTGAAC
TGAAATACCTGGGTCTGGACACCCACTCTCGTAAAAAACGTCAGCTG
TACTCTGCTCTGGCTAACAAATGCTGCCACGTTGGTTGCACCAAACGT
TCTCTGGCTCGTTTCTGCGGCGGAGGTGGGAGTTCTTATACCTACAAT
TATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGT
CTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCT
GCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTC
TCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGC
CCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTG
GCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCA
GGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAA
AGTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAAACTCACACA
TGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT
CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA
GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTC
AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTA
CAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA
CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC
CCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG
GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCG
TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG
CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC
TCCGGGTAAATGATAA
BLV1H12-beta294CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
Relaxin2GACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
(GGSIEGR)AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
HCGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCGACTCTTGGATGGAAGAAGTTATCAAACTGTGCGGTCG
TGAACTGGTTCGTGCTCAGATCGCTATCTGCGGTATGTCTACCTGGTC
TGGCGGAAGCATCGAGGGCCGCCAGCTGTACTCTGCTCTGGCTAACA
AATGCTGCCACGTTGGTTGCACCAAACGTTCTCTGGCTCGTTTCTGCG
GCGGAGGTGGGAGTTCTTATACCTACAATTATGAATGGCATGTGGAT
GTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAAC
TGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCT
CTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAG
CCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCA
CACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTC
AATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTA
ATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAA
CCCAAATCTTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACC
TGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG
GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG
CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG
GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG
AGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC
TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA
GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGATAA
BLV1H12-beta295CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
Relaxin2GACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
(IEGRCpepIEGR)AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
HCGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCGACTCTTGGATGGAAGAAGTTATCAAACTGTGCGGTCG
TGAACTGGTTCGTGCTCAGATCGCTATCTGCGGTATGTCTACCTGGTC
TATCGAGGGCCGCTCTCTGTCTCAGGAAGACGCTCCGCAGACCCCGC
GTCCGGTTGCTGAAATCGTTCCGTCTTTCATCAACAAAGACACCGAA
ACCATCAACATGATGTCTGAATTCGTTGCTAACCTGCCGCAGGAACT
GAAACTGACCCTGTCTGAAATGCAGCCGGCTCTGCCGCAGCTGCAGC
AGCACGTTCCGGTTCTGAAAGACTCTTCTCTGCTGTTCGAAGAATTCA
AAAAACTGATCCGTAACCGTCAGTCTGAAGCTGCTGACTCTTCTCCGT
CTGAACTGAAATACCTGGGTCTGGACACCCACTCTATCGAGGGCCGC
CAGCTGTACTCTGCTCTGGCTAACAAATGCTGCCACGTTGGTTGCACC
AAACGTTCTCTGGCTCGTTTCTGCGGCGGAGGTGGGAGTTCTTATACC
TACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGT
GACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGT
CAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGC
CTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTC
AGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGT
CCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGT
ACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCC
ACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAAACTC
ACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA
GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC
TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGT
GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGG
AGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG
AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGC
CTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC
TGTCTCCGGGTAAATGATAA
BLV1H12-beta296CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
hGH HCGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCTTCCCAACCATTCCCTTATCCAGGCTTTTTGACAACGCT
ATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAG
GAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCT
GCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACC
CTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTC
CGCATCTCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTC
CTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTGACAG
CAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGC
TGATGGGGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTC
AAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACG
CACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATG
GACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGA
GGGCAGCTGTGGCTTCGGCGGAGGTGGGAGTTCTTATACCTACAATT
ATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTC
TCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTG
CTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCT
CAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCC
CTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGG
CCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAG
GGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAA
GTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTC
CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT
GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG
ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA
GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC
CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC
CTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGAC
CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT
GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGA
CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC
ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT
CCGGGTAAATGATAA
BLV1H12297CAGGCCGTCCTGAACCAGCCAAGCAGCGTCTCCGGGTCTCTGGGGCA
immunoglobulinGCGGGTCTCAATCACCTGTAGCGGGTCTTCCTCCAATGTCGGCAACG
fusion proteinGCTACGTGTCTTGGTATCAGCTGATCCCTGGCAGTGCCCCACGAACC
LCCTGATCTACGGCGACACATCCAGAGCTTCTGGGGTCCCCGATCGGTT
CTCAGGGAGCAGATCCGGAAACACAGCTACTCTGACCATCAGCTCCC
TGCAGGCTGAGGACGAAGCAGATTATTTCTGCGCATCTGCCGAGGAC
TCTAGTTCAAATGCCGTGTTTGGAAGCGGCACCACACTGACAGTCCT
GGGGCAGCCCAAGAGTCCCCCTTCAGTGACTCTGTTCCCACCCTCTAC
CGAGGAACTGAACGGAAACAAGGCCACACTGGTGTGTCTGATCAGC
GACTTTTACCCTGGATCCGTCACTGTGGTCTGGAAGGCAGATGGCAG
CACAATTACTAGGAACGTGGAAACTACCCGCGCCTCCAAGCAGTCTA
ATAGTAAATACGCCGCCAGCTCCTATCTGAGCCTGACCTCTAGTGATT
GGAAGTCCAAAGGGTCATATAGCTGCGAAGTGACCCATGAAGGCTC
AACCGTGACTAAGACTGTGAAACCATCCGAGTGCTCC

TABLE 20
Immunoglobulin Fusion Protein Nucleotide and Amino Acid Sequences
NameSEQ ID NOSequence
Trastuzumab-298EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV
beta hGHARIETKKYQSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAY
(CDR2H) HCIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLE
PVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQ
IFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVE
GSCGFGGGGSSYTYNYETRYADSVKGRFTISADTSKNTAYLQMNSLRAE
DTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKS
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPP
VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS
IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK
BLV1H12-beta299CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
Fab hGHGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
(CDR3H)AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCTTCCCAACCATTCCCTTATCCAGGCTTTTTGACAACGCT
ATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAG
GAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCT
GCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACC
CTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTC
CGCATCTCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTC
CTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTGACAG
CAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGC
TGATGGGGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTC
AAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACG
CACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATG
GACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGA
GGGCAGCTGTGGCTTCGGCGGAGGTGGGAGTTCTTATACCTACAATT
ATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTC
TCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTG
CTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCT
CAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCC
CTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGG
CCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAG
GGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAA
GTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAAACTCACACAC
ATCACCATCATCATCACTAGTGA
BLV1H12-beta300QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEWL
Fab hGHGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCTSV
(CDR3H)HQETKKYQSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAY
IPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLE
PVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQ
IFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVE
GSCGFGGGGSSYTYNYEWHVDVWGQGLLVTVSSASTTAPKVYPLSSCC
GDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYS
LSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKSCDKTHTHHHHHH
BLV1H12-beta301CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCA
hFc (IgG) hGHGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACA
(CDR3H)AGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATG
GCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGA
CTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGT
GTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACT
ATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGGGGGT
GGCGGAAGCTTCCCAACCATTCCCTTATCCAGGCTTTTTGACAACGCT
ATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAG
GAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCT
GCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACC
CTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTC
CGCATCTCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTC
CTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTGACAG
CAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGC
TGATGGGGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTC
AAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACG
CACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATG
GACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGA
GGGCAGCTGTGGCTTCGGCGGAGGTGGGAGTTCTTATACCTACAATT
ATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTC
TCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTG
CTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCT
CAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCC
CTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGG
CCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAG
GGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAA
GTGGACAAAGCAGTGGAACCCAAATCTTGCGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTC
CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT
GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG
ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA
GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC
CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC
CTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGAC
CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT
GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGA
CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC
ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT
CCGGGTAAATGATAA
BLV1H12-beta302QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEWL
hFc (IgG) hGHGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCTSV
(CDR3H)HQETKKYQSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAY
IPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLE
PVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQ
IFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVE
GSCGFGGGGSSYTYNYEWHVDVWGQGLLVTVSSASTTAPKVYPLSSCC
GDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYS
LSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK
Trastuzumab-303GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCAGCCTGGGG
beta CDRH3GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCAATATTAAGGAC
EPOACTTACATCCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG
GGTCGCACGTATTTATCCTACCAATGGTTACACACGCTACGCAGACT
CCGTGAAGGGCCGATTCACCATCTCCGCAGACACTTCCAAGAACACG
GCGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTA
TTACTGTTCGAGAGAAACTAAGAAATACCAGAGCGGGGGTGGCGGA
AGCGCCCCACCACGCCTCATCTGTGACAGCCGAGTCCTGGAGAGGTA
CCTCTTGGAGGCCAAGGAGGCCGAGAATATCACGACGGGCTGTGCTG
AACACTGCAGCTTGAATGAGAATATCACTGTCCCAGACACCAAAGTT
AATTTCTATGCCTGGAAGAGGATGGAGGTCGGGCAGCAGGCCGTAG
AAGTCTGGCAGGGCCTGGCCCTGCTGTCGGAAGCTGTCCTGCGGGGC
CAGGCCCTGTTGGTCAACTCTTCCCAGCCGTGGGAGCCCCTGCAGCT
GCATGTGGATAAAGCCGTCAGTGGCCTTCGCAGCCTCACCACTCTGC
TTCGGGCTCTGGGAGCCCAGAAGGAAGCCATCTCCCCTCCAGATGCG
GCCTCAGCTGCTCCACTCCGAACAATCACTGCTGACACTTTCCGCAA
ACTCTTCCGAGTCTACTCCAATTTCCTCCGGGGAAAGCTGAAGCTGTA
CACAGGGGAGGCCTGCAGGACAGGGGACAGAGGCGGAGGTGGGAGT
TCTTATACCTACAATTATGAAGACTACTGGGGCCAAGGAACCCTGGT
CACCGTCTCCTCAGCCAGCACTAAAGGTCCATCTGTGTTCCCTCTGGC
TCCTTGCAGCCGGAGCACCTCCGAGTCCACAGCCGCTCTGGGATGTC
TGGTGAAAGATTACTTCCCCGAGCCCGTCACCGTGAGCTGGAATAGC
GGAGCACTGACCTCCGGCGTCCACACATTCCCCGCCGTGCTCCAAAG
CTCCGGCCTGTACAGCCTCTCCTCCGTGGTCACCGTGCCCAGCAGCTC
TCTGGGCACAAAGACCTATACCTGTAACGTGGATCACAAGCCTAGCA
ACACCAAAGTGGATAAGCGGGTGGAGAGCAAGTACGGCCCTCCCTG
TCCCCCTTGCCCCGCTCCTGAGGCCGCTGGCGGACCTTCCGTGTTCCT
GTTTCCCCCTAAGCCCAAGGACACCCTCATGATTAGCCGGACACCCG
AAGTGACCTGCGTGGTCGTGGATGTGTCCCAGGAGGACCCTGAAGTG
CAATTTAACTGGTACGTGGACGGCGTCGAGGTGCACAACGCCAAGAC
CAAGCCTCGGGAAGAGCAGTTCAACAGCACCTACCGGGTGGTCAGC
GTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACA
AGTGCAAGGTGAGCAACAAGGGCCTGCCCAGCTCCATCGAGAAGAC
CATCAGCAAGGCCAAGGGCCAGCCCAGGGAACCCCAGGTGTATACC
CTGCCCCCTAGCCAGGAGGAAATGACCAAAAACCAGGTGAGCCTGA
CCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG
GAGAGCAACGGCCAGCCCGAGAACAATTACAAGACCACCCCTCCTGT
GCTGGACAGCGACGGCTCCTTCTTTCTGTATAGCCGGCTGACCGTGG
ACAAGAGCAGGTGGCAGGAGGGCAACGTGTTCTCCTGTAGCGTGATG
CACGAGGCCCTGCACAACCATTACACCCAGAAGAGCTTGAGCCTGAG
CCTGGGCAAA
Trastuzumab-304EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV
beta CDRH3ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCS
EPORETKKYQSGGGGSAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE
NITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNS
SQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTIT
ADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRGGGGSSYTYNYEDYWG
QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN
TKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK