[0001] This application is a continuation in part of co-pending, commonly assigned patent applications PCT/US00/41422 filed 20 Oct. 2000 and U.S. Ser. No. 09/501,097, filed 09 Feb. 2000, both of which were continuations-in-part of U.S. Ser. No. 09/421,608, filed 20 Oct. 1999 (now abandoned). This application also claims priority to provisional application U.S. Ser. No. 60/281,003, filed 04 Apr. 2001. All of the above applications are incorporated by reference in their entirety.
[0003] 1. Field of the Invention
[0004] The present invention in the fields of molecular biology, immunology and medicine relates to a chimeric nucleic acid, preferably DNA, encoding a fusion protein and its use as a vaccine to enhance immune responses, primarily cytotoxic T lymphocyte (CTL) responses to specific antigens such as tumor or viral antigens. The fusion protein comprises an antigenic polypeptide fused to a bacterial toxin translocation protein that promotes processing via the MHC class I pathway and selective induction of immunity mediated by CD8
[0005] 2. Description of the Background Art
[0006] Cytotoxic T lymphocytes (CTL) are critical effectors of antitumor responses (reviewed in Refs 1-3). Activated CTL are effector cells that mediate antitumor immunity by direct lysis of their target tumor cells or by releasing of cytokines that orchestrate immune and inflammatory responses that interfere with tumor growth or metastasis. Depletion of CD8
[0007] Naked DNA vaccines have emerged recently as attractive approaches for vaccine development (reviewed in 6-11). DNA vaccines generated long-term cell-mediated immunity (reviewed in 12). In addition, DNA vaccines can generate CD8
[0008] The present inventors and their colleagues recently demonstrated that linkage of HPV-16 E7 antigen to Mtb heat shock protein 70 (Hsp70) leads to the enhancement of DNA vaccine potency (5). (See also U.S. Ser. No. 09/501,097, filed 09 Feb. 2000; and U.S. Ser. No. 099/421,608, filed 20 Oct. 1999, from which the present application claims priority) Immunization with HSP complexes isolated from tumor or virus-infected cells induced potent anti-tumor immunity (Janetzki, S et al., 1998.
[0009] 1. Chen, C H et a., J Biomed Sci. 5: 231-252, 1998
[0010] 2. Pardoll, D M. Nat Med. 4: 525-531, 1998
[0011] 3. Wang, R F et al., Immunol Rev. 170. 85-100, 1999
[0012] 4. Lin, K -Y et al.., Canc Res. 56: 21-26, 1996
[0013] 5. Chen, C -H et al., Canc Res. 60: 1035-42, 2000
[0014] 6. Hoffman, S L et al., Ann N Y Acad Sci. 772: 88-94, 1995
[0015] 7. Robinson, H L. Vaccine. 15: 785-787, 1997
[0016] 8. Donnelly, J J et al.., Annu Rev Immunol. 15: 617-648, 1997
[0017] 9. Klinman, D M et al., Immunity. 11: 123-129, 1999
[0018] 10. Restifo, N P et al., Gene Ther. 7: 89-92, 2000
[0019] 11. Gurunathan, S et al., Annu Rev Immunol. 18: 927-974, 2000
[0020] 12. Gurunathan, S et al., Curr Opin Immunol. 12: 442-447, 2000
[0021] 13. Wang, R et al. Science. 282: 476-480, 1998.
[0022] The growing understanding of the antigen presentation pathway creates the potential for designing novel strategies to enhance vaccine potency. One strategy taken by the present inventors in the present invention to enhance the presentation of antigen through the MHC class I pathway to CD8
[0023] The present inventors created a novel fusion of the translocation domain (domain II) of
[0024] Thus the present invention is directed to a nucleic acid encoding a chimeric or fusion polypeptide which polypeptide comprises:
[0025] (a) a first domain comprising a translocation polypeptide; and
[0026] (b) a second domain comprising at least one antigenic peptide.
[0027] In the above nucleic acid ,the translocation polypeptide is preferably a bacterial toxin translocation polypeptide, more preferably domain II of
[0028] The above nucleic acid is preferably SEQ ID NO:3 or a homologue thereof.
[0029] The above nucleic preferably comprises a nucleotide sequence that encodes a translocation polypeptide which sequence is included in SEQ ID NO:1.
[0030] In the above nucleic acids, the antigenic peptide preferably comprises an epitope that binds to and is presented on the cell surface by MHC class I proteins. The epitope is preferably between about 8 and about 11 amino acid residues in length.
[0031] Preferably the antigen (i) is derived from a pathogen selected from the group consisting of a mammalian cell, a microorganism or a virus; or (ii) cross-reacts with an antigen of the pathogen. The virus may be a human papilloma virus and the antigen is preferably the HPV-16 E7 peptide. It is preferred that HPV-16 E7 polypeptide not be oncogenic.
[0032] The pathogen may be a bacterium.
[0033] In another embodiment, the antigen is a tumor-specific or tumor-associated antigen.
[0034] The above nucleic acid may be operatively linked to a promoter, preferably one which is expressed in an antigen presenting cell (APC), more preferably in a dendritic cell.
[0035] The present invention also provides an expression vector comprising any of the above nucleic acid molecules, operatively linked to a promoter and, optionally, to one or more regulatory elements that enhance expression of the nucleic acid in a cell.
[0036] The above expression vector may be a viral vector or a plasmid, including a self-replicating RNA replicon.
[0037] In the above expression vector, the translocation polypeptide is preferably ETA(dII).
[0038] Also provided is a particle comprising the above nucleic acid or expression vector. The particle preferably comprises a material, such as gold, that is suitable for introduction into a cell or an animals by particle bombardment.
[0039] The present invention is also directed to a cell which has been modified to comprise the above nucleic acid or the above the expression vector, and which cell expresses the nucleic acid. Preferably, the cell is an APC, such as a dendritic cell, a keratinocyte, a macrophage, a monocyte, a B lymphocyte, a microglial cell, an astrocyte, or an activated endothelial cell.
[0040] Also provided is a chimeric polypeptide comprising
[0041] (a) a first domain comprising a translocation polypeptide; and
[0042] (b) a second domain comprising at least one antigenic peptide.
[0043] The translocation polypeptide is preferably a bacterial toxin translocation polypeptide, more preferably, ETA(dII). Preferably, the translocation polypeptide comprises SEQ ID NO:3 or a homologue thereof.
[0044] The above chimeric polypeptide is preferably encoded by a nucleic acid as described above.
[0045] Preferably, in the chimeric polypeptide, the antigenic peptide comprises an epitope that binds to and is presented on the cell surface by MHC class I proteins.
[0046] In the above chimeric peptide, the translocation domain and the antigenic peptide may be linked by a chemical linker.
[0047] Preferably, the chimeric polypeptide above is a fusion polypeptide.
[0048] The first domain may be either N-terminal or C-terminal to the second domain.
[0049] The present invention is also directed to a pharmaceutical composition capable of inducing or enhancing an antigen specific immune response, comprising a pharmaceutically acceptable carrier or excipient and any one or more of:
[0050] (a) the above nucleic acid;
[0051] (b) the above expression vector;
[0052] (c) the above particle
[0053] (d) the above cell; or.
[0054] (e) the above chimeric polypeptide.
[0055] In another embodiment, the invention is directed to a method of enhancing an antigen specific immune response comprising administering an effective amount of a composition comprising
[0056] (a) the above nucleic acid;
[0057] (b) the above expression vector;
[0058] (c) the above particle
[0059] (d) the above cell; or.
[0060] (e) the above chimeric polypeptide.
[0061] thereby inducing or enhancing the antigen specific immune response.
[0062] In the above method, the antigen specific immune response is preferably mediated at least in part by CD8
[0063] In the above methods, the composition may be administered ex vivo, for example, o APCs, preferably human APCs, such as ones from a live subject. Preferred APCs are DCs. This method may further comprise administering the ex vivo-treated APCs to a histocompatible subject.
[0064] In another embodiment of the above methods, the composition is administered in vivo, preferably to a human. Preferred routes of administration are intramuscularly, intradermally, or subcutaneously. In administering the composition to a subject with a tumor, the route may be intratumoral or peritumoral.
[0065] Also provided is a method of increasing the numbers of CD8
[0066] (a) the above nucleic acid;
[0067] (b) the above expression vector;
[0068] (c) the above particle
[0069] (d) the above cell; or.
[0070] (e) the above chimeric polypeptide.
[0071] wherein the antigenic peptide comprises an epitope that binds to and is presented on the cell surface by MHC class I proteins, thereby increasing the numbers of antigen-specific CD8
[0072] In another embodiment, the invention provides a method of inhibiting the growth of a tumor in a subject comprising administering an effective amount of a composition comprising
[0073] (a) the above nucleic acid;
[0074] (b) the above expression vector;
[0075] (c) the above particle
[0076] (d) the above cell; or.
[0077] (e) the above chimeric polypeptide.
[0078] thereby inhibiting growth of the tumor. In this method the administering may be intratumoral or peritumoral.
[0079]
[0080]
[0081]
[0082]
[0083] The ability of the ETA(dII) polypeptide to facilitate translocation from the endosomal/lysosomal compartments to the cytoplasm suggested to the present inventors that at it may lead to the enhancement of MHC class I presentation of exogenous antigen if physically linked to the antigen. They therefore engineered a DNA vaccine encoding ETA(dII) linked to a model antigen, which was predicted to enhance MHC class I presentation of this antigen to CD8
[0084] The results presented herein indicate that vaccination with a chimeric ETA(dII)/E7 DNA vaccine enhanced MHC class I presentation of E7, leading to a dramatic increase in the number of E7-specific CD8
[0085] The invention provides compositions and methods for enhancing the immune responses, particularly cytotoxic T cell immune responses, induced by ex vivo or in vivo administration of chimeric polypeptides or, preferably, nucleic acid vaccines that encode these chimeric polypeptides. The preferred chimeric or fusion polypeptide comprises(l) at least one first polypeptide or peptide that, upon introduction to cells of the host immune system, in vitro or in vivo, promotes (a) processing via the MHC class I pathway and/or (b) development or activity of APCs, primarily DCs, and (2) at least one second polypeptide or peptide that is an antigenic polypeptide or peptide in the host.
[0086] As noted, in a preferred embodiment, the chimeric or fusion polypeptides are “indirectly” administered by administration of a nucleic acid that encodes the chimeric molecule; the nucleic acid construct, and thus the fusion protein, is expressed in vivo. The chimeric nucleic acids are administered in the form of DNA vaccines, either naked DNA or suicidal DNA, or a self-replicating RNA replicons.
[0087] The fusion protein comprises at least two domains or repeats thereof. A preferred embodiment of the first type of domain is a polypeptide that facilitates translocation from the endosomal/lysosomal compartments to the cytoplasm, thereby promoting processing via the MHC class I pathway. The most preferred polypeptide is ETA(dII). Other useful translocation polypeptides may be similar pathogenic bacterial toxins from Diptheria, Clostridium, Botulinum, Bacillus, Yersinia,
[0088] The second domain comprises a peptide or polypeptide, that includes one or several epitopes, derived from an antigen against which it is desired to induce an immune response, preferably a tumor antigen. In a preferred embodiment, the peptide comprises at least one MHC class I-binding peptide epitope that helps stimulate CD8+ CTLs and is recognized by such cells and their precursors.
[0089] The order in which the two (or more) component polypeptides of the fusion protein are arranged, and therefore, the order of the encoding nucleic acid fragments in the nucleic acid vector, can be altered without affecting immunogenicity of the fusion polypeptides proteins and the utility of the composition. For example, the ETA(dII)-encoding DNA sequences may be located 5′ or 3′ to the target antigen-encoding sequences. In one embodiment, these polypeptide-encoding nucleic acid domains are in-frame so that the DNA construct encodes a recombinant fusion polypeptide in which the antigen is located N-terminal to the ETA(dII)-derived polypeptide.
[0090] The vaccines of the present invention include, the antigenic epitope itself and a translocation polypeptide such as ETA(dII). In addition to the specific antigens and vectors employed in the Examples, the present invention is intended to encompass a vector such as naked RNA, self replicating RNA replicons and viruses including vaccinia, adenoviruses, adeno-associated virus (AAV), lentiviruses and RNA alphaviruses.
[0091] In addition to the translocation polypeptide, the vaccine construct of the present invention optionally, may also include
[0092] (a) an additional antigen targeting or processing signal such as proteins that promote intercellular transport, e.g., VP22 protein from herpes simplex virus and related herpes viruses (see, for example, commonly assigned International patent application published as WO 02/09645, 07-Feb.-2002, incorporated by reference in its entirety); an endoplasmic reticulum chaperone polypeptide such as calreticulin, ER60, GRP94 or gp96, well-characterized ER chaperone polypeptide that representatives of the HSP90 family of stress-induced proteins (see, co-pending commonly assigned International patent application published as WO 02/09645, 14-Feb.-2002, incorporated by reference in its entirety; see also Argon (1999)
[0093] (b) an immunostimulatory cytokine, preferably those that target APCs, preferably DC's, such as granulocyte macrophage colony stimulating factor (GM-CSF), or active fragments or domains thereof; and
[0094] (C) a costimulatory signal, such as a B7 family protein, including B7-DC (see commonly assigned U.S. patent application Ser. No. 09/794,210), B7.1, B7.2, soluble CD40, etc.).
[0095] (For description of some of the foregoing, see, for example, commonly owned International patent applications PCT/US01/23966, PCT/US01/24134, PCTUS/00/41422))
[0096] Naked DNA vaccines represent an attractive approach for generating antigen-specific immunity because of their stability and simplicity of delivery. Concerns with DNA vaccines include potential integration into the host genome, cell transformation, and limited potency. The use of DNA-based alphaviral RNA replicons (“suicidal DNA vectors”), as disclosed herein, may alleviate concerns surrounding DNA integration or cell transformation since suicidal DNA vectors eventually cause lysis of the cells they transfect.
[0097] To further improve the potency of suicidal DNA vaccines, ETA(dII) is linked to an antigen such as E7 as a model antigen, using DNA-based Semliki Forest virus (SFV) RNA vector, pSCA1. This suicidal DNA vaccine containing ETA(dII)/E7/fusion DNA produces significantly greater E7-specific T cell-mediated immune response in mice than do vaccines containing the wild type E7 DNA alone. Importantly, this fusion converts a less effective vaccine into one with significant therapeutic potency against established E7-expressing metastatic tumors. The antitumor effect is dependent upon CD8+ T cells. Thus, linkage of ETA(dII) to an antigen enhances the potency of a suicidal DNA vaccine.
[0098] In the methods of the invention, the chimeric polypeptide or nucleic acid that encodes it are employed to induce or enhance immune responses. In one embodiment, the compositions of the invention synergistically enhance immune responses and antitumor effects through both immunological and anti-angiogenic mechanisms.
[0099] The experiments described herein demonstrate that the methods of the invention can enhance a cellular immune response, particularly, tumor-destructive CTL reactivity, induced by a DNA vaccine encoding an epitope of a human pathogen. Human HPV-16 E7 was used as a model antigen for vaccine development because human papillomaviruses (HPVs), particularly HPV-16, are associated with most human cervical cancers. The oncogenic HPV protein E7 is important in the induction and maintenance of cellular transformation and co-expressed in most HPV-containing cervical cancers and their precursor lesions. Therefore, cancer vaccines, such as the compositions of the invention, that target E7 can be used to control of HPV-associated neoplasms (Wu (1994)
[0100] In one embodiment, the antigen (e.g., the MHC class I-binding peptide epitope) is derived from a pathogen, e.g., it comprises a peptide expressed by a pathogen. The pathogen can be a virus, such as, e.g., a papilloma virus, a herpesvirus, a retrovirus (e.g., an immunodeficiency virus, such as HIV-1), an adenovirus, and the like. The papilloma virus can be a human papilloma virus; for example, the antigen (e.g., the Class I-binding peptide) can be derived from an HPV-16 E7 polypeptide. In one embodiment, the HPV-16 E7 polypeptide is substantially non-oncogenic, i.e., it does not bind retinoblastoma polypeptide (pRB) or binds pRB with such low affinity that the HPV-16 E7 polypeptide is effectively non-oncogenic when expressed or delivered in vivo.
[0101] In alternative embodiments, the pathogen is a bacteria, such as
[0102] In another embodiment, the MHC class I-binding peptide epitope is derived from a tumor cell. The tumor cell-derived peptide epitope can comprise a tumor associated antigen, e.g., a tumor specific antigen, such as, e.g., a HER-2/neu antigen.
[0103] In one embodiment, the isolated or recombinant nucleic acid molecule is operatively linked to a promoter, such as, e.g., a constitutive, an inducible or a tissue-specific promoter. The promoter can be expressed in any cell, including cells of the immune system, including, e.g., antigen presenting cells (APCs), e.g., in a constitutive, an inducible or a tissue-specific manner.
[0104] In alternative embodiments, the APCs are dendritic cells, keratinocytes, astrocytes, monocytes, macrophages, B lymphocytes, a microglial cell, or activated endothelial cells, and the like.
[0105] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art of this invention. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
[0106] The term “antigen” or “immunogen” as used herein refers to a compound or composition comprising a peptide, polypeptide or protein which is “antigenic” or “immunogenic” when administered (or expressed in vivo by an administered nucleic acid, e.g., a DNA vaccine) in an appropriate amount (an “immunogenically effective amount”), i.e., capable of inducing, eliciting, augmenting or boosting a cellular and/or humoral immune response either alone or in combination or linked or fused to another substance (which can be administered at once or over several intervals). An immunogenic composition can comprise an antigenic peptide of at least about 5 amino acids, a peptide of 10 amino acids in length, a polypeptide fragment of 15 amino acids in length, 20 amino acids in length or longer. Smaller immunogens may require presence of a “carrier” polypeptide e.g., as a fusion protein, aggregate, conjugate or mixture, preferably linked (chemically or otherwise) to the immunogen. The immunogen can be recombinantly expressed from a vaccine vector, which can be naked DNA comprising the immunogen's coding sequence operably linked to a promoter, e.g., an expression cassette as described herein. The immunogen includes one or more antigenic determinants or epitopes which may vary in size from about 3 to about 15 amino acids.
[0107] The term “epitope” as used herein refers to an antigenic determinant or antigenic site that interacts with an antibody or a T cell receptor (TCR), e.g., the MHC class I-binding peptide compositions (or expressed products of the nucleic acid compositions of the invention) used in the methods of the invention. An “antigen” is a molecule or chemical structure that either induces an immune response or is specifically recognized or bound by the product or mediator of an immune response, such as an antibody or a CTL. The specific conformational or stereochemical “domain” to which an antibody or a TCR bind is an “antigenic determinant” or “epitope.” TCRs bind to peptide epitopes which are physically associated with a third molecule, a major histocompatibility complex (MHC) class I or class II protein.
[0108] The term “recombinant” refers to (1) a nucleic acid or polynucleotide synthesized or otherwise manipulated in vitro, (2) methods of using recombinant DNA technology to produce gene products in cells or other biological systems, or (3) a polypeptide encoded by a recombinant nucleic acid. For example, the ETA(dII)-encoding nucleic acid or polypeptide, the nucleic acid encoding an MHC class I-binding peptide epitope (antigen) or the peptide itself can be recombinant. “Recombinant means” includes ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into a single unit in the form of an expression cassette or vector for expression of the coding sequences in the vectors resulting in production of the encoded polypeptide.
[0109] The term “self-replicating RNA replicon” refers to a construct based on an RNA viruses, such as alphavirus genome RNAs (e.g., Sindbis virus, Semliki Forest virus, etc.), that have been engineered to allow expression of heterologous RNAs and proteins. These recombinant vectors are self-replicating (“replicons”) which can be introduced into cells as naked RNA or DNA, as described in detail in co-pending, commonly assigned U.S. and PCT patent applications by the present inventors, having Ser. Nos. 10/060,274, and PCT/US02/______, both filed on 10 Feb. 2002, and entitled “
[0110] The section that follows lists the sequences of the ETA(dII) polypeptides alone or in fusion with E7 antigen, the nucleic acids encoding some of these peptides and nucleic acids of the vectors into which the sequences encoding these polypeptides are cloned.
[0111] The complete coding sequence for
1 ctgcagctgg tcaggccgtt tccgcaacgc ttgaagtcct ggccgatata ccggcagggc 61 cagccatcgt tcgacgaata aagccacctc agccatgatg ccctttccat ccccagcgga 121 accccgacat ggacgccaaa gccctgctcc tcggcagcct ctgcctggcc gccccattcg 181 ccgacgcggc gacgctcgac aatgctctct ccgcctgcct cgccgcccgg ctcggtgcac 241 cgcacacggc ggagggccag ttgcacctgc cactcaccct tgaggcccgg cgctccaccg 301 gcgaatgcgg ctgtacctcg gcgctggtgc gatatcggct gctggccagg ggcgccagcg 361 ccgacagcct cgtgcttcaa gagggctgct cgatagtcgc caggacacgc cgcgcacgct 421 gaccctggcg gcggacgccg gcttggcgag cggccgcgaa ctggtcgtca ccctgggttg 481 tcaggcgcct gactgacagg ccgggctgcc accaccaggc cgagatggac gccctgcatg 541 tatcctccga tcggcaagcc tcccgttcgc acattcacca ctctgcaatc cagttcataa 601 atcccataaa agccctcttc cgctccccgc cagcctcccc gcatcccgca ccctagacgc 661 cccgccgctc tccgccggct cgcccgacaa gaaaaaccaa ccgctcgatc agcctcatcc 721 ttcacccatc acaggagcca tcgcgatgca cctgataccc cattggatcc ccctggtcgc 781 cagcctcggc ctgctcgccg gcggctcgtc cgcgtccgcc gccgaggaag ccttcgacct 841 ctggaacgaa tgcgccaaag cctgcgtgct cgacctcaag gacggcgtgc gttccagccg 901 catgagcgtc gacccggcoa tcgccgacac caacggccag ggcgtgctgc actactccat 961 ggtcctggag ggcggcaacg acgcgctcaa gctggccatc gacaacgccc tcagcatcac 1021 cagcgacggc ctgaccatcc gcctcgaagg cggcgtcgag ccgaacaagc cggtgcgcta 1081 cagctacacg cgccaggcgc gcggcagttg gtcgctgaac tggctggtac cgatcggcca 1141 cgagaagccc tcgaacatca aggtgttcat ccacgaactg aacgccggca accagctcag 1201 ccacatgtcg ccgatctaca ccatcgagat gggcgacgag ttgctggcga agctggcgcg 1261 cgatgccacc ttcttcgtca gggcgcacga gagcaacgag atgcagccga cgctcgccat 1321 cagccatgcc ggggtcagcg tggtcatggc ccagacccag ccgcgccggg aaaagcgctg 1381 gagcgaatgg gccagcggca aggtgttgtg cctgctcgac ccgctggacg gggtctacaa 1441 ctacctcgcc cagcaacgct gcaacctcga cgatacctgg gaaggcaaga tctaccgggt 1501 gctcgccggc aacccggcga agcatgacct ggacatcaaa cccacggtca tcagtcatcg 1561 cctgcacttt cccgagggcg gcagcctggc cgcgctgacc gcgcaccagg cttgccacct 1621 gccgctggag actttcaccc gtcatcgcca gccgcgcggc tgggaacaac tggagcagtg 1681 cggctatccg gtgcagcggc tggtcgccct ctacctggcg gcgcggctgt cgtggaacca 1741 ggtcgaccag gtgatccgca acgccctggc cagccccggc agcggcggcg acctgggcga 1801 agcgatccgc gagcagccgg agcaggcccg tctggccctg accctggccg ccgccgagag 1861 cgagcgcttc gtccggcagg gcaccggcaa cgacgaggcc ggcgcggcca acgccgacgt 1921 ggtgagcctg acctgcccgg tcgccgccgg tgaatgcgcg ggcccggcgg acagcggcga 1981 cgccctgctg gagcgcaact atcccactgg cgcggagttc ctcggcgacg gcggcgacgt 2041 cagcttcagc acccgcggca cgcagaactg gacggtggag cggctgctcc aggcgcaccg 2101 ccaactggag gagcgcggct atgtgttcgt cggctaccac ggcaccttcc tcgaagcggc 2161 gcaaagcatc gtcttcggcg gggtgcgcgc gcgcagccag gacctcgacg cgatctggcg 2221 cggtttctat atcgccggcg atccggcgct ggcctacggc tacgcccagg accaggaacc 2281 cgacgcacgc ggccggatcc gcaacggtgc cctgctgcgg gtctatgtgc cgcgctcgag 2341 cctgccgggc ttctaccgca ccagcctgac cctggccgcg ccggaggcgg cgggcgaggt 2401 cgaacggctg atcggccatc cgctgccgct gcgcctggac gccatcaccg gccccgagga 2461 ggaaggcggg cgcctggaga ccattctcgg ctggccgctg gccgagcgca ccgtggtgat 2521 tccctcggcg atccccaccg acccgcgcaa cgtcggcggc gacctcgacc cgtccagcat 2581 ccccgacaag gaacaggcga tcagcgccct gccggactac gccagccagc ccggcaaacc 2641 gccgcgcgag gacctgaagt aactgccgcg accggccggc tcccttcgca ggagccggcc 2701 ttctcggggc ctggccatac atcaggtttt cctgatgcca gcccaatcga atatgaattc 2760
[0112] The amino acid sequence of ETA (SEQ ID NO:2), GenBank Accession No. K01397, is shown below
MHLIPHWIPL VASLGLLAGG SSASAAEEAF DLWNECAKAC VLDLKDGVRS SRMSVDPATA 60 DTNGQGVLHY SMVLEGGNDA LKLATDNALS TTSDGLTIRL EGGVEPNKPV RYSYTRQARG 120 SWSLNWLVPT GHEKPSNIKV FTHELNAGNQ LSHMSPIYTT EMGDELLAKL ARDATFFVRA 180 HESNEMQPTL ATSHAGVSVV MAQTQPRREK RWSEWASGKV LCLLDPLDGV YNYLAQQRCN 240 LDDTWEGKTY RVLAGNPAK PARSQDLDAI WRGFYIAGDP ALAYGYAQDQ EPDARGRTRN GALLRVYVPR SSLPGFYRTS 540 LTLAAPEAAG EVERLTGHPL PLRLDAITGP EEEGGRLETT LGWPLAERTV VIPSAIPTDP 600 RNVGGDLDPS STPDKEQAIS ALPDYASQPG KPPREDLK 638
[0113] Residues 1-25 (italicized) represent the signal peptide; the start of thw mature plypeptide is shown as a bold/underlined A. The mature polypeptide is residules 26-638 of SEQ ID NO:2. The ETA(dII) translocation domain (underscored above) spans residues 247-417 of the mature polypeptide (corresponding to residues 272-442 of SEQ ID NO:2) and is presented below separately as SEQ ID NO:3.
[0114] The sequences shown below (nucleotide is SEQ ID NO:4 and amino acid is SEQ ID NO:5) are the construct in which ETA(dII) is fused to the HPV-16 E7 polypeptide. The ETA(dII) sequence appears in plain font, extra codons from pcDNA3 are italicized; those between the ETA(dII) and E7 sequence are also bolded (and result in the interpositio of two amino acids between ETA(dII) and E7. The E7 sequence is underscored. The E7 sequence ends in Gln.
1/1 31/11 atg cgc ctg cac ttt ccc gag ggc ggc agc ctg gcc gcg ctg acc gcg cac cag gct tgc Met arg leu his phe pro glu gly gly ser leu ala ala leu thr ala his gln ala cys 61/21 91/31 cac ctg ccg ctg gag act ttc acc cgt cat cgc cag ccg cgc ggc tgg gaa caa ctg gag his leu pro leu glu thr phe thr arg his arg gln pro arg gly trp glu gln leu glu 121/41 151/51 cag tgc ggc tat ccg gtg cag cgg ctg gtc gcc ctc tac ctg gcg gcg cgg ctg tcg tgg gln cys gly tyr pro val gln arg leu val ala leu tyr leu ala ala arg leu ser trp 181/61 211/71 aac cag gtc gac cag gtg atc cgc aac gcc ctg gcc agc ccc ggc agc ggc ggc gac ctg asn gln val asp gln val ile arg asn ala leu ala ser pro gly ser gly gly asp leu 241/81 271/91 ggc gaa gcg atc cgc gag cag ccg gag cag gcc cgt ctg gcc ctg acc ctg gcc gcc gcc gly glu ala ile arg glu gln pro glu gln ala arg leu ala leu thr leu ala ala ala 301/101 331/111 gag agc gag cgc ttc gtc cgg cag ggc acc ggc aac gac gag gcc ggc gcg gcc aac gcc glu ser glu arg phe val arg gln gly thr gly asn asp glu ala gly ala ala asn ala 361/121 391/131 gac gtg gtg agc ctg acc tgc ccg gtc gcc gcc ggt gaa tgc gcg ggc ccg gcg gac agc asp val val ser leu thr cys pro val ala ala gly glu cys ala gly pro ala asp ser 421/141 451/151 ggc gac gcc ctg ctg gag cgc aac tat ccc act ggc gcg gag ttc ctc ggc gac ggc ggc gly asp ala leu leu glu arg asn tyr pro thr gly ala glu phe leu gly asp gly gly 481/161 511/171 gac gtc agc ttc agc acc cgc ggc acg cag asp val ser phe ser thr arg gly thr gln 541/181 571/191 ttg cat gaa tat atg tta gat ttg caa cca gag aca act gat ctc tac tgt tat gag caa 601/201 631/211 tta aat gac agc tca gag gag gag gat gaa ata gat ggt cca gct gga caa gca gaa ccg 661/221 691/231 gac aga gcc cat tac aat att gta acc ttt tgt tgc aag tgt gac tct acg ctt cgg ttg 721/241 751/251 tgc gta caa agc aca cac gta gac att cgt act ttg gaa gac ctg tta atg ggc aca cta 781/261 811/271 gga att gtg tgc ccc atc tgt tct caa gga tcc gag ctc ggt acc aag ctt aag ttt aaa 841/281 ccg ctg atc agc ctc gac tgt gcc ttc tag pro leu ile ser 2eu asp cys ala phe AMB
[0115] Compared to the GenBank sequence of E7 (SEQ ID NO:6 & 7) shown below, three C-terminal amino acids have been deleted.
[0116] The HPV E7 sequence (nucleotide sequence is SEQ ID NO:6 and amino acid sequence is SEQ ID NO:7) is shown below:
1/1 31/11 atg cat gga gat aca cct aca ttg cat gaa tat atg tta gat ttg caa cca gag aca act Met his gly asp thr pro thr leu his glu tyr met leu asp leu gln pro glu thr thr 61/21 91/31 gat ctc tac tgt tat gag caa tta aat gac agc tca gag gag gag gat gaa ata gat ggt asp leu tyr cys tyr glu gln leu asn asp ser ser glu glu glu asp glu ile asp gly 121/41 151/51 cca gct gga caa gca gaa ccg gac aga gcc cat tac aat att gta acc ttt tgt tgc aag pro ala giy gln aia glu pro asp arg ala his tyr asn ile val thr phe cys cys iys 181/61 211/71 tgt gac tct acg ctt cgg ttg tgc gta caa agc aca cac gta gac att cgt act ttg gaa cys asp ser thr leu arg leu cys val gln ser thr his val asp ile arg thr leu glu 241/81 271/91 gac ctg tta atg ggc aca cta gga att gtg tgc ccc atc tgt tct cag gat aag ctt asp leu leu met gly thr leu gly ile val cys pro ile cys ser gln asp lys leu
[0117] The sequence of the pcDNA3 plasmid vector (SEQ ID NO:8) is:
GACGGATCGG GAGATCTCCC GATCCCCTAT GGTCGACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAC TATrTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGC GTTTAAACGG GCCCTCTAGA CTCGAGCGGC CGCCACTGTG CTGGATATCT GCAGAATTCC ACCACACTGG ACTAGTGGAT CCGAGCTCGG TACCAAGCTT AAGTTTAAAC CGCTGATCAG CCTCGACTGT GCCTTCTAGT TGCCAGCCAT CTGTTGTTTG CCCCTCCCCC GTGCCTTCCT TGACCCTGGA AGGTGCCACT CCCACTGTCC TTTCCTAATA AAATGAGGAA ATTGCATCGC ATTGTCTGAG TAGGTGTCAT TCTATTCTGG GGGGTGGGGT GGGGCAGGAC AGCAAGGGGG AGGATTGGGA AGACAATAGC AGGCATGCTG GGGATGCGGT GGGCTCTATG GCTTCTGAGG CGGAAAGAAC CAGCTGGGGC TCTAGGGGGT ATCCCCACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG TGTGGTGGTT ACGCGCAGCG TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC CCTTCCTTTC TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG GGGCATCCCT TTAGGGTTCC GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA TTAGGGTGAT GGTTCACGTA GTGGGCCATC GCCCTGATAG ACGGTTTTTC GCCCTTTGAC GTTGGAGTCC ACGTTCTTTA ATAGTGGACT CTTGTTCCAA ACTGGAACAA CACTCAACCC TATCTCGGTC TATTCTTTTG ATTTATAAGG GATTTTGGGG ATTTCGGCCT ATTGGTTAAA AAATGAGCTG ATTTAACAAA AATTTAACGC GAATTAATTC TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG CTCCCCAGGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCAGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCATAGT CCCGCCCCTA ACTCCGCCCA TCCCGCCCCT AACTCCGCCC AGTTCCGCCC ATTCTCCGCC CCATGGCTGA CTAATTTTTT TTA1TTATGC AGAGGCCGAG GCCGCCTCTG CCTCTGAGCT ATTCCAGAAG TAGTGAGGAG GCTTTTTTGG AGGCCTAGGC TTTTGCAAAA AGCTCCCGGG AGCTTGTATA TCCATTTTCG GATCTGATCA AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAGGA CGAGGCAGCG CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCT GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAA AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG AACTGTTCGC CAGGCTCAAG GCGCGCATGC CCGACGGCGA GGATCTCGTC GTGACCCATG GCGATGCCTG CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC CAACCTGCCA TCACGAGATT TCGATTCCAC CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCCAACTTGT TTATTGCAGC TTATAATGGT TACAAATAAA GCAATAGCAT CACAAATTTC ACAAATAAAG CATTTTTTTC ACTGCATTCT AGTTGTGGTT TGTCCAAACT CATCAATGTA TCTTATCATG TCTGTATACC GTCGACCTCT AGCTAGAGCT TGGCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAATTG TTATCCGCTC ACAATTCCAC ACAACATACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA GTGAGCTAAC TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT GAGCAAAAGG CCAGCAATAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA ATGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAGTAT ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGACC CACGCTCACC GGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCATCG TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATA ATACCGCGCC ACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTTTCA ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGACGT C
[0118] The nucleic acid sequence of plasmid construct pcDNA3-ETA(dII)/E7 (SEQ ID NO:9) is shown below. ETA(dII)/E7 is ligated in the EcoRI/BamHI sites of pcDNA3 vector. The nucleotides encoding ETA(dII)/E7 are shown in lower case bold.
| 10 | 20 | 30 | 40 | 50 | 60 | 70 | 80 1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTCGACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGTAT 80 81 CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA 160 161 CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT 240 241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA 320 321 CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT 400 401 AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAC TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT 480 481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA 560 561 TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA 640 641 TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC 720 721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG 800 801 GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG 880 881 GGAGACCCAA GCTGGCTAGC GTTTAAACGG GCCCTCTAGA CTCGAGCGGC CGCCACTGTG CTGGATATCT GCAGAATTCa 960 961 1041 1121 1201 1281 1361 1441 1521 1601 1681 1761 1841 CTGTTGTTTG CCCCTCCCCC GTGCCTTCCT TGACCCTGGA AGGTGCCACT CCCACTGTCC TTTCCTAATA AAATGAGGAA 1920 1921 ATTGCATCGC ATTGTCTGAG TAGGTGTCAT TCTATTCTGG GGGGTGGGGT GGGGCAGGAC AGCAAGGGGG AGGATTGGGA 2000 2001 AGACAATAGC AGGCATGCTG GGGATGCGGT GGGCTCTATG GCTTCTGAGG CGGAAAGAAC CAGCTGGGGC TCTAGGGGGT 2080 2081 ATCCCCACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG TGTGGTGGTT ACGCGCAGCG TGACCGCTAC ACTTGCCAGC 2160 2161 GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC CCTTCCTTTC TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG 2240 2241 GGGCATCCCT TTAGGGTTCC GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA TTAGGGTGAT GGTTCACGTA 2320 2321 GTGGGCCATC GCCCTGATAG ACGGTTTTTC GCCCTTTGAC GTTGGAGTCC ACGTTCTTTA ATAGTGGACT CTTGTTCCAA 2400 2401 ACTGGAACAA CACTCAACCC TATCTCGGTC TATTCTTTTG ATTTATAAGG GATTTTGGGG ATTTCGGCCT ATTGGTTAAA 2480 2481 AAATGAGCTG ATTTAACAAA AATTTAACGC GAATTAATTC TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG 2560 2561 CTCCCCAGGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCAGGTGTG GAAAGTCCCC AGGCTCCCCA 2640 2641 GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCATAGT CCCGCCCCTA ACTCCGCCCA TCCCGCCCCT 2720 2721 AACTCCGCCC AGTTCCGCCC ATTCTCCGCC CCATGGCTGA CTAATTTTTT TTATTTATGC AGAGGCCGAG GCCGCCTCTG 2800 2801 CCTCTGAGCT ATTCCAGAAG TAGTGAGGAG GCTTTTTTGG AGGCCTAGGC TTTTGCAAAA AGCTCCCGGG AGCTTGTATA 2880 2881 TCCATTTTCG GATCTGATCA AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG CAGGTTCTCC 2960 2961 GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA TCGGCTGCTC TGATGCCGCC GTGTTCCGGC 3040 3041 TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAGGA CGAGGCAGCG 3120 3121 CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCT 3200 3201 GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAA AGTATCCATC ATGGCTGATG 3280 3281 CAATGCGGCG GCTGCATACG CTTGATCCGG CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT 3360 3361 ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG AACTGTTCGC 3440 3441 CAGGCTCAAG GCGCGCATGC CCGACGGCGA GGATCTCGTC GTGACCCATG GCGATGCCTG CTTGCCGAAT ATCATGGTGG 3520 3521 AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC 3600 3601 CGTGATATTG CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC CCGATTCGCA 3680 3681 GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC 3760 3761 CAACCTGCCA TCACGAGATT TCGATTCCAC CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG 3840 3841 GCTGGATGAT CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCCAACTTGT TTATTGCAGC TTATAATGGT 3920 3921 TACAAATAAA GCAATAGCAT CACAAATTTC ACAAATAAAG CATTTTTTTC ACTGCATTCT AGTTGTGGTT TGTCCAAACT 4000 4001 CATCAATGTA TCTTATCATG TCTGTATACC GTCGACCTCT AGCTAGAGCT TGGCGTAATC ATGGTCATAG CTGTTTCCTG 4080 4081 TGTGAAATTG TTATCCGCTC ACAATTCCAC ACAACATACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA 4160 4161 GTGAGCTAAC TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG TCGTGCCAGC TGCATTAATG 4240 4241 AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG 4320 4321 TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA 4400 4401 AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG 4480 4481 CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT 4560 4561 TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG 4640 4641 GGAAGCGTGG CGCTTTCTCA ATGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT 4720 4721 GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT 4800 4801 TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG 4880 4881 TGGCCTAACT ACGGCTACAC TAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT 4960 4961 TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA 5040 5041 AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT 5120 5121 TTGGTCATGA GATTATCAAA AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAGTAT 5200 5201 ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA TTTCGTTCAT 5280 5281 CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA 5360 5361 CCGCGAGACC CACGCTCACC GGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC 5440 5441 TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT AATAGTTTGC 5520 5521 GCAACGTTGT TGCCATTGCT ACAGGCATCG TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA 5600 5601 CGATCAAGGC GAGTTACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG 5680 5681 TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC GTAAGATGCT 5760 5761 TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA 5840 5841 ATACGGGATA ATACCGCGCC ACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC 5920 5921 AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT TTTACTTTCA 6000 6001 CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA 6080 6081 CTCATACTCT TCCTTTTTCA ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT 6160 6161 TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGACGT C 6221 | 10 | 20 | 30 | 40 | 50 | 60 | 70 | 80
[0119] Basic texts disclosing general methods of molecular biology, all of which are incorporated by reference, include: Sambrook, J et al.,
[0120] Unless otherwise indicated, a particular nucleic acid sequence is intended to encompasses conservative substitution variants thereof (e.g., degenerate codon substitutions) and a complementary sequence. The term “nucleic acid” is synonymous with “polynucleotide” and is intended to include a gene, a cDNA molecule, an mRNA molecule, as well as a fragment of any of these such as an oligonucleotide, and further, equivalents thereof (explained more fully below). Sizes of nucleic acids are stated either as kilobases (kb) or base pairs (bp). These are estimates derived from agarose or polyacrylamide gel electrophoresis (PAGE), from nucleic acid sequences which are determined by the user or published. Protein size is stated as molecular mass in kilodaltons (kDa) or as length (number of amino acid residues). Protein size is estimated from PAGE, from sequencing, from presumptive amino acid sequences based on the coding nucleic acid sequence or from published amino acid sequences.
[0121] Specifically, cDNA molecules encoding the amino acid sequence corresponding to the fusion polypeptide of the present invention or fragments or derivatives thereof can be synthesized by the polymerase chain reaction (PCR) (see, for example, U.S. Pat. No. 4,683,202) using primers derived the sequence of the protein disclosed herein. These cDNA sequences can then be assembled into a eukaryotic or prokaryotic expression vector and the resulting vector can be used to direct the synthesis of the fusion polypeptide or its fragment or derivative by appropriate host cells, for example COS or CHO cells.
[0122] This invention includes isolated nucleic acids having a nucleotide sequence encoding the novel fusion polypeptides that comprise a translocation polypeptide and an antigen, fragments thereof or equivalents thereof. The term nucleic acid as used herein is intended to include such fragments or equivalents. The nucleic acid sequences of this invention can be DNA or RNA.
[0123] A cDNA nucleotide sequence the fusion polypeptide can be obtained by isolating total mRNA from an appropriate cell line. Double stranded cDNA is prepared from total mRNA. cDNA can be inserted into a suitable plasmid, bacteriophage or viral vector using any one of a number of known techniques.
[0124] In reference to a nucleotide sequence, the term “equivalent” is intended to include sequences encoding structurally homologous and/or a functionally equivalent proteins. For example, a natural polymorphism in ETA(dII) nucleotide sequence (especially at the third base of a codon) may be manifest as “silent” mutations which do not change the amino acid sequence. Furthermore, there may be one or more naturally occurring isoforms or related, immunologically cross-reactive family members of these proteins. Such isoforms or family members are defined as proteins that share function amino acid sequence similarity to, for example, ETA(dII)
[0125] A fragment of the nucleic acid sequence is defined as a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length translocation polypeptide, antigenic polypeptide or the fusion thereof.. This invention includes such nucleic acid fragments that encode polypeptides which retain (1) the ability of the fusion polypeptide to induce increases in frequency or reactivity of T cells, preferably CD8+ T cells, that are specific for the antigen part of the fusion polypeptide.
[0126] For example, a nucleic acid fragment as intended herein encodes a ETA(dII) polypeptide that retains the ability to improve the immunogenicity of an antigen when administered as a fusion polypeptide with an antigenic polypeptide or peptide.
[0127] Generally, the nucleic acid sequence encoding a fragment of a ETA(dII) polypeptide comprises of nucleotides from the sequence encoding the mature protein (or an active fragment thereof).
[0128] Nucleic acid sequences of this invention may also include linker sequences, natural or modified restriction endonuclease sites and other sequences that are useful for manipulations related to cloning, expression or purification of encoded protein or fragments. These and other modifications of nucleic acid sequences are described herein or are well-known in the art.
[0129] The techniques for assembling and expressing DNA coding sequences for translocation types of proteins, and DNA coding sequences for antigenic polypeptides, include synthesis of oligonucleotides, PCR, transforming cells, constructing vectors, expression systems, and the like; these are well-established in the art such that those of ordinary skill are familiar with standard resource materials, specific conditions and procedures.
[0130] This invention includes an expression vector comprising a nucleic acid sequence encoding a translocation polypeptide/antigen fusion polypeptide, preferably a ETA(dII)/antigen fusion polypeptide operably linked to at least one regulatory sequence.
[0131] The term “expression vector” or “expression cassette” as used herein refers to a nucleotide sequence which is capable of affecting expression of a protein coding sequence in a host compatible with such sequences. Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be included, e.g., enhancers.
[0132] “Operably linked” means that the coding sequence is linked to a regulatory sequence in a manner that allows expression of the coding sequence. Known regulatory sequences are selected to direct expression of the desired protein in an appropriate host cell. Accordingly, the term “regulatory sequence” includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in, for example, Goeddel,
[0133] Thus, expression cassettes include plasmids, recombinant viruses, any form of a recombinant “naked DNA” vector, and the like. A “vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. The vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include, but are not limited to replicons (e.g., RNA replicons (see Example 1, below), bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA, e.g., plasmids, viruses, and the like (U.S. Pat. No. 5,217,879), and includes both the expression and nonexpression plasmids. Where a recombinant microorganism or cell culture is described as hosting an “expression vector” this includes both extrachromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
[0134] Those skilled in the art appreciate that the particular design of an expression vector of this invention depends on considerations such as the host cell to be transfected and/or the type of protein to be expressed.
[0135] The present expression vectors comprise the full range of nucleic acid molecules encoding the various embodiments of the fusion polypeptide and its functional derivatives (defined herein) including polypeptide fragments, variants, etc.
[0136] Such expression vectors are used to transfect host cells (in vitro, ex vivo or in vivo) for expression of the DNA and production of the encoded proteins which include fusion proteins or peptides. It will be understood that a genetically modified cell expressing the fusion polypeptide may transiently express the exogenous DNA for a time sufficient for the cell to be useful for its stated purpose.
[0137] The present in invention provides methods for producing the fusion polypeptides, fragments and derivatives. For example, a host cell transfected with a nucleic acid vector that encodes the fusion polypeptide is cultured under appropriate conditions to allow expression of the polypeptide.
[0138] Host cells may also be transfected with one or more expression vectors that singly or in combination comprise DNA encoding at least a portion of the fusion polypeptide and DNA encoding at least a portion of a second protein, so that the host cells produce yet further fusion polypeptides that include both the portions.
[0139] A culture typically includes host cells, appropriate growth media and other byproducts. Suitable culture media are well known in the art. The fusion polypeptide can be isolated from medium or cell lysates using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, fractionation column chromatography (e.g. ion exchange, gel filtration, affinity chromatography, etc.) and/or electrophoresis (see generally, “Enzyme Purification and Related Techniques”,
[0140] The term “isolated” as used herein, when referring to a molecule or composition, such as a translocation polypeptide or a nucleic acid coding therefor, means that the molecule or composition is separated from at least one other compound (protein, other nucleic acid, etc.) or from other contaminants with which it is natively associated or becomes associated during processing.. An isolated composition can also be substantially pure. An isolated composition can be in a homogeneous state and can be dry or in aqueous solution. Purity and homogeneity can be determined, for example, using analytical chemical techniques such as polyacrylamide gel electrophoresis (PAGE) or high performance liquid chromatography (HPLC). Even where a protein has been isolated so as to appear as a homogenous or dominant band in a gel pattern, there are trace contaminants which co-purify with it.
[0141] Prokaryotic or eukaryotic host cells transformed or transfected to express the fusion polypeptide or a homologue or functional derivative thereof are within the scope of the invention. For example, the fusion polypeptide may be expressed in bacterial cells such as
[0142] Expression in eukaryotic cells leads to partial or complete glycosylation and/or formation of relevant inter- or intra-chain disulfide bonds of the recombinant protein.
[0143] Although preferred vectors are described in the Examples, other examples of expression vectors are provided here. Examples of vectors for expression in yeast
[0144] Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the reporter group and the target protein to enable separation of the target protein from the reporter group subsequent to purification of the fusion protein. Proteolytic enzymes for such cleavage and their recognition sequences include Factor Xa, thrombin and enterokinase.
[0145] Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein.
[0146] Inducible non-fusion expression vectors include pTrc (Amann et al, (1988)
[0147] One embodiment of this invention is a transfected cell which expresses novel fusion polypeptide.
[0148] Construction of suitable vectors containing the desired coding and control sequences employs standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and re-ligated in the form desired.
[0149] The DNA sequences which form the vectors are available from a number of sources. Backbone vectors and control systems are generally found on available “host” vectors which are used for the bulk of the sequences in construction. For the pertinent coding sequence, initial construction may be, and usually is, a matter of retrieving the appropriate sequences from cDNA or genomic DNA libraries. However, once the sequence is disclosed it is possible to synthesize the entire gene sequence in vitro starting from the individual nucleotide derivatives. The entire gene sequence for genes of sizeable length, e.g., 500-1000 bp may be prepared by synthesizing individual overlapping complementary oligonucleotides and filling in single stranded nonoverlapping portions using DNA polymerase in the presence of the deoxyribonucleotide triphosphates. This approach has been used successfully in the construction of several genes of known sequence. See, for example, Edge, M. D.,
[0150] Synthetic oligonucleotides are prepared by either the phosphotriester method as described by references cited above or the phosphoramidite method as described by Beaucage, S. L., and Caruthers, M. H.,
[0151] Once the components of the desired vectors are thus available, they can be excised and ligated using standard restriction and ligation procedures. Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog. In general, about 1 mg of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ml of buffer solution; in the examples herein, typically, an excess of restriction enzyme is used to insure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37° C. are workable, although variations can be tolerated. After each incubation, protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction, and