DETAILED DESCRIPTION OF THE INVENTION

[0028] The invention relates to immunoconjugates useful for killing a malignant or virus-infected target cell. The immunoconjugate includes a conjugate of an antibody portion having a specificity for a surface antigen on a virus-infected or malignant cell, and a cytokine.

[0029] FIG. 1 shows a schematic view of a representative immunoconjugate 10. In this embodiment, cytokine molecules 2 and 4 are peptide bonded to the carboxy termini 6 and 8 of CH3 regions 10 and 12 of antibody heavy chains 14 and 16. V _L regions 26 and 28 are shown paired with V _H regions 18 and 20 in a typical IgG configuration, thereby providing two antigen binding sites 30 and 32 at the amino ends of immunoconjugate 10 and two cytokine receptor-binding sites 40 and 42 at the carboxy ends of immunoconjugate 10. Of course, in their broader aspects, the immunoconjugates need not be paired as illustrated.

[0030] The immunoconjugates of this invention can be produced by genetic engineering techniques; i.e., by forming a nucleic acid construct encoding the chimeric immunoconjugate. Preferably, the gene construct encoding the immunoconjugate of the invention includes, in 5′ to 3′ orientation, a DNA segment which encodes a heavy chain variable region, a DNA segment encoding the heavy chain constant region, and DNA coding for the cytokine. The fused gene is assembled in or inserted into an expression vector for transfection of the appropriate recipient cells where it is expressed. The hybrid chain can be combined with a light (or heavy) chain counterpart to form monovalent and divalent immunoconjugates.

[0031] The cytokine can be any cytokine or analog or fragment thereof which has a therapeutically valuable biological function. Useful cytokines include the interleukins and hematopoietic factors such as interleukin-2 (IL-2) and granulocyte-macrophage colony stimulating factor (GMCSF). Lymphokines such as LT and TNF∝, which require the formation of multimeric structures to function,-can also be used. The gene encoding the lymphokine or cytokine can be cloned de novo, obtained from an available source, or synthesized by standard DNA synthesis from a known nucleotide sequence. For example, the DNA sequence of LT is known (see, e.g. Nedwin et al. (1985) Nucleic Acids Res. 13:6361), as are the sequences for interleukin-2 (see, e.g., Taniguchi et al. (1983) Nature 302:305-318), granulocyte-macrophage colony stimulating factor (see, e.g., Gasson et al. (1984) Science 266:1339-1342), and tumor necrosis factor alpha (see, e.g., Nedwin et al. 1. Ibid.)

[0032] The heavy chain constant region for the conjugates can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu. Heavy chains or various subclasses (such as the IgG subclasses 1-4) can be used. The light chains can-have either a kappa or lambda constant chain. DNA sequences for these immunoglobulin regions are well known in the art. (See, e.g., Gillies et al. (1989) J. Immunol. Meth. 125:191).

[0033] In preferred embodiments, the variable region is derived from an antibody specific for the target antigen (an antigen associated with a diseased cell such as a cancer cell or virus-infected cell), and the constant region includes the CH1, CH2 and CH3 domains. The gene encoding the cytokine is joined, (e.g., by appropriate linkers, e.g., by DNA encoding (Gly ₄ -Ser) ₃ in frame to the 3′ end of the gene encoding the constant region (e.g., CH3 exon), either directly or through an intergenic region. In certain embodiments, the intergenic region can comprise a nucleotide sequence coding for a proteolytic cleavage site. This site, interposed between the immunoglobulin and the cytokine, can be designed to provide for proteolytic release of the cytokine at the target site. For example, it is well known that plasmin and trypsin cleave after lysine and arginine residues at sites that are accessible to the proteases. Many other site-specific endoproteases and the amino acid sequences they attack are well-known.

[0034] The nucleic acid construct can include the endogenous promoter and enhancer for the variable region-encoding gene to regulate expression of the chimeric immunoglobulin chain. For example, the variable region encoding genes can be obtained as DNA fragments comprising the leader peptide, the VJ gene (functionally rearranged variable (V) regions with joining (J) segment) for the light chain or VDJ gene for heavy chain, and the endogenous promoter and enhancer for these genes. Alternatively, the gene coding for the variable region can be obtained apart from endogenous regulatory elements and used in an expression vector which provides these elements.

[0035] Variable region genes can be obtained by standard DNA cloning procedures from cells that produce the desired antibody. Screening of the genomic library for a specific functionally rearranged variable region can be accomplished with the use of appropriate DNA probes such as DNA segments containing the J region DNA sequence and sequences downstream. Identification and confirmation of correct clones are then achieved by DNA sequencing of the cloned genes and comparison of the sequence to the corresponding sequence of the full length, properly spliced mRNA.

[0036] The target antigen can be a cell surface antigen of a tumor cell, a virus-infected cell or another diseased cell. Genes encoding appropriate variable regions can be obtained generally from Ig-producing lymphoid cells. For example, hybridoma cell lines producing Ig specific for tumor associated antigens or viral antigens can be produced by standard somatic cell hybridization techniques. (See, e.g., U.S. Pat. No. 4,96,265.) These Ig-producing cell lines provide the source of variable region genes in functionally rearranged form. The variable region genes will typically be of murine origin because this murine system lends itself to the production of a wide variety of Igs of desired specificity.

[0037] The DNA fragment containing the functionally rearranged variable region gene is linked to a DNA fragment containing the gene encoding the desired constant region (or a portion thereof). Ig constant regions (heavy and light chain) can be obtained from antibody-producing cells by standard gene cloning techniques. Genes for the two classes of human light chains and the five classes of human heavy chains have been cloned, and thus, constant regions of human origin are readily available from these clones.

[0038] The fused gene encoding the hybrid IgH chain is assembled or inserted into expression vectors for incorporation into a recipient cell. The introduction of gene construct into plasmid vectors can be accomplished by standard gene splicing procedures.

[0039] The chimeric IgH chain can be co-expressed in the same cell with a corresponding L chain so that a complete immunoglobulin can be expressed and assembled simultaneously. For this purpose, the heavy and light chain constructs can be placed in the same or separate vectors.

[0040] Recipient cell lines are generally lymphoid cells. The preferred recipient cell is a myeloma (or hybridoma). Myelomas can synthesize, assemble, and secrete immunoglobulins encoded by transfected genes and they can glycosylate protein. A particularly preferred recipient cell is the Sp2/0 myeloma which normally does not produce endogenous immunoglobulin. When transfected, the cell will produce only Ig encoded by the transfected gene constructs. Transfected myelomas can be grown in culture or in the peritoneum of mice where secreted immunoconjugate can be recovered from ascites fluid. Other lymphoid cells such as B lymphocytes can be used as recipient cells.

[0041] There are several methods for transfecting lymphoid cells with vectors containing the nucleic acid constructs encoding the chimeric Ig chain. A preferred way of introducing a vector into lymphoid cells is by spheroblast fusion. (see, Gillies et al. (1989) Biotechnol. 7:798-804). Alternative methods include electroporation or calcium phosphate precipitation.

[0042] Other useful methods of producing the immunoconjugates include the preparation of an RNA sequence encoding the construct and its translation in an appropriate in vivo or in vitro system.

[0043] The immunoconjugate of this invention can be used to deliver selectively a cytokine to a target cell in vivo so that the cytokine can exert a localized biological effect such as a local inflammatory response, stimulation of T cell growth and activation, and ADCC activity. A therapeutically effective amount of the immunoconjugate is administered into the circulatory system of a subject harboring the target cell.

[0044] The invention is illustrated further by the following non-limiting Examples.

[0045] 1. Plasmid Construction

[0046] Described below is the construction of PdHL2, a plasmid which contains the human Cγl heavy and kappa light chain gene sequences as well as insertion sites for V region cDNA cassettes (Gillies et al. (1989) J. Immunol. Meth. 125:191). This plasmid may be used as a starter plasmid for constructing any IgH chain cytokine fusion. For example, PdHL2 was used for the expression of Ig/LT fusion proteins. A LT cDNA was isolated from a human peripheral blood leukocyte library cloned in λgt10. The sequence was identical to that reported in the literature by Nedwin et al. (Nucleic Acids Res (1985) 13:6361). The cDNA was inserted into vector pDEM (Gillies et al., ibid) as an XhoI fragment after first removing most of the 3′ untranslated region with Bal31 nuclease. The resulting plasmid, pDEM-LT ( FIG. 2 ), expresses (in transfected cells) a fusion mRNA with a 5′ untranslated sequence derived from the metallothionein (MT) promoter, the LT coding sequence and a 3′ untranslated sequence and a poly A addition signal from the mouse Cκ gene. Fusion protein-encoding vectors-were constructed by ligating HindIII to TaqI (CH2-LT) or HindIII to NsiI (CH3-LT) fragments of the human Cγl gene to HindIII and PvuII digested PDEM-LT using synthetic DNA linkers ( FIG. 2 ). These linkers: 1

[0047] provide the protein coding sequence from the unique site (NsiI or TaqI) to the end of the heavy-chain domain (indicated by the slash), and join them to the amino terminus of the mature form of LT (up to the unique PvuII site). The linker for the CH3 fusion protein also includes a silent mutation that creates a SmaI site close to the end of the domain for future use in constructing fusion proteins. The DNA sequences at the junction of each construct were confirmed and each HindIII to EcoRI fragment was inserted into plasmid pdHL2-VCγlκ (14.18). This plasmid contains the V cassettes for the ch14.18 anti-ganglioside GD2 antibody (Gillies et al., ibid.).

[0048] 2. Cell Culture and Transfection

[0049] Sp2/0 Ag14 mouse hybridoma cells were maintained and transfected as described by Gillies et al. (BioTechnology (1989) 7:8799). Drug selection in methotrexate (MTX) was initiated 24 hours after transfection by adding an equal volume of medium containing MTX at 0.1 μM. Two additional feedings with selection medium were done at 3 day intervals. Transfectants secreting human Ig determinants were identified by ELISA (Gillies et al., 1989. ibid), grown in medium containing increasing concentrations of MTX, and subcloned by limiting dilution in medium containing MTX at 5 μM.

[0050] 3. Purification and Characterization of Fusion Proteins

[0051] Proteins were biosynthetically labeled by incubating transfected cells (1×10 ^{6/mL) for} 16 hr in growth medium containing ³⁵ S-methionine (50 μCi/mL-Amersham). Culture supernatants were then clarified by centrifugation in a microcentrifuge and the labeled proteins were immmunoprecipitated with polyclonal anti-human κ chain antisera (Jackson Immunoresearch, Bar Harbor, Me.) and protein A Sepharose (Repligen, Corp., Cambridge, Mass.). Protein samples were boiled for 5 min. in gel sample buffer in the presence or absence of 2-mercaptoethanol and analyzed on a 7% polyacrylamide gel. Proteins were detected by fluorography (diphenyloxazole in DMSO) and autoradiography.

[0052] Unlabeled proteins were purified from spent suspension culture medium by either immunoaffinity chromatography with a monoclonal anti-human κ antibody for the CH2-LT protein or by protein A Sepharose chromatography for the CH3-LT protein. All materials were concentrated by membrane dialysis into PBS. An alternative procedure for purification of the CH3-LT protein was developed to prevent the loss of LT activity during elution from the protein A column. Spent culture media was diluted with three volumes of 10 mM sodium phosphate buffer (pH 6.5) and loaded onto a Bakerbond AbX (J. T. Baker) column at room temperature. The column was washed with 10 mM sodium phosphate buffer until the absorbance returned to baseline and then with PBS, pH 6.5 (150 mM NaCl, 10 mM sodium phosphate, pH 6.5). The CH3-LT protein was eluted with 150 mM NaCl, 50 mM sodium phosphate, pH 6.5.

[0053] 4. Activity Assay

[0054] The antigen binding activity of the Ig-LT proteins was measured as described in Gillies et al. (J. Immunol. Meth. (1989) 125:191), and LT activity was determined in the cytolytic or cytostatic assay (Kahn et al. (1982)) utilizing the 159124T2.5 subclone of the mouse L929 cell line (provided by Dr. H. Schreiber, University of Chicago). Cells were seeded into 96-well plates at 4×10 ⁴ cells per well, with (cytolytic) or without (cytostatic) mitomycin C (2 μg/mL), and 10 μL of the test sample was added after 24 hr. Cells were stained either 24 or 48 hr later (see FIG. descriptions) with crystal violet and the amounts of dye retained in the wells were compared to those of untreated wells and those receiving the LT standard (R&D Systems). The same assay was also carried out with the GD2-bearing human melanoma line M21, originally provided by D. L. Morton, University of California, Los Angeles. The latter cell line was also used for measuring CDC and ADCC activity as described earlier (Gillies et al. (1990) Human Antibody. Hybridomas 1:47)

[0055] 5. Expression of Ig/LT Immunoconjugates

[0056] The Ig/LT immunoconjugates were made by directly fusing the cDNA sequence encoding the mature form of LT to the end of either the CH2 or CH3 exon of the human Cγl gene ( FIG. 2 ) with the appropriate synthetic linkers. This gene fusion was then combined in a vector together with the V regions of murine antibody 14.18 and the human Cκ gene, and expressed in transfected Sp2/0 cells. These immunoconjugates were then expressed and tested for antigen binding activity and Ig chain assembly. The immunoconjugates retained antigen binding when measured in a competitive antigen binding ELISA (see below), and were assembled. Cells expressing these immunoconjugates were labeled with ³⁵ S-methionine, and the secreted proteins were analyzed by SDS-PAGE in the presence or absence of reducing agent.

[0057] As seen in FIG. 3 , the CH2-LT immunoconjugate was expressed as a mixture of whole (approximately 180 Kd) and half (90 Kd) molecules. The CH3-LT fusion protein, on the other hand, consisted entirely of fully assembled molecules. This result is not surprising since the CH3 domain is most responsible for Ig chain assembly. The reason why a portion of the CH2-LT did assemble, i.e. formed disulfide bonds in the hinge-domain of the antibody, is likely due to the dimerization of the carboxy-terminal LT domains.

[0058] 6. Biological Activity of Ig/LT Conjugates

[0059] The LT activities of the CH2-LT and CH3-LT conjugates were compared in the standard cytolytic assay (Kahn, A. et al. (1982) “A standardized automated computer assisted micro-assay for lymphotoxin.” In: Human Lymphokines, Biological response modified; (Kahn and Hill, eds.) Academic Press, New York, p. 23), using a mouse L929 subclone. This assay measures the ability of the immunoconjugate to bind to the TNF/LT receptor and trigger the active cell killing process in this cell line. When crude preparations (culture supernatants) were compared ( FIG. 4A ), CH3-LT was found to be much more active (nearly 100 fold by this assay) than CH2-LT and exhibited approximately the same specific activity per mole as the LT standard. This higher activity of CH3-LT is likely due to the increased proportion of fully assembled H-chain fusion proteins. Thus, the presence of the CH3 exon in the immunoconjugate may allow the H-chains to associate more efficiently, perhaps positioning the LT domains in a manner that allows for dimerization and, as a consequence, more LT receptor binding.

[0060] When purified preparations were compared, the difference in activities between CH2-LT and CH3-LT was still evident, but the activity of the conjugates, especially CH3-LT, was greatly reduced compared to the LT control ( FIG. 4B ). Since both proteins had been purified by using elution steps at acidic pH (i.e., less than pH4), the pH sensitivity of the culture supernatants was examined, and the LT activity was found to be very acid labile.

[0061] An alternative purification scheme was developed in which the pH was not reduced to below 6.5. The material from this preparation was compared to that purified by protein A, the original starting material, and the LT standard. The results of the LT cytostatic assay, in the absence of mitomycin C, shown in FIG. 5 , demonstrate that full LT activity can be maintained during purification provided low pH is avoided. This assay was used to give a better dose response for the LT control and to demonstrate that the relationship between CH2-LT and CH3-LT is consistent for both assay systems. The same results were obtained in the cytolytic assay.

[0062] The results show that full activity (as measured by this assay) can be maintained when LT is fused to an Ig H chain. The fact that the LT amino terminus is covalently bound to the carboxy-terminus of the antibody apparently does not prevent LT receptor binding or the steps subsequent to binding that are required for activating the cell killing process.

[0063] 7. Antigen Binding and Effector Functions of Ig/LT Immunoconjugates

[0064] The antigen binding activity of the immunoconjugates was measured on antigen-coated plates in either a direct binding or competition assay format. In the direct binding assay antigen binding activity was found to be much higher than that of the control ch14.18 antibody. Since the source of the GD2 antigen was a crude membrane extract from neuroblastoma cells, it is possible that the TNF/LT receptor is present in the preparation and that binding of the conjugate through the LT domain is responsible for this increased activity. When antigen binding was measured in a competition assay, the conjugate was found to compete with the labeled ch14.18 antibody for antigen only slightly more efficiently than the unlabeled ch14.18 antibody ( FIG. 7 ).

[0065] The results show that it is possible to-combine the antigen binding activity of an anti-tumor cell antibody with the potent biological activity of a cytokine. The presence of the CH3 exon in the immunoconjugate results in complete H-chain assembly and, as a consequence, higher LT and effector activities. The assembly of H chains may likely result in LT dimerization.

[0066] In addition, a free amino terminus is not necessary for LT binding to its receptor since in the highly active CH3-LT immunoconjugate, the amino terminus of the LT domain is peptide bonded to the Ig H chain.

[0067] 8. Construction and Expression of Ig/GM-CSF Immunoconjugates

[0068] Ig/GM-CSF conjugates were made by joining a nucleotide sequence encoding GM-CSF to a nucleotide sequence encoding an Ig heavy chain, such that the encoded protein includes a heavy chain fused via the carboxy terminus to GM-CSF. The construct was made as follows. The mature protein coding sequence of GM-CSF was linked to the end of the CH3 exon of the human Cγ ₁ gene using PdHDL2 and appropriate oligonucleotide linkers, as described above for the LT conjugate and according to procedures well-known in the art. Also as described above for LT conjugates, the Ig heavy chain GM-CSF fused gene was combined with the heavy chain V region gene of the 14.18 anti-GD2 heavy chain, and carried on the same vector as the human Cκ gene and the light chain V region gene of the 14.18 antibody. After transfection of the DNA into hybridoma cells and consequent expression of the H and L genes, a complete ch14.18 antibody with GM-CSF attached to the end of each H chain was produced. The fusion protein was purified from conditioned medium using adsorbtion to and elution from protein A Sepharose. The peak material was diafiltered using an Amicon stirred cell into PBS and concentrated to approximately 1mg/mL.

[0069] The fusion protein was analyzed by electrophoresis on a 10% SDS-polyacrylamide gel ( FIG. 8 ) under reducing (R) or non-reducing (NR) conditions and the proteins were visualized by staining with Coomassie Blue. Lane 1, ch14.18-CH3-GMCSF; Lane 2, ch14.18; M, molecular weight markers of the indicated sizes in kD. The relative molecular weight of the fused H chain of 75 kD in lane 1 (R) is consistent with a glycosylated GM-CSF (˜25 kD) being fused to the H chain (50 kD). Note in the non-reduced lane 1 that the fusion protein is assembled into a single high molecular weight species of ˜200 kD.

[0070] 9. Biological Activity of Iq/GM-CSF Conjugates

[0071] The GM-CSF activity of the ch14.18-GM-CSF fusion protein was examined in a proliferation assay using the GM-CSF-dependent cell line AML-193 (human acute myelogenous leukemia) (obtained from Daniel Santoli, Wistar Institute, Philadelphia, Pa.). Cells are cultured for 2 days in serum-free medium containing insulin and transferrin (but no GM-CSF), at which time GM-CSF or fusion protein sample dilutions are added. After five more days, 5 μCi of ³ H-thymidine is added to each well and after 16 hr, the cells are harvested in 10% trichloroacetic acid. After 30 min. on ice the precipitated material is collected on GF/C filters, dried and counted by liquid scintillation.

[0072] In FIG. 9 , the proliferation obtained with varying amounts of GM-CSF, conditioned medium containing the secreted fusion protein, or ch14.18-GM-CSF purified by protein A Sepharose are compared. The results show that significant GM-CSF activity is maintained once the molecule is fused to the H-chain but that the activity is-either 20% (conditioned medium) or 10% that of (purified fusion protein) GM-CSF standard. Maximum incorporation was obtained with less than 10 ng/mL of the purified fusion protein (GM-CSF equivalents or 50 ng of total protein). This slight loss of activity is not likely to affect the utility of-this fusion protein, especially if large amounts of ch14.18-GM-CSF accumulate at the site of solid tumors expressing the GD2 antigen.

[0073] The in vivo half-life of the immunoconjugate was determined by injecting mice (20 μg injected in the tail vein) with ch14.18-GM-CSF. Samples of blood were collected at the indicated times and the amount of fusion protein in the serum was determined by ELISA. The capture antibody was a polyclonal goat anti-human IgG (Fc-specific) and the detecting antibody was a horseradish peroxidase-conjugated goat anti-human K. As seen in Table 1, the half-life (calculated between the 24 hr and 4 day time points) was nearly 3 days. This compares to the published value of 85 min. in humans (Herrmann et al. (1989) J. Clin Oncol. 7:159-167). This increased half-life may compensate for the reduced activity of the fusion protein, especially since the local concentration of the immunoconjugate at the tumor site is likely to be increased by antibody targeting. 2

[0074] 10. Construction, Expression, and Activity of Ig/TNF Immunoconjugates

[0075] Ig/TNF immunoconjugates were made by fusing nucleotide sequences encoding TNFα and immunoglobulin heavy chain such that TNFα is fused to the carboxy terminus of the heavy chain. Briefly, the mature TNFα coding sequence was fused to the end of the human Cγl CH3 exon using oligonucleotides. The recombined fragment was joined downstream of the heavy chain V region encoding gene from the anti-GD2 mouse antibody 14.18; also contained in this vector was the human κ gene, including both the V region gene encoding the light chain V region from the anti-GD2 mouse antibody 14.18 and the C region encoding gene. Hybridoma cells were transfected and selected as described above. Clones secreting human antibody determinants were expanded and used for the production and purification of the ch14.18-CH3-TNFα fusion protein by protein A Sepharose chromatography. The activity of the fusion protein was tested as described above for the CH3-LT fusion proteins.

[0076] As seen in FIG. 10 , the amount of cytotoxicity obtained with the fusion protein met or exceeded that of native TNF( at either early (20 hr) or late (24 hr) points in the assay. This fusion protein appears to be fully functional with respect to TNFα activity, even though it was purified using protein A Sepharose. The CH3-LT construct was partially inactivated by the elution at acidic pH using the same protocol.

[0077] The results described above for the Ig/LT, Ig/GM-CSF, andIg/TNFα immunoconjugates demonstrate that an antibody can be genetically fused to a cytokine without the loss of antigen binding activity and effector functions of the antibody, or the receptor binding and biological activity of a cytokine.

[0078] 11. Dosage

[0079] Immunoconjugates of the invention may be administered at a therapeutically effective dosage within the range of lug-100mg/kg body weight per day. The immunoconjugate may be administered in physiologic saline or any other biologically compatible buffered solution. This solution may be administered systemically (e.g., by injection intravenously or intramuscularly).

[0080] Other Embodiments

[0081] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore considered to be in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.