Bispecific trivalent antibodies binding claudin-6 or claudin-18.2 and CD3 for the treatment of claudin-expressing cancer diseases

Abstract

To provide novel drugs and methods for treatment of cancer diseases.SOLUTION: The present disclosure provides: binding agents comprising at least three binding domains, where a first binding domain binds to a T cell-specific antigen, and where second and third binding domains bind to a claudin; and methods of using these binding agents or nucleic acids encoding the same for treating cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to novel agents and methods for the treatment of cancer diseases. [Background technology]

[0002] Claudins are integral membrane proteins located within epithelial and endothelial tight junctions. Claudins are predicted to have four transmembrane segments with two extracellular loops and cytoplasmic N- and C-termini. The claudin (CLDN) family of transmembrane proteins plays an important role in maintaining epithelial and endothelial tight junctions and may also play a role in cytoskeletal maintenance and cell signaling.

[0003] CLDN18 has been described in mice and humans (Niimi, Mol. Cell. Biol. 21:7380-90, 2001) and exists as two different splice variants. The splice variants (Genbank accession numbers: splice variant 1 (CLDN18.1): NP_057453, NM_016369 and splice variant 2 (CLDN18.2): NM_001002026, NP_001002026) have molecular weights of approximately 27.9 / 27.72 kD. The splice variants CLDN18.1 and CLDN18.2 differ in the N-terminal portion, including the first transmembrane (TM) region and loop 1, but have identical C-terminal primary protein sequences.

[0004] In normal tissues, CLDN18.2 is expressed only in short-lived differentiated gastric epithelial cells, and CLDN18.2 expression is undetectable outside the stomach. CLDN18.2 is maintained during malignant transformation and is therefore frequently displayed on the surface of human gastric cancer cells. This pan-tumor antigen is also ectopically activated at significant levels in esophageal, pancreatic, and lung adenocarcinomas. CLDN18.2 protein has also been localized in lymph node metastases of gastric adenocarcinoma, particularly distant metastases to the ovary (so-called Krukenberg tumors).

[0005] CLDN6 is expressed in a range of different human cancer cells, but its expression in normal tissues is restricted to the placenta.

[0006] The differential expression of claudins, such as CLDN18.2 and CLDN6, between cancer and normal cells, their membrane localization, and their absence in most normal tissues involved in injury make these molecules attractive targets for cancer immunotherapy. The use of antibody-based therapeutics targeting claudins in cancer treatment promises a high level of therapeutic specificity. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent Application Publication No. 2003 / 0118592 [Patent Document 2] U.S. Patent Application Publication No. 2003 / 0133939 [Patent Document 3] International Publication No. 2004 / 035607 [Patent Document 4] International Publication No. 87 / 04462 [Patent Document 5] International Publication No. 89 / 01036 [Patent Document 6] European Patent Application Publication No. 338 841 [Patent Document 7] U.S. Patent Application Publication No. 2009004213 [Non-patent literature]

[0008] [Non-Patent Document 1] Niimi, Mol. Cell. Biol. 21:7380-90, 2001 [Non-patent document 2] Lin and Weiss, Journal of Cell Science 114, pp. 243-244 (2001) [Non-patent document 3] Ward et al. (1989) Nature 341:544-546 [Non-patent document 4] Bird et al. (1988) Science 242:423-426 [Non-patent document 5] Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883 [Non-patent document 6] Chang, C.-H. et al., In: Bispecific Antibodies. Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 199-216 (2011) [Non-Patent Document 7] Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc., 1985). [Non-patent document 8] Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc., 1987). [Non-Patent Document 9] Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985) [Non-Patent Document 10] "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin (editor), pages 303~16 (Academic Press, 1985) [Non-licensed Document 11] Thorpe, "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 pages (1982) [Non-licensed Document 12] Scatchard, Ann NY Acad. ScL, 51:660 pages (1949) [Non-licensed Document 13] Smith and Waterman, 1981, Ads App. Math. 2, page 482 [Non-licensed Document 14] Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443 pages [Non-licensed Document 15] Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, page 2444 [Non-licensed Document 16] Kohler and Milstein, Nature 256: 495 pages [Non-licensed Document 17] Spieker-Polet, Proc. Natl. Acad. Sci. USA 92:9348 (1995) [Non-licensed Document 18] Rossi, Am. J. Clin. Pathol. 124:295 (2005) [Non-licensed Document 19] Morrison, S. (1985) Science, p. 229 [Non-licensed Document 20] Verma, R., (1998) J. Immunol. Meth. 216:165-181 pages [Non-licensed Document 21] Pollock, (1999) J. Immunol. Meth. 231:147-157 pages [Non-licensed Document 22] Fischer, R., (1999) Biol. Chem. 380:825-839 pages [Non-licensed Document 23] Riechmann, L.ら, (1998) Nature 332:323-327 pages [Non-licensed Document 24] Jones, P.ら, (1986) Nature 321:522-525 pages [Non-licensed Document 25] Queen, C., (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033 pages [Non-licensed Document 26] Andersen, 2008: Cancer treatment: the combination of vaccination with other therapies. Cancer Immunology Immunotherapy, 57(11):1735-1743 [Non-licensed Document 27] Quoixら, 2011: Therapeutic vaccination with TG4010 and first-line chemotherapy in advanced non-small-cell lung cancer: a controlled phase 2B trial. Lancet Oncol. 12(12):1125-33 pages [Non-licensed Document 28] Liseth et al., 2010:Combination of intensive chemotherapy and anticancer vaccines in the treatment of human malignancies:the hematological experience. J Biomed Biotechnol. 2010:6920979 pages [Non-Patent Document 29] Hirooka et al., 2009:A combination therapy of gemcitabine with immunotherapy for patients with inoperable locally advanced pancreatic cancer. Pancreas 38(3):pp.e69-74 [Non-Patent Document 30] Baskar et al., 2012: Cancer and radiation therapy: current advances and future directions. Int. J Med Sci. 9(3):193-199 [Non-Patent Document 31] Gadri et al., 2009: Synergistic effect of dendritic cell vaccination and anti-CD20 antibody treatment in the therapy of murine lymphoma. J Immunother. 32(4):333-40 [Non-Patent Document 32] Lechner et al., 2011: Chemokines, costimulatory molecules and fusion proteins for the immunotherapy of solid tumors. Immunotherapy 3(11), pp. 1317-1340 [Non-Patent Document 33] Zhang et al., 2009: Targeting cancer with small molecule kinase inhibitors. Nature Reviews Cancer 9, pp. 28-39 [Non-Patent Document 34] van Duin et al., 2005: Triggering TLR signaling in vaccination. Trends in Immunology, 27(1):49-55 [Non-Patent Document 35] Schoenfeld and Dranoff 2011: Anti-angiogenesis immunotherapy. Hum Vaccin. (9): pp. 976-81 [Non-Patent Document 36] Sorensen and Thompsen 2007:Virus-based immunotherapy of cancer:what do we know and where are we going? APMIS 115(11):1177-93 [Non-Patent Document 37] Buonaguro et al., 2011:Developments in virus-like particle-based vaccines for infectious diseases and cancer. 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Nihon Rinsho 69(9):1657-61 Summary of the Invention [Problem to be solved by the invention]

[0009] The object of the present invention is to provide novel agents and methods for the treatment of cancer diseases.

[0010] The solution to the problem underlying the present invention is based on the concept of generating tumor-associated claudin molecules, i.e., binding agents containing two binding domains specific for cancer cells. The binding agent contains a binding domain specific for a T cell-specific antigen, such as CD3, and can bind to T cells, attracting them to a complex and thus targeting the cytotoxic effect of the T cells against cancer cells. The formation of this complex, either alone or in combination with accessory cells, can induce signaling in cytotoxic T cells, leading to the release of cytotoxic mediators.

[0011] We report for the first time that a binding factor containing two binding domains targeting claudins and another binding domain targeting a T cell-specific antigen such as CD3 can induce potent T cell-mediated lysis and is effective in treating tumor diseases. [Means for solving the problem]

[0012] The present invention provides a binding agent comprising at least three binding domains, wherein a first binding domain binds to a T cell-specific antigen, and a second binding domain and a third binding domain bind to a claudin. The binding agent of the present invention can bind to T cells (e.g., by engaging the CD3 receptor) and can bind to cancer cells expressing claudins to be targeted for destruction.

[0013] In one embodiment, the binding agent comprises six antibody variable domains with at least three binding domains, where at least two binding domains bind to a claudin and at least one binding domain binds to a T cell-specific antigen.

[0014] In one embodiment, each of the first, second and third binding domains comprises a variable domain of an immunoglobulin heavy chain (VH) and a variable domain of an immunoglobulin light chain (VL).

[0015] In one embodiment, the first binding domain comprises a heavy chain variable (VH) domain of an immunoglobulin with specificity for a T cell-specific antigen (VH(T)) and a light chain variable (VL) domain of an immunoglobulin with specificity for a T cell-specific antigen (VL(T)), and the second binding domain and the third binding domain each comprise a heavy chain variable (VH) domain of an immunoglobulin with specificity for a claudin antigen (VH(CLDN)) and a light chain variable (VL) domain of an immunoglobulin with specificity for a claudin antigen (VL(CLDN)).

[0016] In one embodiment, the heavy chain variable domain (VH) and the corresponding light chain variable domain (VL) of one or more binding domains are connected via a peptide linker, in particular a flexible peptide linker such as a glycine-serine peptide linker. In one embodiment, the peptide linker has the amino acid sequence (G4S) x (where x is 3, 4, 5 or 6).

[0017] In one embodiment, the heavy chain variable domain (VH) and the corresponding light chain variable domain (VL) of one or more binding domains have the format of a Fab and / or scFv molecule.

[0018] In one embodiment, the first binding domain has the format of a Fab molecule and / or the second and third binding domains have the format of an scFv molecule.

[0019] In one embodiment, a binding agent of the invention is a dimer composed of two polypeptide chains, where a first polypeptide comprises an scFv linked to an additional VL domain through the constant domain (CL) of an immunoglobulin light chain, and a second polypeptide comprises an scFv linked to an additional VH domain through constant domain 1 (CH1) of an immunoglobulin heavy chain. The two polypeptide chains are preferably linked together by a disulfide bridge. The disulfide bridge is preferably formed between a Cys residue in the CL domain and a Cys residue in the CH1 domain, such that in the antigen-binding configuration, the additional VL domain of the first polypeptide and the additional VH domain of the second polypeptide associate, and the binding agent as a whole comprises three antigen-binding domains. According to the invention, the VH and VL domains of the scFv moiety preferably have the amino acid sequence (G4S) x (where x is 3, 4, 5, or 6), and the Fab chain and scFv are preferably linked by a peptide linker, such as a peptide linker comprising the amino acid sequence DVPG2S or SGPG3RS(G4S)2. In one embodiment, the scFv portion binds to a claudin, and the Fab portion binds to a T cell-specific antigen.

[0020] In one embodiment, the first binding domain is constituted by a Fab fragment, and the second and third binding domains are each constituted by an scFv, wherein each chain of the Fab fragment is linked to one scFv, and the scFv is preferably linked to the C-terminus of the Fab fragment.

[0021] In one embodiment, the binding agent comprises first and second polypeptides, wherein the first and second polypeptides comprise a VH domain with specificity for a T cell-specific antigen (VH(T)), a VL domain with specificity for a T cell-specific antigen (VL(T)), a first VH domain with specificity for a claudin (VH(CLDN)), a second VH domain with specificity for a claudin (VH(CLDN)), a first VL domain with specificity for a claudin (VL(CLDN)), and a second VL domain with specificity for a claudin (VL(CLDN)), wherein the first and second polypeptides associate to form the binding agent.

[0022] In one embodiment, the binding agent is (a) a first polypeptide comprising a VH domain (VH(T)) having specificity for a T cell-specific antigen, a VH domain (VH(CLDN)) having specificity for a claudin, and a VL domain (VL(CLDN)) having specificity for a claudin; (b) a second polypeptide comprising a VL domain (VL(T)) with specificity for a T cell-specific antigen, a VH domain (VH(CLDN)) with specificity for a claudin, and a VL domain (VL(CLDN)) with specificity for a claudin; wherein the first polypeptide and the second polypeptide associate to form a binding agent.

[0023] In one embodiment, the first polypeptide further comprises a constant domain 1 (CH1) of an immunoglobulin heavy chain and the second polypeptide further comprises a constant domain (CL) of an immunoglobulin light chain, wherein both domains can associate.

[0024] In one embodiment, the first and second polypeptides are covalently linked via a disulfide bridge between the CH1 and CL domains.

[0025] In one embodiment of the binding agent of the invention, in the first polypeptide and the second polypeptide, the VH domain, VL domain, CH1 domain and CL domain are arranged from N-terminus to C-terminus as follows: VH(T)-CH1-VH(CLDN)-VL(CLDN) and VL(T)-CL-VH(CLDN)-VL(CLDN), or VH(T)-CH1-VL(CLDN)-VH(CLDN) and VL(T)-CL-VL(CLDN)-VH(CLDN), or VH(T)-CH1-VH(CLDN)-VL-(CLDN) and VL(T)-CL-VL(CLDN)-VH-(CLDN), or VH(T)-CH1-VL(CLDN)-VH-(CLDN) and VL(T)-CL-VH(CLDN)-VL-(CLDN), or VH(CLDN)-CH1-VH(T)-VL(T) and VL(CLDN)-CL-VH(CLDN)-VL(CLDN), or - VH(CLDN)-CH1-VL(T)-VH(T) and VL(CLDN)-CL-VL(CLDN)-VH(CLDN), or - VH(CLDN)-CH1-VL(T)-VH(T) and VL(CLDN)-CL-VH(CLDN)-VL(CLDN), or VH(CLDN)-CH1-VH(T)-VL(T) and VL(CLDN)-CL-VL(CLDN)-VH(CLDN), or - VH(CLDN)-CH1-VH(CLDN)-VL(CLDN) and VL(CLDN)-CL-VH(T)-VL(T), or - VH(CLDN)-CH1-VL(CLDN)-VH(CLDN) and VL(CLDN)-CL-VL(T)-VH(T), or - VH(CLDN)-CH1-VL(CLDN)-VH(CLDN) and VL(CLDN)-CL-VH(T)-VL(T), or - VH(CLDN)-CH1-VH(CLDN)-VL(CLDN) and VL(CLDN)-CL-VL(T)-VH(T) are arranged in the order of

[0026] In one embodiment, the N-terminal most VH domain of one chain associates with the N-terminal most VL domain of the other chain to form a binding domain, and each of the VH-VL or VL-VH domains within a chain forms a binding domain.

[0027] In one embodiment, the VH-VL or VL-VH domain is linked to the CH1 domain or CL domain via a peptide linker, such as a peptide linker comprising the amino acid sequence DVPG2S or SGPG3RS(G4S)2.

[0028] In one embodiment, the VH and VL domains have the amino acid sequence (G4S) x (where x is 3, 4, 5, or 6) to form a VH-VL or VL-VH domain.

[0029] In the binding agents of the present invention, a VH domain (VH(T)) with specificity for a T cell-specific antigen and a VL domain (VL(T)) with specificity for a T cell-specific antigen can associate to form a binding domain that binds to the T cell-specific antigen.

[0030] Furthermore, in the binding factors of the present invention, the VH domain (VH(CLDN)) with specificity for a claudin and the VL domain (VL(CLDN)) with specificity for a claudin can associate to form a binding domain that binds to a claudin. According to the present invention, the VH domain (VH(CLDN)) with specificity for a claudin and the VL domain (VL(CLDN)) with specificity for a claudin can be the same or different. In the case of binding factors of the present invention comprising different VH domains with specificity for a claudin (VH(CLDN), VH(CLDN)*) and / or different VL domains with specificity for a claudin (VL(CLDN), VL(CLDN)*), the VH(CLDN) and VL(CLDN) can associate to form a first binding domain that binds to a claudin, and the VH(CLDN)* and VL(CLDN)* can associate to form a second binding domain that binds to a claudin.

[0031] In one embodiment, a binding agent of the invention is a bispecific dimeric binding agent.

[0032] In one embodiment, the T cell specific antigen is one that is expressed on the surface of a T cell.

[0033] In one embodiment, binding of the binding agent to a T cell-specific antigen on a T cell leads to proliferation and / or activation of the T cell.

[0034] In one embodiment, the T cell-specific antigen is CD3.

[0035] In one embodiment, the first binding domain binds to the epsilon chain of CD3.

[0036] In one embodiment, CD3 is expressed on the surface of a T cell. In one embodiment, binding of a binding agent to CD3 on a T cell leads to proliferation and / or activation of the T cell, wherein the T cell preferably releases cytotoxic factors, such as perforin and granzymes, to initiate cytolysis and apoptosis of cancer cells.

[0037] In one embodiment, the claudin is expressed on the surface of a cancer cell.

[0038] In one embodiment, the claudin is selected from the group consisting of claudin 6 and claudin 18.2.

[0039] In one embodiment, the binding agent binds to the extracellular domain of a claudin.

[0040] In one embodiment, the binding agent of the present invention binds to a native epitope of a claudin present on the surface of a living cell, hi one embodiment, the binding agent binds to the first extracellular loop of the claudin.

[0041] In one embodiment, the binding to a T cell-specific antigen and / or the binding to a claudin is specific binding.

[0042] In one embodiment, the binding agent induces T cell-mediated cytotoxicity against cancer cells that express claudins.

[0043] In one embodiment, the binding agent induces T cell-mediated cytotoxicity against claudin-expressing cancer cells with an EC50 of ≦10 nM, or ≦1 nM, or ≦500 pM, or ≦250 pM, or ≦100 pM, or ≦50 pM.

[0044] In one embodiment, the claudin is claudin 6 and the cancer cells are selected from the group consisting of bladder cancer, ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly lung cancer including lung squamous cell carcinoma and adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, particularly basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, particularly malignant pleomorphic adenoma, sarcoma, particularly synovial sarcoma and carcinosarcoma, bile duct cancer, and bladder cancer. , in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in particular renal cell carcinoma including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer including cancer of the ileum, in particular small intestinal adenocarcinoma and adenocarcinoma of the ileum, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, in particular testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors such as teratocarcinoma or embryonal carcinoma, in particular germ cell tumors of the testis, and metastatic forms thereof.

[0045] In one embodiment, the claudin is claudin 18.2 and the cancer cells are derived from a cancer selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and metastases thereof, Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis.

[0046] In one embodiment, the T cell-specific antigen is CD3, and VH(T) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5 or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or VL(T) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6 or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0047] In one embodiment, the claudin is claudin 6, and VH(CLDN) comprises or consists of the amino acid sequence represented by SEQ ID NO: 8 or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or VL(CLDN) comprises or consists of the amino acid sequence represented by SEQ ID NO: 10 or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0048] In one embodiment, the T cell-specific antigen is CD3 and the claudin is claudin 6; - the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14 or a fragment thereof or a variant of said amino acid sequence or fragment, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 or a fragment thereof or a variant of said amino acid sequence or fragment; - the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 or a fragment thereof or a variant of said amino acid sequence or fragment, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 or a fragment thereof or a variant of said amino acid sequence or fragment; - the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17 or a fragment thereof or a variant of said amino acid sequence or fragment, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or a fragment thereof or a variant of said amino acid sequence or fragment; or - the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18 or a fragment thereof or a variant of said amino acid sequence or fragment, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or a fragment thereof or a variant of said amino acid sequence or fragment.

[0049] In one embodiment, the claudin is claudin 18.2, and (i) VH(CLDN) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or VL(CLDN) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or a fragment thereof, or a variant of said amino acid sequence or fragment; or (ii) VH(CLDN) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or VL(CLDN) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0050] In one embodiment, the T cell-specific antigen is CD3 and the claudin is claudin 18.2; - the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30 or a fragment thereof or a variant of said amino acid sequence or fragment, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32 or a fragment thereof or a variant of said amino acid sequence or fragment; - the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31 or a fragment thereof or a variant of said amino acid sequence or fragment, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32 or a fragment thereof or a variant of said amino acid sequence or fragment; - the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33 or a fragment thereof or a variant of said amino acid sequence or fragment, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35 or a fragment thereof or a variant of said amino acid sequence or fragment; - the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34 or a fragment thereof or a variant of said amino acid sequence or fragment, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35 or a fragment thereof or a variant of said amino acid sequence or fragment; - the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36 or a fragment thereof or a variant of said amino acid sequence or fragment, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37 or a fragment thereof or a variant of said amino acid sequence or fragment; - the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38 or a fragment thereof or a variant of said amino acid sequence or fragment, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39 or a fragment thereof or a variant of said amino acid sequence or fragment; - the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40 or a fragment thereof or a variant of said amino acid sequence or fragment, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41 or a fragment thereof or a variant of said amino acid sequence or fragment; - the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 42 or a fragment thereof or a variant of said amino acid sequence or fragment, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43 or a fragment thereof or a variant of said amino acid sequence or fragment.

[0051] In different embodiments, the binding agent of the invention or one or more polypeptide chains of a binding agent of the invention comprises or does not comprise a secretory signal, such as an N-terminal secretory signal, in particular an immunoglobulin, e.g. an IgG secretory signal, such as the sequence MGWSCIILFLVATATGVHS, and / or comprises or does not comprise a tag, in particular a C-terminal tag, such as a His tag, in particular the sequence Gly-Gly-Ser-(His)6 or (His)6 or a Strep tag.

[0052] The invention also provides nucleic acids encoding the binding agents of the invention.

[0053] The present invention also provides nucleic acids encoding the first and / or second polypeptides defined herein.

[0054] In one embodiment, the nucleic acid of the present invention is in the form of a vector or in the form of RNA.

[0055] In one embodiment, the nucleic acid of the invention is a recombinant nucleic acid.

[0056] The present invention also provides a host cell comprising a nucleic acid of the present invention.

[0057] The present invention also provides a binding agent of the present invention, a nucleic acid of the present invention, or a host cell of the present invention for use as a pharmaceutical.

[0058] The present invention also provides a binding agent of the present invention, a nucleic acid of the present invention, or a host cell of the present invention for use in treating or preventing cancer.

[0059] The present invention also provides pharmaceutical compositions comprising a binding agent of the present invention, a nucleic acid of the present invention, or a host cell of the present invention.

[0060] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0061] The present invention also provides a method of treating a disease, comprising administering a binding agent of the present invention, a nucleic acid of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention to a subject in need thereof. In one embodiment, the disease is cancer.

[0062] The present invention also provides a method for treating or preventing cancer, comprising the step of administering a binding agent of the present invention, a nucleic acid of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention to a subject in need thereof.

[0063] The present invention also provides use of a binding agent of the present invention, a nucleic acid of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention for the manufacture of a medicament. In one embodiment, the medicament is for the treatment of cancer.

[0064] In one embodiment, cells of the cancer express a claudin to which the binding agent can bind.

[0065] In one embodiment, the claudin is claudin 6 and the cancer is selected from the group consisting of bladder cancer, ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly lung cancer including lung squamous cell carcinoma and adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, particularly basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, particularly malignant pleomorphic adenoma, sarcoma, particularly synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, urinary tract ... The cancer is selected from the group consisting of cancer of the bladder, particularly transitional cell carcinoma and papillary carcinoma, kidney cancer, particularly renal cell carcinoma including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer including cancer of the ileum, particularly small intestinal adenocarcinoma and ileal adenocarcinoma, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, particularly testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors such as teratocarcinoma or embryonal carcinoma, particularly testicular germ cell tumors, and metastatic forms thereof.

[0066] In one embodiment, the claudin is claudin 18.2, and the cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and metastases thereof, Krukenberg tumor, peritoneal metastasis, and / or lymph node metastasis.

[0067] The invention also provides a binding agent, nucleic acid, or host cell described herein for use in a method of treatment described herein. In one embodiment, the invention provides a pharmaceutical composition described herein for use in a method of treatment described herein.

[0068] According to the present invention, claudin 18.2 preferably has the amino acid sequence according to SEQ ID NO: 1, and claudin 6 preferably has the amino acid sequence according to SEQ ID NO: 2 or 3.

[0069] Other features and advantages of the invention will be apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0070] [Figure 1A] Figures 1A and 1B show a modular scheme for the DNA construct and bstb protein targeting the TAA CLDN6. (A) Design of the bstb chain at the DNA level. (B) Schematic model of the bstb molecule. CH1 is derived from IgG1 of bstb_369 / 367 and IgG2 of bstb_371 / 367. C: constant region; CMV: cytomegalovirus promoter; Fd: digestible fragment / heavy chain portion of Fab (antigen-binding fragment); H: heavy chain; His: 6xHis tag; L: light chain; L1: SGPG3RS(G4S)2 linker; L2: DVPG2S linker; L3: (G4S)4 linker; SS: disulfide bridge; scFv: single-chain variable fragment; Sec: secretion signal; Strp: Strep tag; and V: variable domain. [Figure 1B] See Figure 1A. [Figure 2A]Figures 2A-2D show the purification of tagged and untagged bstb proteins from cell culture supernatants. Expi293F™ cells were transiently transfected with each bstb construct. Supernatants were collected 7 days post-transfection and purified. (A) Chromatogram showing the first purification step of tagged bstb via Ni-NTA affinity chromatography (immobilized metal ion affinity chromatography - IMAC). mAU (milli-absorbency units) on the y-axis are plotted against volume (ml) on the x-axis. On the left are the IMAC peaks for bstb_369 / 367, and on the right are the IMAC peaks for bstb_371 / 367. Each right peak contains HMW species, and each center peak contains monomeric species. Each left peak represents an impurity. 1) Identifies the pooled fractions as the main peak containing primarily monomeric species, and 2) identifies the pooled fractions as HMW species. (B) Chromatogram showing the second purification step of tagged bstb. The pool of the IMAC main peak was subjected to Strep-Tactin® affinity chromatography. The plot on the left is the bstb_369 / 367 peak, and the plot on the right is the bstb_371 / 367 peak. (C) Separation of the HMW and monomeric species of bstb_369 / 367 by size exclusion chromatography (SEC). The pooled HMW and pooled monomeric fractions are identified in square brackets. (D) SE-HPLC analysis of untagged bstb_5726 / 5725 after purification. mAU (milli-absorbance units) on the y-axis are plotted against time (minutes) on the x-axis. bstb denotes bispecific TriMAB; HMW denotes high molecular weight species; LMW denotes low molecular weight species. [Figure 2B] See Figure 2A. [Figure 2C] See Figure 2A. [Figure 2D] See Figure 2A. [Figure 3A]Figures 3A and 3B show SDS-PAGE analysis of the CLDN6 x CD3 protein bstb_369 / 367. Supernatant from Expi293F™ cells transiently expressing bstb_369 / 367 was purified via IMAC. HMW species eluted separately from the main peak. The main peak species was then subjected to Strep-Tactin® affinity chromatography. The eluted pool was further separated by SEC to collect highly monomeric bstb. Aliquots of cell culture supernatant, reference, and different purification steps were loaded onto a 4-15% Tris-glycine stain-free gel under non-reducing (left) and reducing (right) conditions. (A) Bands were visualized by fluorescence on the stain-free gel. (B) Western blot analysis using an anti-His tag detection antibody (top blot) or StrepMAB detection antibody. The left arrow indicates the non-reduced monomer and HMW, and the right arrow indicates the reduced bstb chain. Fd: digestible fragment / heavy chain portion of Fab (antigen-binding fragment); HMW: high molecular weight species; His: 6xHis tag; IMAC: immobilized metal affinity chromatography; L: light chain; scFv: single-chain variable fragment; SEC: size-exclusion chromatography; Strp: Strep tag. [Figure 3B] See Figure 3A. [Figure 4A]Figures 4A-4D show in vitro cytotoxicity assays to determine specific lysis mediated by the CLDN6 x CD3 proteins bstb_369 / 367 and bstb_371 / 367. Specific concentration-dependent lysis was determined using human PBMCs as effector cells and human stably luciferase-transduced cancer cells as target cells at a 5:1 effector:target ratio in luciferase-based cytotoxicity assays. CLDN6+ cell lines PA-1 (ovarian teratocarcinoma) and / or OV-90 (ovarian cancer) were used as positive targets, and the CDLN6- cell line MDA-MB-231 (breast cancer) was used as a negative target. Mean values ​​from triplicates are shown, including standard deviations. Half-maximal lysis values ​​(EC50) are indicated below the corresponding graphs. (A) Specific lysis (standard gradient) mediated by bstb_369 / 367 and bstb_371 / 367 of CLDN6+ ovarian cancer cells OV-90 after 48 hours of incubation. (B) Comparison of specific lysis (standard gradient) mediated by bstb_369 / 367 and the CDLN6×CD3-specific bi(scFv)2 reference protein. Cell lines and incubation times are shown on a single graph. (C) Specific lysis (variable gradient) of bstb_369 / 367 and the effect of 5% HMW on the activity of bstb_369 / 367. Left: 16 hours of incubation with PA-1; Right: 48 hours of incubation with OV-90. The solid line shows the lysis curve for monomeric bstb_369 / 367, and the dotted line shows the lysis curve for monomeric bstb_369 / 367 spiked with 5% HMW species. (D) Specific lysis of OV-90 after 48 h of incubation (standard gradient). Left plot: lysis mediated by bstb_369 / 367 as monomers and untagged analogues bstb_5726 / 5725 spiked with 5% HMW. Right plot: lysis mediated by bstb_369 / 367 as reference and CH1 (IgG2)-bearing variant bstb_5727 / 5725 as monomers spiked with 5% HMW. bstb indicates bispecific TriMAB; bi(scFv)2 indicates bispecific single-chain variable fragment; EC50 indicates half-maximal effective concentration; HMW indicates high molecular weight species. [Figure 4B] See Figure 4A. [Figure 4C] See Figure 4A. [Figure 4D] See Figure 4A. [Figure 5A] Figures 5A and 5B show target-dependent T cell modulation mediated by the CLDN6 × CD3 proteins bstb_369 / 367 and bstb_371 / 367. CLDN6+ OV-90 and PA-1, and CLDN6- MDA-MB-231 cancer cell lines were used as target cells. Human PBMCs were used as effector cells at an E:T ratio of 5:1. Anti-CD3 IgG2a OKT3 was applied at a concentration of 100 ng / ml as an activation control. Mock samples were incubated with DPBS, and background signals were subtracted from the analyzed sample values. PBMCs without target cells were used as an additional specificity control. All samples were set up in duplicate in a 24-well format. Increasing concentrations (0.005–5000 ng / ml) of the bstb proteins bstb_369 / 367 or bstb_371 / 367 were applied. (A) T cell activation: PBMCs were collected after 48 hours of co-incubation and labeled with anti-CD5-PE-Cy7, anti-CD25-PE, anti-CD69-APC, and eFluor506, and live T cell activation was analyzed by flow cytometry. (B) T cell proliferation: Human PBMCs were CFSE stained prior to assay setup. PBMCs were collected after 72 hours of co-incubation and labeled with anti-CD5-APC and eFluor506 to exclude non-lymphoid and dead cells. The decrease in CFSE signal, indicative of T cell proliferation, was analyzed by flow cytometry. bstb indicates bispecific TriMAB; ctrl indicates control. [Figure 5B] See Figure 5A. [Figure 6A]Figures 6A and 6B show the binding of various CLDN6 x CD3 bispecific antibodies to the tumor-specific antigen CLDN6. (A) The relative binding affinities of the bispecific (scFv)2 reference, bstb_369 / 367, and bstb_5726 / 5725, were examined by flow cytometry on endogenously CLDN6-expressing PA-1 human cancer cells over a concentration range of 9.77 ng / ml to 10 μg / ml. Primary antibodies were detected with protein L-FITC (4 μg / ml). Data are shown as mean ± standard deviation (n = 2 replicates). (B) The effect of high molecular weight (HMW) species on the binding of monomeric bstb_5726 / 5725 was analyzed by flow cytometry using monomeric bstb_5726 / 5725 or monomeric bstb_5726 / 5725 spiked with approximately 3% or 5% HMW species (concentration range 9.77 ng / ml to 10 μg / ml). Primary antibodies were detected with protein L-FITC (4 μg / ml). Data are shown as mean ± standard deviation (n = 2 replicates). bstb is bispecific TriMAB; MFI is median fluorescence intensity. [Figure 6B] See Figure 6A. [Figure 7A]Figures 7A-7C show the in vivo efficacy of the CLDN6 × CD3 protein bstb_369 / 367 in a mouse xenograft tumor model. Male and female immunodeficient NSG mice were used. (A) Injection schedule scheme. Mice were subcutaneously inoculated with CLDN6+ human ovarian cancer cells OV-90 as target cells and engrafted intraperitoneally (ip) with human PBMCs as effector cells. Treatment began at a mean tumor volume of approximately 35 mm3 per group and was administered ip three times per week. Group 1 (G1) received the vehicle buffer DPBS, Group 2 (G2) received a low dose of bstb_369 / 367 at 31 μg / kg, and Group 3 (G3) received a high dose of 308 μg / kg. (B) Tumor growth over time for all mice and groups. Treatment was administered ip during the periods highlighted by the boxes. The upper panel is the vehicle group G1; the lower left panel is the bstb_369 / 367 low-dose group G2, and the lower right panel is the bstb_369 / 367 high-dose group G3. Each line represents an individual mouse. (C) Kaplan-Meier survival plot for all groups from the start of treatment to the day of euthanasia. The table below shows the median survival time per group and the significance of survival in G2 and G3 compared to G1 by log-rank (Mantel-Cox) test. bstb = bispecific TriMAB; d = number of days; G = group; GVHD = graft-versus-host disease; ip = intraperitoneal; PBMC = peripheral blood mononuclear cells; sc = subcutaneous. [Figure 7B] See Figure 7A. [Figure 7C] See Figure 7A. [Figure 8] Estimation of in vivo pharmacokinetics of CLDN6xCD3 protein bstb_369 / 367. 5 mg / kg bstb_369 / 367 was injected i.p. into female immunodeficient NSG mice on day 0. DPBS injection ("0 hours") served as a pre-injection control. Plasma was collected at 0.25, 1, 2, 3, 6, and 8 hours after injection. bstb_369 / 367 plasma concentrations were detected by ELISA. Concentrations are plotted on a logarithmic scale on the y-axis. Each point represents the mean and standard deviation of three mice. i.p. indicates intraperitoneal. [Figure 9A]Figure 1 shows in vivo dosing findings using the CLDN6 × CD3-bstb protein bstb_5726 / 5725 in a mouse xenograft tumor model. Male and female immunodeficient NSG mice were inoculated subcutaneously with CLDN6+ human ovarian cancer cells OV-90 as target cells and engrafted intraperitoneally (ip) with human PBMCs as effector cells. Treatment began at a mean tumor volume of approximately 150 mm3 per group, with three ip applications per week. (A) Injection schedule scheme. (B) Tumor growth plot. Doses for groups (n = 6) are indicated by individual plots. The control group G7 (n = 8) received DPBS. The plot shows tumor growth over time for all mice and groups. Treatment periods are highlighted by boxes. Each line represents an individual mouse (mouse ID = BIO-####). bstb: bispecific TriMAB; DPBS: Dulbecco's phosphate buffered saline; ip: intraperitoneal application; PBMC: peripheral blood mononuclear cells; sc: subcutaneous. [Figure 9B] See Figure 9A. [Figure 10A]Figures 10A and 10B show a modular scheme depicting DNA constructs and bstb proteins targeting the TAA CLDN18.2. (A) Design of the bstb chain at the DNA level. The anti-CLDN18.2-scFv is oriented in either VH-VL order (upper scheme) or VL-VH order (lower scheme). Constructs were designed with or without disulfide bridges (SS) ("+ / -"). (B) Theoretical models of exemplary bstb molecules carrying anti-CLDN18.2 scFv in VH-VL or VL-VH order, with tags but without disulfide bridges (SS) in the scFv portion (upper diagram) and without tags but with SS (lower diagram). CH1 is derived from IgG1 or IgG2. C: constant region; CMV: cytomegalovirus promoter; Fd: digestible fragment / heavy chain portion of Fab (antigen-binding fragment); H: heavy chain; His: 6xHis tag; L: light chain; L1: SGPG3RS(G4S)2 linker; L2: DVPG2S linker; L3: (G4S)4 linker; L4: (G4S)5 linker; SS: disulfide bridge; scFv: single-chain variable fragment; Sec: secretion signal; Strp: Strep tag; V: variable domain. [Figure 10B] See Figure 10A. [Figure 11]Figure 1 shows an in vitro cytotoxicity assay comparing specific lysis mediated by CLDN18.2 × CD3-bstb proteins. Human PBMCs were used as effector cells and human stably luciferase-transduced cancer cells as target cells at an effector:target ratio of 5:1 in a luciferase-based cytotoxicity assay. IMAC- and Strep-Tactin®-purified bstb test items were used without further enrichment of monomeric species via SEC. Left graph: Specific and concentration-dependent lysis of CLDN18.2+ gastric cancer cells NUGC-4_hCLDN18.2 mediated by bstb_5730 / 5728, bstb_5731 / 5729, bstb_5732 / 5728, and bstb_5733 / 5729 after 48 hours of incubation. Right graph: Lysis of CDLN18.2-control cell line MDA-MB-231. Mean values ​​of triplicates are shown with standard deviations. bstb indicates bispecific TriMAB. [Figure 12A]Figures 12A and 12B show SE-HPLC analysis of various purified CLDN18.2xCD3-bstb proteins. Expi293F™ cells were transiently transfected with bstb_5745 / 5747, bstb_5749 / 5751, bstb_5746 / 5748, or bstb_5750 / 5752 constructs. Supernatants were collected 7 days post-transfection and subjected to purification. (A) SE-HPLC analysis of bstb_5745 / 5747 (upper plot) and bstb_5749 / 5751 (lower plot) after purification. mAU (milli-absorbance units) on the y-axis are plotted against time (minutes) on the x-axis. The monomer content is greatly increased in bstb_5749 / 5751, which contains a residual disulfide bond within the anti-CLDN18.2 scFv portion. (B) SE-HPLC analysis of bstb_5746 / 5748 (upper plot) and bstb_5750 / 5752 (lower plot) after purification (lower plot). mAU (milli-absorbance units) on the y-axis are plotted against time (minutes) on the x-axis. The monomer content is greatly increased in bstb_5749 / 5751, which contains a residual disulfide bond within the anti-CLDN18.2 scFv portion. bstb: bispecific TriMAB; HMW: high molecular weight species; LMW: low molecular weight species. [Figure 12B] See Figure 12A. [Figure 13A]Figures 13A and 13B show in vitro cytotoxicity assays comparing the specific lysis mediated by highly monomeric disulfide bridge-containing CLDN18.2 x CD3-bstb and dual (scFv)2 proteins. Human PBMCs were used as effector cells and human stably luciferase-transduced CLDN18.2+ gastric cancer cells NUGC-4_hCLDN18.2 as target cells at an effector:target ratio of 5:1 in the luciferase-based cytotoxicity assay. Monomeric bstb test items isolated by SEC and their dual (scFv)2 protein analogs were used in 10-point, 5-fold serial dilutions. (A) Concentration-dependent specific lysis mediated by bstb_5749 / 5751 and bstb_5750 / 5752 (left graph) and dual scFv_5506 and dual scFv_5538 (right graph) after 48 hours of incubation. EC50 values ​​are summarized in the table below. The EC50 value of "plate reference" was used for normalization. (B) Fold difference of bivalent bstb compared to the relevant bi(scFv)2 analog after normalization to "plate reference". The relevant bi(scFv)2 EC50 value was set to 1 for calculations. Bi-scFv is bispecific single-chain variable fragment; bstb is bispecific TriMAB; EC50 indicates half-maximal effective concentration. [Figure 13B] See Figure 13A. [Figure 14] Figure 1 shows a modular scheme illustrating the RNA construct for encoding CLDN6xCD3bstb. Design of the bstb Fd chain (upper panel) and L chain (lower panel) at the RNA level. C indicates the constant region; CMV indicates the cytomegalovirus promoter; Fd indicates the heavy chain portion of the digestible fragment / Fab (antigen-binding fragment); H indicates the heavy chain; His indicates a 6xHis tag; L indicates the light chain; L1 indicates the SGPG3RS(G4S)2 linker; L2 indicates the DVPG2S linker; L3 indicates the (G4S)4 linker; SS indicates a disulfide bridge; scFv indicates a single-chain variable fragment; Sec indicates a secretion signal; Strp indicates a Strep tag; and V indicates a variable domain. [Figure 15A]Figures 15A-15C show Western blot analysis of IVT-mRNA-encoded CLDN6xCD3 bstb_435 / 434 and bstb_436 / 434 in producer cells. The human chronic myeloid leukemia cell line K-562 was transiently transfected via electroporation with equal amounts of IVT-mRNA for Fd- and L-chains or HO alone (mock). Forty-eight hours after electroporation, K-562 supernatants were collected, and cell lysates were generated. As references, purified protein analogs bstb_369 / 367 (A) or bstb_371 / 367 (B) were loaded onto the gel as monomers and HMW preparations. Gradient SDS-PAGE and Western blot analysis were performed to detect the translated and purified protein products. Both bstb variants ((A) bstb_435 / 434, (B) bstb_436 / 434) were detected in K-562 supernatants and cell lysates with anti-6xHis-tag HRP (Fd portion) and anti-Strep-MAB-HRP (L portion). Anti-β-actin immunoblotting was used as a loading control for cell lysates. Samples were loaded under non-reducing and reducing conditions as indicated in the attached sample loading table. Arrowheads indicate the protein bands of interest. (A) bstb_435 / 434, (B) bstb_436 / 434. In (C), both bstb variants were detected under reducing conditions with a mixture of anti-6xHis-tag HRP and anti-Strep-MAB-HRP, and the heterodimeric state of the antibody derivatives was visualized. bstb: bispecific TriMAB; Fd: digestible fragment / heavy chain portion of Fab (antigen-binding fragment); His: 6xHis tag; HMW: high molecular weight species; HRP: horseradish peroxidase; L: light chain portion of bstb; scFv: single-chain variable fragment; SN: supernatant. [Figure 15B] See Figure 15A. [Figure 15C] See Figure 15A. [Figure 16]Figure 1 shows an in vitro cytotoxicity assay to determine specific lysis mediated by CLDN6xCD3 bstb_435 / 434 and bstb_436 / 434. Human PBMCs were used as effector cells and human stably luciferase-transduced cancer cells as target cells at an effector:target ratio of 5:1 in a luciferase-based cytotoxicity assay. Seven-point, 5-fold serial dilutions of K-562SN-containing RNA-encoded bstb_435 / 434 or bstb_436 / 434 were applied. Concentration-dependent specific lysis of CLDN6+ ovarian cancer cells OV-90 after 48 hours of incubation is shown. CDLN6- breast cancer cells MDA-MB-231 were used as a negative control. Mean values ​​of triplicates are shown, including standard deviations. bstb indicates bispecific TriMAB, and EC50 indicates half-maximal effective concentration. [Figure 17A]Figures 17A-17C show a comparison of the in vivo efficacy of CLDN6xCD3 RNA and protein bstb and dual(scFv)2 in a mouse xenograft tumor model. Male (black symbols) and female (white symbols) immunodeficient NSG mice were used. (A) Injection schedule scheme. Mice were inoculated subcutaneously (sc) with CLDN6+ human ovarian cancer cells OV-90 as target cells and engrafted intraperitoneally (ip) with human PBMCs as effector cells. Treatment was initiated at a mean tumor volume of approximately 250 mm3 per group and administered intravenously (iv) once per week. Group 1 (G1) received an RNA control complex (luciferase RNA in TransIT), G2 received a bstb RNA complex, and G3 received a dual(scFv)2 RNA complex. Each injection contained a total of 3 μg of RNA. G4-6 served as protein control / reference groups. G4 received vehicle buffer (protein formulation buffer), G5 received 100 μg / kg of bstb reference, and G6 received 200 μg / kg of dual(scFv)2 reference. (B) Tumor growth over time for all mice and groups. Treatments were administered intravenously as indicated by the arrows. Treatments per group are indicated in the graph titles. Each line represents an individual mouse. (C) Flow cytometry analysis of human T cells infiltrating xenograft tumor tissue from mice treated with control RNA (G1), bstb RNA (G2), or dual(scFv)2 RNA (G3). Each symbol represents an individual mouse, and the line represents the mean value per group. Symbols follow (B). Dual-scFv, bispecific single-chain fragment; bstb, bispecific TriMAB; d, days; G, group; GVHD, graft-versus-host disease; ip, intraperitoneal; iv, intravenous; PBMC, peripheral blood mononuclear cells; sc, subcutaneous. [Figure 17B] See Figure 17A. [Figure 17C] See Figure 17A. [Figure 18A]Figures 18A and 18B show VH and VL amino acid sequences according to the IMGT nomenclature. The amino acid sequences of the VH and VL domains used in the bstb molecules described herein are shown in standard single-letter code. A) VH and VL sequences of anti-CLDN18.2 and anti-CLDN6, B) VH and VL sequences of anti-CD3. CDRs are complementarity-determining regions; FRs are framework regions; IMGT is the international ImMunoGeneTics information system; VH is variable heavy chain; and VL is variable light chain. [Figure 18B] See Figure 18A. DETAILED DESCRIPTION OF THE INVENTION

[0071] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are used to describe specific embodiments only, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0072] The elements of the present invention are described below. While these elements are listed in specific embodiments, it should be understood that they can be combined in any manner and in any number to produce additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description herein should be understood to support and cover embodiments combining the explicitly described embodiments with any number of the described and / or preferred elements. Furthermore, any order and combination of all elements described in this application should be construed as disclosed by the description in this application, unless the context dictates otherwise.

[0073] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0074] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0075] Throughout this specification and the claims that follow, unless the context requires otherwise, the term "comprise" and variations thereof, such as "comprises" and "comprising," refer to the inclusion of a stated member, integer, or step or group of members, integers, or steps, but not the exclusion of any other members, integers, or steps or group of members, integers, or steps. However, in some embodiments, such other members, integers, or steps or group of members, integers, or steps may be excluded, i.e., the subject matter is understood to consist of the inclusion of a stated member, integer, or step or group of members, integers, or steps. The terms "a," "an," and "the" and similar references used in the context of describing the invention (particularly in the context of the claims) are to be construed to encompass both the singular and the plural, unless otherwise stated in the specification or clearly contradicted by the content. Recitation of ranges of numerical values ​​herein is merely intended as a shorthand method of reciting each separate value falling within the range individually. Unless otherwise stated herein, each individual value is incorporated herein as if each such value were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise stated herein or clearly contradicted by content. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better illustrate the invention and are not intended to impose limitations on the scope of the invention not expressly stated in the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0076] Several publications are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0077] Claudins are a family of proteins that are essential components of tight junctions, where they establish a paracellular barrier that controls the flow of molecules through the gap between epithelial cells. Claudins are transmembrane proteins that span the membrane four times and have both their N- and C-termini located in the cytoplasm. The first extracellular loop, designated EC1 or ECL1, consists of an average of 53 amino acids, and the second extracellular loop, designated EC2 or ECL2, consists of approximately 24 amino acids. Cell surface proteins of the claudin family, such as CLDN6 and CLDN18.2, are expressed in tumors of various origins. Their selective expression (absence of expression in normal tissues associated with cytotoxicity) and localization to the plasma membrane make them particularly suitable as target structures for antibody-mediated cancer immunotherapy.

[0078] In the context of the present invention, preferred claudins are CLDN6 and CLDN18.2. CLDN6 and CLDN18.2 have been identified as being differentially expressed in tumor tissues, with the only normal tissue in which CLDN18.2 is expressed being the stomach, and the only normal tissue in which CLDN6 is expressed being the placenta.

[0079] CLDN18.2 is selectively expressed in normal tissues in differentiated epithelial cells of the gastric mucosa. CLDN18.2 is expressed in cancers of various origins, such as pancreatic cancer, esophageal cancer, gastric cancer, bronchial cancer, breast cancer, and ENT tumors. CLDN18.2 is a valuable target for the prevention and / or treatment of primary tumors, such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer, as well as their metastasis, particularly metastasis of gastric cancer such as Krukenberg tumor, peritoneal metastasis, and lymph node metastasis.

[0080] CLDN6 has been found to be expressed in, for example, ovarian cancer, lung cancer, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, melanoma, head and neck cancer, sarcoma, bile duct cancer, renal cell carcinoma, and bladder cancer. CLDN6 is also found to be expressed in ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly lung cancer including lung squamous cell carcinoma and adenocarcinoma, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, particularly basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, particularly malignant pleomorphic adenoma, sarcoma, particularly synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, particularly transitional cell carcinoma and papillary carcinoma, and kidney cancer. CLDN6 is a particularly preferred target for the prevention and / or treatment of renal cell carcinoma, particularly renal cell carcinoma including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer including cancer of the ileum, particularly small intestinal adenocarcinoma and ileal adenocarcinoma, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, particularly testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors such as teratocarcinoma or embryonal carcinoma, particularly testicular germ cell tumors, and metastatic forms thereof. In one embodiment, the cancer disease associated with CLDN6 expression is selected from the group consisting of ovarian cancer, lung cancer, metastatic ovarian cancer, and metastatic lung cancer. Preferably, the ovarian cancer is carcinoma or adenocarcinoma. Preferably, the lung cancer is carcinoma or adenocarcinoma, preferably bronchiole carcinoma, such as bronchiole carcinoma or bronchial adenocarcinoma.

[0081] The term "claudin" or "CLDN" includes CLDN18.2 and CLDN6. Preferably, the claudin is a human claudin.

[0082] The term "claudin 18" or "CLDN18" includes any variants, such as claudin 18 splice variant 1 (claudin 18.1 (CLDN18.1)) and claudin 18 splice variant 2 (claudin 18.2 (CLDN18.2)).

[0083] The term "claudin 18.2" or "CLDN18.2" preferably relates to human CLDN18.2, and in particular to a protein comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 in the Sequence Listing or a variant of said amino acid sequence. The first extracellular loop of CLDN18.2 preferably comprises amino acids 27 to 81, more preferably amino acids 29 to 78, of the amino acid sequence shown in SEQ ID NO: 1. The second extracellular loop of CLDN18.2 preferably comprises amino acids 140 to 180 or 144 to 167 of the amino acid sequence shown in SEQ ID NO: 1. The first and second extracellular loops preferably form the extracellular portion of CLDN18.2.

[0084] The term "claudin 6" or "CLDN6" preferably relates to human CLDN6, and in particular to a protein comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 in the Sequence Listing, or a variant of said amino acid sequence. The first extracellular loop of CLDN6 preferably comprises amino acids 28 to 80 or 29 to 81, more preferably amino acids 28 to 76, of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3. The second extracellular loop of CLDN6 preferably comprises amino acids 138 to 160, preferably amino acids 141 to 159, more preferably amino acids 145 to 157, of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3. The first and second extracellular loops preferably form the extracellular portion of CLDN6.

[0085] The term "variant" according to the present invention particularly relates to mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs, especially naturally occurring variants of these. Allelic variants involve changes in the normal sequence of a gene, the significance of which is often unknown. Complete gene sequencing often identifies multiple allelic variants for a given gene. Species homologs are nucleic acid or amino acid sequences that differ from a given nucleic acid or amino acid sequence of species origin. The term "variant" encompasses all post-translationally modified variants and conformational variants.

[0086] The second target molecule of the binding agent described herein is a T cell-specific antigen. A T cell-specific antigen is an antigen on the surface of a T cell. A preferred embodiment of such a T cell-specific antigen is the CD3 (cluster of differentiation 3) complex.

[0087] The CD3 complex refers to an antigen expressed on a subset of mature human T cells, thymocytes, and natural killer cells as part of the multimolecular T cell receptor (TCR) complex. The T cell coreceptor is a protein complex composed of four distinct chains. In mammals, the complex contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with a molecule known as the T cell receptor (TCR) and a ζ chain to generate an activation signal for T lymphocytes. The TCR, ζ chain, and CD3 molecule together comprise the TCR complex.

[0088] Human CD3 epsilon is represented by GenBank accession number NM_000733 and comprises SEQ ID NO: 4. Human CD3 gamma is represented by GenBank accession number NM_000073. Human CD3 delta is represented by GenBank accession number NM_000732. CD3 is responsible for TCR signaling. As described in Lin and Weiss, Journal of Cell Science 114, pp. 243-244 (2001), activation of the TCR complex by binding of an MHC-presented specific antigen epitope results in phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) by Src family kinases, resulting in the activation of Ca. 2+ This induces the recruitment of additional kinases leading to T cell activation, including release. For example, clustering of CD3 on T cells by immobilized anti-CD3 antibodies leads to T cell activation similar to T cell receptor engagement, but independent of the specificity typical of that clone.

[0089] As used herein, "CD3" includes human CD3, which refers to an antigen expressed on human T cells as part of the multimolecular T cell receptor complex.

[0090] With regard to CD3, the binding agents of the invention preferably recognize the epsilon chain of CD3, and in particular recognize an epitope corresponding to the first 27 N-terminal amino acids of CD3 epsilon or a functional fragment of this 27 amino acid stretch.

[0091] According to the present invention, the term "claudin-positive cancer" or similar terms refers to a cancer comprising cancer cells that express claudins, preferably cancer cells that express claudins on the surface of said cancer cells.

[0092] "Cell surface" is used in accordance with its normal meaning in the art, and thus includes the exterior of the cell that is accessible to binding by proteins and other molecules.

[0093] When claudins are located on the surface of a cell, they are expressed on the surface of the cell and available for binding by claudin-specific antibodies added to the cell.

[0094] The term "extracellular portion" in the context of the present invention refers to that part of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from the outside of said cell, for example by an antigen-binding molecule, such as an antibody, that is located on the outside of said cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0095] The terms "portion" and "fragment" are used interchangeably herein and refer to a contiguous element. For example, a portion of a structure, such as an amino acid sequence or protein, refers to a contiguous element of said structure. A portion, part, or fragment of a structure preferably comprises one or more functional properties of said structure. For example, a portion, part, or fragment of an epitope is preferably immunologically equivalent to the epitope or peptide from which it is derived. A portion or fragment of a protein sequence preferably comprises a sequence of at least 4, particularly at least 6, at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 contiguous amino acids of the protein sequence.

[0096] According to the present invention, CLDN18.2 is not substantially expressed in cells when the expression level is low compared to its expression in gastric cells or gastric tissue. Preferably, the expression level is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of the expression level in gastric cells or gastric tissue. Preferably, CLDN18.2 is not substantially expressed in cells when the expression level exceeds the expression level in non-cancerous tissue other than the stomach by no more than 2-fold, preferably no more than 1.5-fold, preferably does not exceed the expression level in said non-cancerous tissue. Preferably, CLDN18.2 is not substantially expressed when the expression level is below the detection limit and / or when the expression level is too low to be bound by a CLDN18.2-specific antibody added to the cells.

[0097] According to the present invention, CLDN18.2 is expressed in a cell when the expression level is preferably more than 2-fold, preferably more than 10-fold, 100-fold, 1000-fold, or 10,000-fold higher than the expression level in non-cancerous tissues other than the stomach. Preferably, CLDN18.2 is expressed in a cell when the expression level is above the detection limit and / or when the expression level is high enough to allow binding of a CLDN18.2-specific antibody added to the cell. Preferably, CLDN18.2 expressed in a cell is expressed or exposed on the surface of the cell.

[0098] According to the present invention, CLDN6 is substantially not expressed when its expression level is low compared to its expression in placental cells or placental tissue. Preferably, the expression level is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of the expression level in placental cells or placental tissue. Preferably, CLDN6 is not substantially expressed in cells when its expression level exceeds the expression level in non-cancerous tissue other than the placenta by no more than 2-fold, preferably no more than 1.5-fold, preferably does not exceed the expression level in said non-cancerous tissue. Preferably, CLDN6 is not substantially expressed when its expression level is below the detection limit and / or when its expression level is too low to be bound by a CLDN6-specific antibody added to the cells.

[0099] According to the present invention, CLDN6 is expressed in a cell when the expression level is preferably more than 2-fold, preferably more than 10-fold, 100-fold, 1000-fold, or 10,000-fold higher than the expression level in non-cancerous tissues other than placenta. Preferably, CLDN6 is expressed in a cell when the expression level is above the detection limit and / or when the expression level is sufficiently high to allow binding of a CLDN6-specific antibody added to the cell. Preferably, CLDN6 expressed in a cell is expressed or exposed on the surface of the cell.

[0100] According to the present invention, the term "disease" refers to any pathological condition, including cancer, particularly the pathological forms of cancer described herein. Any reference herein to cancer or a particular form of cancer also includes metastasis of that cancer. In a preferred embodiment, the disease treated in accordance with the present application involves cells expressing claudins (CLDNs), such as CLDN18.2 and / or CLDN6.

[0101] According to the present invention, a "disease associated with claudin-expressing cells" or similar expression means that claudins are expressed in cells of a diseased tissue or organ. In one embodiment, expression of claudins in cells of a diseased tissue or organ is increased compared to the state of a healthy tissue or organ. Increase refers to an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or more. In one embodiment, expression is found only in diseased tissue, while expression in healthy tissue is suppressed. According to the present invention, diseases associated with claudin-expressing cells include cancer diseases. Furthermore, according to the present invention, cancer diseases are preferably cancer diseases in which cancer cells express claudins.

[0102] As used herein, "cancer disease" or "cancer" includes diseases characterized by dysregulated cell growth, proliferation, differentiation, adhesion, and / or migration. "Cancer cells" refer to abnormal cells that grow by rapid, uncontrolled cell proliferation and continue to grow after the stimulus that initiated the new growth has ceased. Preferably, "cancer diseases" are characterized by cells that express claudins, and cancer cells express claudins. Claudin-expressing cells are preferably cancer cells, preferably cells of a cancer described herein.

[0103] The term "cancer" according to the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, small intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, and lung cancer, as well as metastases thereof. Examples include lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer, or metastases of the aforementioned cancer types or tumors. The term cancer according to the present invention also includes metastases of cancer.

[0104] According to the present invention, "cancer" is a malignant tumor derived from epithelial cells. This group represents the most common cancers, including common forms of breast, prostate, lung and colon cancer.

[0105] "Adenocarcinoma" is a cancer that originates in glandular tissue, which is part of a larger tissue category known as epithelial tissue. Epithelial tissue includes skin, glands, and various other tissues that line body cavities and organs. Epithelium is embryologically derived from ectoderm, endoderm, and mesoderm. To be classified as an adenocarcinoma, cells do not necessarily have to be part of a gland, as long as they have secretory properties. This form of cancer can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while less differentiated adenocarcinomas may not. By staining cells from a biopsy, a pathologist determines whether a tumor is an adenocarcinoma or another type of cancer. Due to the ubiquitous nature of glands in the body, adenocarcinoma can occur in many tissues. Although each gland may not secrete the same substances, as long as the cells have an exocrine function, they are considered glands, and therefore their malignant forms are called adenocarcinomas. Malignant adenocarcinomas invade other tissues and often metastasize, given sufficient time. Ovarian adenocarcinoma is the most common type of ovarian cancer and includes serous and mucinous adenocarcinoma, clear cell adenocarcinoma, and endometrial adenocarcinoma.

[0106] "Metastasis" refers to the spread of cancer cells from their original site to another. The formation of metastasis is a highly complex process, involving the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and vessels, and subsequent transport by the blood followed by infiltration of the target organ. Ultimately, the growth of new tumors at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or portions remain and may develop metastatic potential. In one embodiment, the term "metastasis" according to the present invention relates to "distant metastasis," which refers to metastasis away from the primary tumor and the regional lymph node system. In one embodiment, the term "metastasis" according to the present invention relates to lymph node metastasis. One particular form of metastasis treatable using the therapeutic methods of the present invention is metastasis arising from gastric cancer as a primary site. In a preferred embodiment, such gastric cancer metastasis is Krukenberg tumor, peritoneal metastasis, and / or lymph node metastasis.

[0107] Krukenberg tumor is a rare metastatic tumor accounting for 1% to 2% of all ovarian tumors. The prognosis for Krukenberg tumor remains very poor, and no established treatment exists for Krukenberg tumor. Krukenberg tumor is a metastatic signet-ring cell adenocarcinoma of the ovary. The stomach is the primary site for most Krukenberg tumor cases (70%). Cancers of the colon, appendix, and breast (primarily invasive lobular carcinoma) are the second most common primary sites. Rare cases of Krukenberg tumor arising from cancer of the gallbladder, biliary tract, pancreas, small intestine, ampulla of Vater, cervix, and bladder / urachus have been reported.

[0108] "Treatment" means administering a compound or composition or combination of compounds or compositions to a subject to prevent or eliminate disease, including reducing tumor size or number of tumors in the subject, to halt or slow disease in the subject, to inhibit or slow the onset of new disease in the subject, to reduce the frequency or severity of symptoms and / or recurrence in a subject who currently has or previously had disease, and / or to extend or increase the longevity of the subject.

[0109] In particular, the term "treating a disease" as used herein includes curing, shortening the duration, alleviating, preventing, slowing or inhibiting the progression or worsening, or preventing or delaying the onset of a disease or its symptoms.

[0110] In the context of the present invention, terms such as "protect," "prevent," "protect," "prophylactic," or "protective" relate to the prevention or treatment, or both, of the occurrence and / or growth of a disease in a subject, and in particular to reducing the chance that a subject will develop a disease or delaying the onset of a disease. For example, a person at risk of cancer is a candidate for a treatment to prevent cancer.

[0111] "At risk" means that a subject is identified as having a higher than normal likelihood of developing a disease, particularly cancer, compared to the general population. Furthermore, subjects who have had or currently have a disease, particularly cancer, are at high risk of developing the disease, as such subjects may continue to develop the disease. Subjects who currently have or have had cancer also have an increased risk of cancer metastasis.

[0112] The term "patient", according to the present invention, refers to the subject of treatment, in particular a diseased subject, including, for example, a human, a non-human primate or another animal, in particular a mammal, cow, horse, pig, sheep, goat, dog, cat, or rodent, such as a mouse or rat. In a particularly preferred embodiment, the patient is a human.

[0113] "Target cells" shall mean any unwanted cells, such as cancer cells. In a preferred embodiment, the target cells express claudins.

[0114] The term "antigen" relates to a molecule, preferably inducing an immune response, such as a protein or peptide comprising an epitope against which an agent is directed and / or intended to be directed. In a preferred embodiment, the antigen is a tumor-associated antigen, e.g., CLDN18.2 or CLDN6, i.e., a component of cancer cells that can originate from the cytoplasm, cell surface and cell nucleus, and in particular those antigens that are produced in large amounts intracellularly or as surface antigens in cancer cells.

[0115] In the context of the present invention, the term "tumor-associated antigen" relates to a protein which is, under normal conditions, preferably specifically expressed in a limited number of tissues and / or organs or at a particular developmental stage, and which is expressed or aberrantly expressed in one or more tumor or cancer tissues. In the context of the present invention, tumor-associated antigens are preferably associated with the cell surface of cancer cells and are preferably not or only rarely expressed in normal tissues.

[0116] The term "epitope" refers to an antigenic determinant of a molecule, e.g., a portion of a molecule that is recognized by the immune system, e.g., by an antibody. For example, an epitope is a discrete, three-dimensional site on an antigen that is recognized by the immune system. Epitopes usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to conformational epitopes, but not to nonconformational epitopes, is lost in the presence of denaturing solvents. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids in length, for example, the epitope is preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.

[0117] The term "binding agent," as used herein, refers to any agent capable of binding to a desired antigen. In certain embodiments of the invention, the binding agent refers to an antibody, an antibody fragment, or a construct thereof. The binding agent may also comprise synthetic, modified, non-naturally occurring moieties, particularly non-peptide moieties. Such moieties may, for example, bind to a desired antigen-binding function or region, such as an antibody or antibody fragment. In one embodiment, the binding agent is a synthetic construct comprising antigen-binding CDRs or variable regions.

[0118] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably antibodies or antigen receptors such as B-cell receptors (BCRs). Immunoglobulins are characterized by structural domains with a characteristic immunoglobulin (Ig) fold, i.e., immunoglobulin domains. The term encompasses membrane-bound immunoglobulins as well as soluble immunoglobulins, which are commonly referred to as antibodies. Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains, which are linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains, e.g., V L (light chain variable) domain, C L (light chain constant) domain, and C H (Heavy chain constant) domain C H 1. C H 2. C H 3 and C H 4. There are five types of immunoglobulin heavy chains in mammals, namely α, δ, ε, γ, and μ, which account for the different classes of antibodies, namely IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxy termini. Mammals have two types of light chains, namely lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved within the various isotypes of immunoglobulins, while the variable portion is highly diverse and is responsible for antigen recognition.

[0119] The term "antibody" refers to an immunoglobulin or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and chimeric antibodies. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The terms "region" and "domain" are used interchangeably herein. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), with the CDR regions being interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen, and the constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0120] The term "monoclonal antibody," as used herein, refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity. In one embodiment, a monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a non-human animal, e.g., a mouse, fused to an immortalized cell.

[0121] The term "recombinant antibody," as used herein, includes all antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells, e.g., transfectomas, that have been transformed to express the antibody, (c) antibodies isolated from recombinant combinatorial antibody libraries, and (d) antibodies prepared, expressed, produced, or isolated by any other means, including splicing of immunoglobulin gene sequences into other DNA sequences.

[0122] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0123] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, with the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise complete variable domains fused to constant domains, or may comprise only the complementarity-determining regions (CDRs) grafted into appropriate framework regions of the variable domains. The antigen-binding site may be wild-type or may be modified by one or more amino acid substitutions, e.g., to more closely resemble human immunoglobulins. Some forms of humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). Other forms have one or more CDRs that are altered relative to the original antibody.

[0124] The term "chimeric antibody" refers to an antibody in which a portion of each of the heavy and light chain amino acid sequences is homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular class, while the remaining segments of the chain are homologous to the corresponding sequence of another species. Typically, the variable regions of both the light and heavy chains mimic the variable regions of antibodies derived from one species of mammal, while the constant regions are homologous to the sequences of antibodies derived from another species. One obvious advantage of such chimeric forms is that the variable regions can be conveniently derived from currently known sources, using readily available B cells or hybridomas of non-human host organisms combined with constant regions derived from, for example, human cell preparations. While the variable regions have the advantage of ease of preparation and specificity is not affected by the source, the constant regions are human and are less likely to elicit an immune response from a human subject when the antibody is injected, compared with constant regions derived from non-human sources. However, the definition is not limited to this specific example.

[0125] Antibodies may be derived from a variety of species, including but not limited to mouse, rat, rabbit, guinea pig, and human.

[0126] Antibodies described herein include IgA, e.g., IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an IgG1 antibody, more particularly an IgG1, kappa, or IgG1, lambda isotype (i.e., IgG1, κ, λ), an IgG2a antibody (e.g., IgG2a, κ, λ), an IgG2b antibody (e.g., IgG2b, κ, λ), an IgG3 antibody (e.g., IgG3, κ, λ), or an IgG4 antibody (e.g., IgG4, κ, λ).

[0127] As used herein, a "heterologous antibody" is defined in relation to the transgenic organism producing such an antibody. The term refers to an antibody having an amino acid sequence or its corresponding encoding nucleic acid sequence found in an organism that does not constitute the transgenic organism, and which generally originates from a species other than the transgenic organism.

[0128] As used herein, a "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.

[0129] Binding agents, such as antibodies, described herein are preferably isolated. As used herein, "isolated" is intended to refer to a binding agent that is substantially free of other agents with different antigen specificities (e.g., an isolated antibody that specifically binds to CLDN18.2 is substantially free of antibodies that specifically bind to antigens other than CLDN18.2). However, an isolated binding agent that specifically binds to an epitope, isoform, or variant of human CLDN18.2 may have cross-reactivity with other related antigens, such as antigens from other species (e.g., CLDN18.2 species homologs). Furthermore, an isolated binding agent may be a binding agent that is substantially free of other cellular material and / or chemicals.

[0130] An "antigen-binding portion" (or simply "binding portion") of an antibody or an "antigen-binding fragment" (or simply "binding fragment") of an antibody or similar terms refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can also be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker that allows them to be formed into a single protein chain in which the pair of VL and VH domains forms a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. A further example is a binding domain immunoglobulin fusion protein comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application Publication Nos. 2003 / 0118592 and 2003 / 0133939.These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0131] Single-chain variable fragments (scFv) are fusion proteins of immunoglobulin heavy chain variable regions (VH) and light chain variable regions (VL) linked by a short linker peptide, usually 10 to about 30 amino acids. The linker is usually rich in glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of VH to the C-terminus of VL, or vice versa. Bivalent (or divalent) single-chain variable fragments (di-scFv, double-scFv) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, resulting in a tandem scFv. The present invention also includes multispecific molecules containing three or more scFv binding domains. One common flexible linking peptide is (Gly4Ser) x (where x can be 3, 4, 5, or 6). Optionally, the association of VH and VL can be stabilized by one or more intermolecular disulfide bonds.

[0132] As used herein, the term "binding domain" or "antigen-binding domain" refers to a site, e.g., the site of an antibody, that binds to an antigen and comprises the antigen-binding portion of an antibody. A binding domain may be composed of heavy and light chain variable domains (VH and VL), each of which contains four conserved framework regions (FR) and three CDRs. The CDRs vary in sequence and determine specificity for a particular antigen. The VH and VL domains can together form a site that specifically binds to a particular antigen.

[0133] A Fab (fragment antigen-binding) antibody fragment is an immunoreactive polypeptide containing a monovalent antigen-binding domain of an antibody composed of a polypeptide consisting of a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1) portion, and a polypeptide consisting of a light chain variable region (VL) and a light chain constant region, wherein the CL and CH1 portions are preferably linked together by a disulfide bond between Cys residues. Preferably, in the Fab fragments described herein, the CH1 and CL regions are of human origin. In one embodiment, the CL region is a kappa-type CL region. In one embodiment, the CH1 region is derived from IgG1 or IgG2.

[0134] For the purposes of the present invention, all antibodies and antibody derivatives, such as antibody fragments, described herein are encompassed by the term "antibody." The term "antibody derivative" refers to any modified form of an antibody, for example, a conjugate of an antibody with another agent or antibody or antibody fragment. Furthermore, the antibodies and antibody derivatives described herein are used to produce binding agents of the invention, such as antibody fragments.

[0135] Naturally occurring antibodies are generally monospecific, i.e., they bind to a single antigen. The present invention provides binding agents that bind to cytotoxic cells such as T cells (e.g., by engaging the CD3 receptor) and to target cells such as cancer cells (e.g., by engaging claudins). The binding agents of the present invention bind to at least two different types of antigens and are at least bispecific, or multispecific, e.g., trispecific, tetraspecific, etc.

[0136] Binding agents of the invention may be at least trivalent. As used herein, "valent," "valence," "valencies," or other grammatical variations refer to the number of antigen-binding sites or binding domains of a binding agent. Antigen-binding sites that bind to the same antigen may recognize the same epitope or different epitopes. Trivalent and tetravalent bispecific antibodies are known in the art. Binding agents of the invention may have a valency greater than four.

[0137] The binding agents described herein are preferably artificial proteins (including protein complexes) that may be composed of fragments of at least two different antibodies (wherein said fragments of at least two different antibodies form at least two different binding domains) and thereby bind to at least two different types of antigens. Binding agents according to the invention are engineered to bind to immune cells, such as immune effector cells, in particular T cells, such as cytotoxic cells (e.g., by binding to CD3), and simultaneously bind to target cells, such as cancer cells, to be destroyed (e.g., by binding to the tumor-associated antigen claudin).

[0138] Several types of trivalent antibodies have been developed, and all are within the scope of the present invention. Triple bodies or single-chain triple antibodies (sctb) are composed of three different scFv regions linked by a linker sequence. Furthermore, the natural in vivo heterodimerization of heavy chains (CH1 domains) and light chains (CL domains) can also be used to form scaffolds to which multiple scFvs can be added. For example, an scFv specific for one antigen can be linked to a CH1 domain that is also linked to an scFv specific for another antigen, and this chain can interact with another chain containing an scFv specific for either antigen linked to a CL domain (scFv3-CH1 / CL). Another example of a trivalent construct includes the use of a Fab fragment specific for one epitope linked at its C-terminus to two scFvs on each chain, each specific for a different epitope (Fab-scFv2). Other examples of trivalent (or tetravalent) molecules include various formats containing additional binding entities attached to the N- or C-terminus of the antibody. For example, one format consists of an intact antibody molecule specific for one antigen with a single-chain Fab (scFab) linked to the C-terminus of the molecule (IgG-scFab). The dock-and-lock (DNL) method has also been used to generate trivalent antibodies (DNL-F(ab)3) (Chang, C.-H. et al., In: Bispecific Antibodies. Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 199-216 (2011)). Each of these antibodies is within the scope of the present invention.

[0139] Tetravalent antibodies have also been constructed, and all types are within the scope of the present invention. Examples of tetravalent antibodies include, but are not limited to, scFv2-Fc, F(ab')2-scFv2, scFv2-H / L, and scFv-dhlx-scFv molecules. Bispecific scFv2-Fc constructs have an Fc domain with two scFvs specific for one molecule linked to the N-terminus of the Fc chain and two other scFvs specific for another molecule linked to the C-terminus of the Fc chain. Bispecific F(ab')2-scFv2 constructs include scFv fragments linked to the C-terminus of the F(ab')2 fragment. scFv2-H / L constructs have scFvs specific for one molecule linked to the heavy chain, while scFvs specific for another molecule are linked to the light chain. Finally, the scFv-dhlx-scFv construct contains one type of scFv linked to a helical dimerization domain followed by another type of scFv, two chains of which can dimerize to generate a tetravalent antibody.

[0140] The binding agents of the present invention may be in the format of an antibody molecule, or an antibody-like molecule, or a protein scaffold with antibody-like properties, or a cyclic peptide with at least two binding specificities. Thus, the binding agent may comprise one or more antibodies or fragments thereof described herein.

[0141] In one embodiment, a binding agent of the invention comprises a heavy chain (Fd fragment) and a light chain (L) of a Fab fragment capable of heterodimerizing, and may further comprise additional binding functions or domains, which may be independently selected from the group consisting of two antibody variable regions, e.g., scFv binding domains, i.e., binding domains comprising VH-VL or VL-VH, and binding domains comprising one antibody variable region, e.g., a VH binding domain and a VHH binding domain.

[0142] In one embodiment, a binding agent of the invention is in the format of a Fab-scFv2 construct, i.e., a Fab fragment with two scFv fragments, preferably at the C-terminus of the constant region of the Fab fragment. In one embodiment, a binding agent of the invention is a dimer, preferably composed of two polypeptide chains linked together by a disulfide bridge, where a first polypeptide comprises an scFv linked to an additional VL domain through a CL polypeptide chain, and a second polypeptide comprises an scFv linked to an additional VH domain through a CH1 polypeptide chain. The disulfide bridge is preferably formed between a Cys residue in the CL and a Cys residue in the CH1, such that in the antigen-binding configuration, the additional VL of the first polypeptide and the additional VH of the second polypeptide associate, resulting in the binding agent as a whole comprising three antigen-binding domains. Thus, in one embodiment, a binding agent of the invention comprises a heavy chain (Fd fragment) and a light chain (L) of a Fab fragment capable of heterodimerization, and further incorporates an scFv binding domain (preferably at the C-terminus of Fd / L). According to the present invention, the VH and VL domains of the scFv portion are preferably linked by a peptide linker, and / or the Fab chain and the scFv are preferably linked by a peptide linker. According to the present invention, the VH and VL domains of the scFv portion preferably have the amino acid sequence (G4S) x (where x is 3, 4, 5, or 6). The Fab chain and scFv are preferably linked by a peptide linker comprising the amino acid sequence DVPG2S or SGPG3RS(G4S)2. In one embodiment, a linker comprising the amino acid sequence SGPG3RS(G4S)2 links the scFv binding domain to the Fd fragment, and a linker comprising the amino acid sequence DVPG2S links the scFv binding domain to the L fragment. In one embodiment, the scFv portion binds to a claudin, and the Fab portion binds to a T cell-specific antigen.

[0143] In one embodiment, a binding agent of the invention comprises a first and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise, from N-terminus to C-terminus, the following domains: VH-CH1-scFv and VL-CL-scFv wherein VH and VL associate to form a binding domain.

[0144] In one embodiment, a binding agent of the invention comprises a first and a second polypeptide, wherein in the first polypeptide and the second polypeptide, the VH domain, VL domain, CH1 domain and CL domain are arranged from N-terminus to C-terminus as follows: - VH(T)-CH1-VH(CLDN)-VL(CLDN) and VL(T)-CL-VH(CLDN)-VL(CLDN); or - VH(T)-CH1-VL(CLDN)-VH(CLDN) and VL(T)-CL-VL(CLDN)-VH(CLDN); or - VH(T)-CH1-VH(CLDN)-VL-(CLDN) and VL(T)-CL-VL(CLDN)-VH-(CLDN); or - VH(T)-CH1-VL(CLDN)-VH-(CLDN) and VL(T)-CL-VH(CLDN)-VL-(CLDN); or - VH(CLDN)-CH1-VH(T)-VL(T) and VL(CLDN)-CL-VH(CLDN)-VL(CLDN); or - VH(CLDN)-CH1-VL(T)-VH(T) and VL(CLDN)-CL-VL(CLDN)-VH(CLDN); or - VH(CLDN)-CH1-VL(T)-VH(T) and VL(CLDN)-CL-VH(CLDN)-VL(CLDN); or - VH(CLDN)-CH1-VH(T)-VL(T) and VL(CLDN)-CL-VL(CLDN)-VH(CLDN); or - VH(CLDN)-CH1-VH(CLDN)-VL(CLDN) and VL(CLDN)-CL-VH(T)-VL(T); or - VH(CLDN)-CH1-VL(CLDN)-VH(CLDN) and VL(CLDN)-CL-VL(T)-VH(T); or - VH(CLDN)-CH1-VL(CLDN)-VH(CLDN) and VL(CLDN)-CL-VH(T)-VL(T); or - VH(CLDN)-CH1-VH(CLDN)-VL(CLDN) and VL(CLDN)-CL-VL(T)-VH(T) are arranged in the order of

[0145] The term "linker" refers to any means useful for linking two different functional units (e.g., antigen-binding moieties). Types of linkers include, but are not limited to, chemical linkers and polypeptide linkers. The sequence of the polypeptide linker is not limited. The polypeptide linker is preferably non-immunogenic and flexible, for example, a linker containing serine and glycine sequences. Depending on the particular construct, the linker may be long or short.

[0146] According to the present invention, the linker linking the VH and VL domains to form the VH-VL or VL-VH scFv domain preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker. In one embodiment, the linker has the amino acid sequence (G4S) x (where x is 3, 4, 5 or 6). In the case of an scFv domain comprising a VH and VL domain in a VH-VL orientation, the linker preferably comprises the amino acid sequence (G4S)4. In the case of an scFv domain comprising a VH and VL domain in a VL-VH orientation, the linker preferably comprises the amino acid sequence (G4S)5.

[0147] According to the present invention, the linker connecting the scFv domain and the Fd domain preferably comprises the amino acid sequence DVPG2S or SGPG3RS(G4S)2, preferably SGPG3RS(G4S)2, preferably at the C-terminus of CH1. According to the present invention, the linker connecting the scFv domain and the L domain preferably comprises the amino acid sequence DVPG2S or SGPG3RS(G4S)2, preferably DVPG2S, preferably at the C-terminus of CL.

[0148] Binding agents according to the invention may also comprise amino acid sequences to facilitate secretion of the molecule, such as an N-terminal secretion signal and / or one or more epitope tags to facilitate binding, purification or detection of the molecule.

[0149] Preferably, the secretory signal is a signal sequence (e.g., the amino acid sequence MGWSCIILFLVATATGVHS) that allows efficient passage through the secretory pathway and / or secretion of the binding agent or its polypeptide chain into the extracellular environment. Preferably, the secretory signal sequence is cleavable and is removed from the mature binding agent. The secretory signal sequence is preferably selected for the cell or organism in which the binding agent is produced.

[0150] The amino acid sequence of the epitope tag may be introduced at any position within the amino acid sequence of the binding agent, may take the form of a loop within the encoded protein structure, or may be fused to the N- or C-terminus of the binding agent. Preferably, the epitope tag is fused to the C-terminus of the binding agent. The epitope tag may comprise a cleavage site that allows removal of the tag from the binding agent. The epitope tag may be any type of epitope tag that is functional under native and / or denatured conditions, and is preferably a histidine tag, most preferably a tag containing six histidines.

[0151] In addition to the first, second, and third binding domains, the binding agents of the invention may comprise one or more further binding domains that serve, for example, to improve selectivity for tumor cells. This can be achieved, for example, by providing binding domains that bind to other antigens expressed on tumor cells.

[0152] In the context of the present invention, the binding agents produced are preferably capable of inducing immune effector functions as described herein, preferably directed against cells bearing the tumor-associated antigen claudin on their surface.

[0153] In the context of the present invention, the term "immune effector function" includes any function mediated by components of the immune system that results in the inhibition of tumor growth and / or the inhibition of tumorigenesis, including, for example, the inhibition of tumor dissemination and metastasis. Preferably, the immune effector function results in the killing of tumor cells. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), the induction of apoptosis in cells bearing tumor-associated antigens, the cytolysis of cells bearing tumor-associated antigens, and / or the inhibition of proliferation of cells bearing tumor-associated antigens. Binding agents can also exert their effects by simply binding to tumor-associated antigens on the surface of cancer cells. For example, an antibody can block the function of a tumor-associated antigen or induce apoptosis simply by binding to a tumor-associated antigen on the surface of cancer cells.

[0154] The binding agents described herein can be conjugated to a therapeutic moiety or agent, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radioisotope. A cytotoxic or cytotoxic agent includes any agent that is detrimental to cells, in particular, that kills cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Suitable therapeutic agents for forming conjugates include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin ) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and antimitotic agents (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic or radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another embodiment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide, or ricin A.

[0155] Binding agents may also be conjugated to radioisotopes, such as iodine-131, yttrium-90, or indium-111, to generate cytotoxic radiopharmaceuticals.

[0156] Techniques for conjugating such therapeutic moieties to antibodies are well known and include, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc., 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospect Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer" See, "Monoclonal Antibodies For Cancer Detection And Therapy," Baldwin et al. (eds.), pp. 303-16 (Academic Press, 1985) and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62:119-58 (1982).

[0157] The term "binding" according to the present invention preferably relates to specific binding.

[0158] According to the present invention, an agent such as an antibody can bind to a predetermined target if it has significant affinity for the target in a standard assay and binds to the predetermined target. "Affinity" or "binding affinity" is often measured using the equilibrium dissociation constant (K D ) Preferably, the term "significant affinity" refers to a -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 The dissociation constant (K D ) to bind to a predetermined target.

[0159] An agent is incapable of (substantially) binding to a target if it has no significant affinity for the target in a standard assay and does not bind significantly, in particular does not bind detectably, to the target. Preferably, the agent does not detectably bind to the target when present at a concentration of up to 2 μg / ml, preferably 10 μg / ml, more preferably 20 μg / ml, in particular 50 μg / ml or 100 μg / ml or more. Preferably, the agent has a K D At least 10 times, 100 times, 10 times 3 double, 10 4 double, 10 5 double, or 10 6 Twice as high as K D For example, if a drug binds to a target at a K D is 10 -7 M, the K for binding to a target for which the drug does not have significant affinity D is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10-2 M or 10 -1 I am M.

[0160] An agent, such as an antibody, is specific for a given target if it can bind to the given target in a standard assay but cannot bind to other targets, i.e., it has no significant affinity for and does not significantly bind to other targets. According to the present invention, an agent is specific for a claudin if it can bind to a claudin but cannot (substantially) bind to other targets. Preferably, an agent is specific for a claudin if its affinity and binding to such other targets do not significantly exceed its affinity or binding to proteins unrelated to claudins, such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or non-claudin transmembrane proteins such as MHC molecules or transferrin receptors, or any other specific polypeptide. Preferably, an agent has a K for binding to a non-specific target. D At least one-tenth, one-hundredth, or tenth of 3 tenths, tenths 4 tenths, tenths 5 1 / 10 or 1 / 10 6 1 / K D For example, a drug is specific for a given target if it binds to the target at a K D is 10 -7 M, K for non-specific target binding D is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 I am M.

[0161] Binding of an agent to a target can be determined experimentally using any suitable method (see, e.g., Berzofsky et al., "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, WE ed., Raven Press, New York, NY (1984); Kuby, Janis, Immunology, WH Freeman and Company, New York, NY (1992)) and the methods described herein. Affinity can be readily determined using conventional techniques, for example, by equilibrium dialysis, by using a BIAcore 2000 instrument, using the general procedures outlined by the manufacturer, by radioimmunoassay using radiolabeled target antigen, or by another method known to those skilled in the art. Affinity data can be analyzed, for example, by the method described in Scatchard et al., Ann NY Acad. ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction can vary when measured under different conditions, such as salt concentration, pH, etc. Thus, affinity and other antigen binding parameters, e.g., K D ,I C 50 Such measurements are preferably carried out using standardized solutions of antibody and antigen, and standardized buffer solutions.

[0162] The term "compete" refers to the competition between two antibodies binding to a target antigen. If two antibodies do not interfere with each other's binding to the target antigen, such antibodies are non-competing, indicating that the antibodies do not bind to the same site, i.e., epitope, of the target antigen. Methods for testing the competition of antibodies binding to a target antigen are well known to those skilled in the art. One example of such a method is a so-called cross-competition assay, which can be performed, for example, as an ELISA or flow cytometry. For example, an ELISA-based assay can be performed by coating an ELISA plate well with each antibody; adding and incubating the competing antibody and the His-tagged extracellular domain of the antigen / target; detecting whether the added antibody inhibits the binding of the His-tagged protein to the coated antibody, which can be performed by adding a biotinylated anti-His antibody followed by streptavidin-polyHRP, further developing the reaction with ABTS, and measuring the absorbance at 405 nm. For example, a flow cytometry assay can be performed by incubating cells expressing the antigen / target with an excess of unlabeled antibody, incubating the cells with a suboptimal concentration of biotin-labeled antibody, followed by incubation with fluorescently labeled streptavidin and analysis by flow cytometry.

[0163] Two antibodies have the same specificity if they bind to the same antigen and the same epitope. Whether a test antibody recognizes the same epitope as a specific antigen-binding antibody, i.e., whether the antibodies bind to the same epitope, can be analyzed based on their competition for the same epitope. Competition between antibodies can be detected by a cross-blocking assay. For example, a competitive ELISA assay can be used as a cross-blocking assay. For example, a target antigen can be coated on the wells of a microtiter plate, and an antigen-binding antibody and a competing candidate test antibody can be added. The amount of antigen-binding antibody bound to the antigen in the well is indirectly correlated with the binding ability of the competing candidate test antibody that competes with the antigen-binding antibody for binding to the same epitope. Specifically, the greater the affinity of the competing candidate test antibody for the same epitope, the less antigen-binding antibody will bind to the well coated with the antigen. The amount of antigen-binding antibody bound to the well can be measured by labeling the antibody with a detectable or measurable label.

[0164] An antibody that competes for binding to an antigen with another antibody, e.g., an antibody comprising the heavy and light chain variable regions described herein, or that has specificity for the antigen of another antibody, e.g., an antibody comprising the heavy and light chain variable regions described herein, can be a variant of the heavy and / or light chain variable regions described herein, e.g., an antibody comprising modified CDRs and / or a degree of identity as described herein.

[0165] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0166] As used herein, "isotype switching" refers to the phenomenon in which the class or isotype of an antibody changes from one Ig class to one of the other Ig classes.

[0167] As used herein, the term "naturally occurring," when applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source, has not been intentionally modified by man in the laboratory, and is present in an organism (including a virus) is naturally occurring.

[0168] As used herein, the term "rearrangement" refers to the configuration of a heavy or light chain immunoglobulin locus in which a V segment is positioned immediately adjacent to a DJ or J segment in a conformation that encodes essentially a complete VH or VL domain, respectively. Rearranged immunoglobulin (antibody) loci can be identified by comparison to germline DNA, and rearranged loci have at least one recombined heptamer / 9amer homology element.

[0169] The term "unrearranged" or "germline configuration" as used herein with respect to a V segment refers to a configuration in which the V segment has not been rearranged so that it is immediately adjacent to a D or J segment.

[0170] In one embodiment, a binding agent of the invention has the ability to bind to CLDN18.2, i.e., the ability to bind to an epitope present in CLDN18.2, preferably an epitope within the extracellular domain of CLDN18.2, particularly an epitope located within the first extracellular loop, preferably amino acids 29 to 78 of CLDN18.2. In a specific embodiment, an agent capable of binding to CLDN18.2 binds to an epitope in CLDN18.2 that is not present in CLDN18.1.

[0171] The agent capable of binding to CLDN18.2 preferably binds to CLDN18.2 but not to CLDN18.1. Preferably, the agent capable of binding to CLDN18.2 is specific to CLDN18.2. Preferably, the agent capable of binding to CLDN18.2 binds to CLDN18.2 expressed on the cell surface. In a particularly preferred embodiment, the agent capable of binding to CLDN18.2 binds to a native epitope of CLDN18.2 present on the surface of living cells.

[0172] In a preferred embodiment, the binding domain of the binding agent of the present invention that binds to CLDN18.2 comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, and 24, or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0173] In a preferred embodiment, the binding domain of the binding agent of the present invention that binds to CLDN18.2 comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 25, or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0174] In certain preferred embodiments, the binding domain that binds to CLDN18.2 of the binding agent of the invention has the following possibilities: (i) VH comprises the amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof, or a variant of the amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 22 or a fragment thereof, or a variant of the amino acid sequence or fragment; (ii) VH comprises the amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof, or a variant of the amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 23 or a fragment thereof, or a variant of the amino acid sequence or fragment; (iii) VH comprises the amino acid sequence represented by SEQ ID NO: 21 or a fragment thereof, or a variant of the amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 22 or a fragment thereof, or a variant of the amino acid sequence or fragment; (iv) VH comprises the amino acid sequence represented by SEQ ID NO: 21 or a fragment thereof, or a variant of the amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 23 or a fragment thereof, or a variant of the amino acid sequence or fragment; (v) VH comprises the amino acid sequence represented by SEQ ID NO: 24 or a fragment thereof, or a variant of the amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 25 or a fragment thereof, or a variant of the amino acid sequence or fragment. It comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from:

[0175] In a particularly preferred embodiment, the binding domain that binds to CLDN18.2 of the binding agent of the invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): VH comprises the amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof, or a variant of said amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 22 or a fragment thereof, or a variant of said amino acid sequence or fragment. Includes.

[0176] In a particularly preferred embodiment, the binding domain that binds to CLDN18.2 of the binding agent of the invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): VH comprises the amino acid sequence represented by SEQ ID NO: 21 or a fragment thereof, or a variant of said amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 23 or a fragment thereof, or a variant of said amino acid sequence or fragment. Includes.

[0177] The term "fragment" particularly refers to one or more complementarity determining regions (CDRs), preferably at least the CDR3 variable region of the heavy chain variable region (VH) and / or light chain variable region (VL). In one embodiment, said one or more complementarity determining regions (CDRs) are selected from the set of complementarity determining regions CDR1, CDR2 and CDR3. In a particularly preferred embodiment, the term "fragment" refers to the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL).

[0178] In one embodiment, a binding domain comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs described herein comprises said CDRs together with their intervening framework regions. Preferably, the portion comprises at least about 50% of one or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of binding agents made by recombinant DNA techniques may lead to the introduction of residues N- or C-terminal to the encoded variable region by a linker introduced to facilitate cloning or other manipulation steps, for example, by introducing a linker that connects the variable region of the invention to further protein sequences, including immunoglobulin heavy chains, other variable domains, or protein tags.

[0179] In one embodiment, a binding domain comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs described herein comprises said CDRs in a human antibody framework.

[0180] In a preferred embodiment, the binding domain that binds to CLDN18.2 of the binding agent of the invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): VH comprises a heavy chain complementarity determining region 3 (HCDR3) comprising the sequence set forth in SEQ ID NO: 58; VL comprises a light chain complementarity determining region 3 (LCDR3) comprising the sequence set forth in SEQ ID NO: 64; Includes.

[0181] In one embodiment, the VH further comprises an HCDR1 comprising the sequence set forth in SEQ ID NO: 56 and / or an HCDR2 comprising the sequence set forth in SEQ ID NO: 57, and / or the VL further comprises an LCDR1 comprising the sequence set forth in SEQ ID NO: 62 and / or an LCDR2 comprising the sequence set forth in SEQ ID NO: 63.

[0182] In a preferred embodiment, the binding domain that binds to CLDN18.2 of the binding agent of the invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): VH comprises HCDR1 comprising the sequence shown in SEQ ID NO: 56, HCDR2 comprising the sequence shown in SEQ ID NO: 57, and HCDR3 comprising the sequence shown in SEQ ID NO: 58; VL comprises LCDR1 comprising the sequence shown in SEQ ID NO: 62, LCDR2 comprising the sequence shown in SEQ ID NO: 63, and LCDR3 comprising the sequence shown in SEQ ID NO: 64; Includes.

[0183] In a preferred embodiment, the binding domain that binds to CLDN18.2 of the binding agent of the invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): VH comprises a heavy chain complementarity determining region 3 (HCDR3) comprising the sequence set forth in SEQ ID NO: 61; VL comprises a light chain complementarity determining region 3 (LCDR3) comprising the sequence set forth in SEQ ID NO: 65; Includes.

[0184] In one embodiment, the VH further comprises an HCDR1 comprising the sequence set forth in SEQ ID NO: 59 and / or an HCDR2 comprising the sequence set forth in SEQ ID NO: 60, and / or the VL further comprises an LCDR1 comprising the sequence set forth in SEQ ID NO: 62 and / or an LCDR2 comprising the sequence set forth in SEQ ID NO: 63.

[0185] In a preferred embodiment, the binding domain that binds to CLDN18.2 of the binding agent of the invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): VH comprises HCDR1 comprising the sequence shown in SEQ ID NO: 59, HCDR2 comprising the sequence shown in SEQ ID NO: 60, and HCDR3 comprising the sequence shown in SEQ ID NO: 61; VL comprises LCDR1 comprising the sequence shown in SEQ ID NO: 62, LCDR2 comprising the sequence shown in SEQ ID NO: 63, and LCDR3 comprising the sequence shown in SEQ ID NO: 65; Includes.

[0186] In one embodiment, the heavy and light chain variable regions comprise the complementarity determining regions interspersed within framework regions. In one embodiment, each variable region comprises three complementarity determining regions (CDR1, 2, and 3) and four framework regions (FR1, 2, 3, and 4). In one embodiment, the complementarity determining regions and the framework regions are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0187] In a further embodiment, the binding domain of the binding agent of the present invention that binds to CLDN18.2 comprises the heavy and light chain variable regions of (i) an antibody that competes for CLDN18.2 binding with an antibody comprising the above-mentioned heavy and light chain variable regions and / or (ii) an antibody that has the specificity for CLDN18.2 of an antibody comprising the above-mentioned heavy and light chain variable regions.

[0188] In one embodiment, the heavy chain variable region (VH) and light chain variable region (VL) of the CLDN18.2-binding domain of the binding agent of the invention have the format of an scFv molecule. In this embodiment, the CLDN18.2-binding domain of the binding agent of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, or fragments thereof, or variants of said amino acid sequences or fragments.

[0189] It should be understood that the CLDN18.2-binding domains of a binding agent of the invention may be identical or essentially identical, or different, and thus may bind to the same or essentially the same epitope or different epitopes. Thus, both CLDN18.2-binding domains of a binding agent of the invention may correspond or essentially correspond to one of the CLDN18.2-binding domains of a binding agent of the invention described herein, or may be independently selected from the CLDN18.2-binding domains of a binding agent of the invention described herein.

[0190] In one embodiment, the binding agent of the present invention has the ability to bind to CLDN6, i.e., the ability to bind to an epitope present in CLDN6, preferably an epitope located within the extracellular domain of CLDN6, particularly the first extracellular loop, preferably within amino acids 28-76 or 29-81 of CLDN6, or the second extracellular loop, preferably within amino acids 141-159 of CLDN6. In a specific embodiment, an agent capable of binding to CLDN6 binds to an epitope of CLDN6 that is not present in CLDN9. Preferably, an agent capable of binding to CLDN6 binds to an epitope of CLDN6 that is not present in CLDN4 and / or CLDN3. Most preferably, an agent capable of binding to CLDN6 binds to an epitope of CLDN6 that is not present in claudin proteins other than CLDN6.

[0191] The agent capable of binding to CLDN6 preferably binds to CLDN6 but does not bind to CLDN9, and preferably does not bind to CLDN4 and / or CLDN3. Preferably, the agent capable of binding to CLDN6 is specific to CLDN6. Preferably, the agent capable of binding to CLDN6 binds to CLDN6 expressed on the cell surface. In a particularly preferred embodiment, the agent capable of binding to CLDN6 binds to a native epitope of CLDN6 present on the surface of living cells.

[0192] In a preferred embodiment, the binding domain of the binding agent of the present invention that binds to CLDN6 comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 8, 11, or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0193] In a preferred embodiment, the binding domain of the binding agent of the present invention that binds to CLDN6 comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 12, or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0194] In certain preferred embodiments, the CLDN6-binding domain of the binding agent of the invention has the following possibilities: (i) VH comprises the amino acid sequence represented by SEQ ID NO: 7 or a fragment thereof, or a variant of the amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 9 or a fragment thereof, or a variant of the amino acid sequence or fragment; (ii) VH comprises the amino acid sequence represented by SEQ ID NO: 7 or a fragment thereof, or a variant of the amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 10 or a fragment thereof, or a variant of the amino acid sequence or fragment; (iii) VH comprises the amino acid sequence represented by SEQ ID NO: 8 or a fragment thereof, or a variant of the amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 9 or a fragment thereof, or a variant of the amino acid sequence or fragment; (iv) VH comprises the amino acid sequence represented by SEQ ID NO: 8 or a fragment thereof, or a variant of the amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 10 or a fragment thereof, or a variant of the amino acid sequence or fragment; (v) VH comprises the amino acid sequence represented by SEQ ID NO: 11 or a fragment thereof, or a variant of the amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof, or a variant of the amino acid sequence or fragment; (vi) VH comprises the amino acid sequence represented by SEQ ID NO: 7 or a fragment thereof, or a variant of the amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof, or a variant of the amino acid sequence or fragment; (vii) VH comprises the amino acid sequence represented by SEQ ID NO: 8 or a fragment thereof, or a variant of the amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof, or a variant of the amino acid sequence or fragment; It comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from:

[0195] In a particularly preferred embodiment, the binding domain that binds to CLDN6 of the binding agent of the present invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): VH comprises the amino acid sequence represented by SEQ ID NO: 8 or a fragment thereof, or a variant of said amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 10 or a fragment thereof, or a variant of said amino acid sequence or fragment. Includes.

[0196] In a particularly preferred embodiment, the binding domain that binds to CLDN6 of the binding agent of the present invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): VH comprises the amino acid sequence represented by SEQ ID NO: 7 or a fragment thereof, or a variant of said amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof, or a variant of said amino acid sequence or fragment. Includes.

[0197] The term "fragment" particularly refers to one or more complementarity determining regions (CDRs), preferably at least the CDR3 variable region of the heavy chain variable region (VH) and / or light chain variable region (VL). In one embodiment, said one or more complementarity determining regions (CDRs) are selected from the set of complementarity determining regions CDR1, CDR2 and CDR3. In a particularly preferred embodiment, the term "fragment" refers to the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL).

[0198] In one embodiment, a binding domain comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs described herein comprises said CDRs together with their intervening framework regions. Preferably, the portion comprises at least about 50% of one or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of binding agents made by recombinant DNA techniques may lead to the introduction of residues N- or C-terminal to the encoded variable region by a linker introduced to facilitate cloning or other manipulation steps, for example, by introducing a linker that connects the variable region of the invention to further protein sequences, including immunoglobulin heavy chains, other variable domains, or protein tags.

[0199] In one embodiment, a binding domain comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs described herein comprises said CDRs in a human antibody framework.

[0200] In a preferred embodiment, the binding domain that binds to CLDN6 of the binding agent of the present invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): the VH comprises a heavy chain complementarity determining region 3 (HCDR3) comprising the sequence Ala Arg Asp Xaa1 Gly Xaa2 Val Xaa3 Asp Tyr, where Xaa1 is any amino acid, preferably an aromatic amino acid, more preferably Phe or Tyr, and most preferably Tyr; Xaa2 is any amino acid, preferably an aromatic amino acid, more preferably Phe or Tyr, and most preferably Tyr; and Xaa3 is any amino acid, preferably Leu or Phe, and most preferably Leu; in one embodiment, the HCDR3 comprises the sequence set forth in SEQ ID NO: 46 or 47; The VL comprises a light chain complementarity determining region 3 (LCDR3) comprising the sequence Gln Gln Arg Xaa1 Xaa2 Xaa3 Pro Pro Trp Thr, where Xaa1 is any amino acid, preferably Ser or Asn, most preferably Ser, Xaa2 is any amino acid, preferably Tyr, Ser, Ile, Asn, or Thr, more preferably Ile, Asn, or Thr, most preferably Ile or Asn, and Xaa3 is any amino acid, preferably Ser or Tyr, more preferably Tyr. In one embodiment, the LCDR3 comprises the sequence set forth in SEQ ID NO: 52 or 53.

[0201] In one embodiment, the VH further comprises an HCDR1 comprising the sequence set forth in SEQ ID NO: 44 and / or an HCDR2 comprising the sequence set forth in SEQ ID NO: 48, wherein Xaa is any amino acid, preferably Thr, Ser or Ile, most preferably Thr, such as the sequence set forth in SEQ ID NO: 45, and / or the VL further comprises an LCDR1 comprising the sequence set forth in SEQ ID NO: 54, wherein Xaa is any amino acid, preferably Ser or Asn, most preferably Ser, such as the sequence set forth in SEQ ID NO: 50 and / or an LCDR2 comprising the sequence set forth in SEQ ID NO: 51.

[0202] In a preferred embodiment, the binding domain that binds to CLDN6 of the binding agent of the present invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): VH comprises HCDR1 comprising the sequence shown in SEQ ID NO: 44, HCDR2 comprising the sequence shown in SEQ ID NO: 45, and HCDR3 comprising the sequence shown in SEQ ID NO: 46; VL comprises LCDR1 comprising the sequence shown in SEQ ID NO: 50, LCDR2 comprising the sequence shown in SEQ ID NO: 51, and LCDR3 comprising the sequence shown in SEQ ID NO: 52; Includes.

[0203] In a preferred embodiment, the binding domain that binds to CLDN6 of the binding agent of the present invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): VH comprises HCDR1 comprising the sequence shown in SEQ ID NO: 44, HCDR2 comprising the sequence shown in SEQ ID NO: 45, and HCDR3 comprising the sequence shown in SEQ ID NO: 47; VL comprises LCDR1 comprising the sequence shown in SEQ ID NO: 50, LCDR2 comprising the sequence shown in SEQ ID NO: 51, and LCDR3 comprising the sequence shown in SEQ ID NO: 53; Includes.

[0204] In a preferred embodiment, the binding domain that binds to CLDN6 of the binding agent of the present invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): VH comprises HCDR1 comprising the sequence shown in SEQ ID NO: 44, HCDR2 comprising the sequence shown in SEQ ID NO: 45, and HCDR3 comprising the sequence shown in SEQ ID NO: 46; VL comprises LCDR1 comprising the sequence shown in SEQ ID NO: 50, LCDR2 comprising the sequence shown in SEQ ID NO: 51, and LCDR3 comprising the sequence shown in SEQ ID NO: 53; Includes.

[0205] In one embodiment, the heavy and light chain variable regions comprise the complementarity determining regions interspersed within framework regions. In one embodiment, each variable region comprises three complementarity determining regions (CDR1, 2, and 3) and four framework regions (FR1, 2, 3, and 4). In one embodiment, the complementarity determining regions and the framework regions are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0206] In a further embodiment, the binding domain of the binding agent of the present invention that binds to CLDN6 comprises the heavy and light chain variable regions of (i) an antibody that competes for CLDN6 binding with an antibody comprising the above-mentioned heavy and light chain variable regions and / or (ii) an antibody that has the specificity for CLDN6 of an antibody comprising the above-mentioned heavy and light chain variable regions.

[0207] In one embodiment, the heavy chain variable region (VH) and light chain variable region (VL) of the CLDN6-binding domain of the binding agent of the present invention have the format of an scFv molecule. In this embodiment, the CLDN6-binding domain of the binding agent of the present invention comprises the amino acid sequence shown in SEQ ID NO: 13 or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0208] It should be understood that the CLDN6-binding domains of a binding agent of the present invention may be identical or essentially identical, or different, and thus may bind to the same or essentially the same epitope or different epitopes. Thus, both CLDN6-binding domains of a binding agent of the present invention may correspond or essentially correspond to one of the CLDN6-binding domains of a binding agent of the present invention described herein, or may be independently selected from the CLDN6-binding domains of a binding agent of the present invention described herein.

[0209] Anti-CD3 antibodies useful for providing binding agents according to the present invention include, but are not limited to, UCHT1-HS (humanized mAB), UCHT1-MM (murine mAB), CLB-T3, TR66, 145-2C11.

[0210] UCHT1 is a monoclonal IgG1 anti-CD3 antibody that detects CD3 in a variety of human and primate specimens. CLB-T3 is a mouse monoclonal anti-CD3 antibody directed against the CD3 antigen and reacts with 80-90% of human peripheral T lymphocytes and medullary thymocytes. TR66 is a mouse IgG1 monoclonal anti-CD3 antibody that recognizes the epsilon chain of human CD3. 145-2C11 is an Armenian hamster monoclonal anti-mouse CD3 antibody.

[0211] Preferably, the VH and VL regions of the CD3-binding domain are derived from antibodies / antibody molecules and antibody-like molecules capable of specifically recognizing human CD3 in the context of other TCR subunits present on activated primary human T cells expressing the TCR in its native configuration. Most preferred are VH and VL regions derived from antibodies specific for the CD3-epsilon chain, said (parent) antibody being capable of specifically binding to an epitope reflecting the native or near-native structure or conformational epitope of human CD3 presented in the context of a TCR complex. In a preferred embodiment of the invention, the VH and VL regions of the CD3-binding domain are derived from a CD3-specific antibody selected from the group consisting of UCHT1-HS, UCHT1-MM, CLB-T3 and TR66, preferably TR66.

[0212] In a preferred embodiment, the CD3-binding domain of a binding agent of the invention comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 5 or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0213] In a preferred embodiment, the CD3-binding domain of a binding agent of the invention comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 6 or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0214] In a preferred embodiment, the CD3-binding domain of a binding agent of the invention comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL): VH comprises the amino acid sequence represented by SEQ ID NO: 5 or a fragment thereof, or a variant of said amino acid sequence or fragment, and VL comprises the amino acid sequence represented by SEQ ID NO: 6 or a fragment thereof, or a variant of said amino acid sequence or fragment. Includes.

[0215] The term "fragment" particularly refers to one or more complementarity determining regions (CDRs), preferably at least the CDR3 variable region of the heavy chain variable region (VH) and / or light chain variable region (VL). In one embodiment, said one or more complementarity determining regions (CDRs) are selected from the set of complementarity determining regions CDR1, CDR2 and CDR3. In a particularly preferred embodiment, the term "fragment" refers to the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL).

[0216] In one embodiment, a binding domain comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs described herein comprises said CDRs together with their intervening framework regions. Preferably, the portion comprises at least about 50% of one or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of binding agents made by recombinant DNA techniques may lead to the introduction of residues N- or C-terminal to the encoded variable region by a linker introduced to facilitate cloning or other manipulation steps, for example, by introducing a linker that connects the variable region of the invention to further protein sequences, including immunoglobulin heavy chains, other variable domains, or protein tags.

[0217] In one embodiment, a binding domain comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs described herein comprises said CDRs in a human antibody framework.

[0218] It should be understood that the binding agents described herein can be delivered to a patient by administering a nucleic acid, such as RNA, encoding the agent and / or by administering host cells containing a nucleic acid, such as RNA, encoding the agent. When a binding agent comprises two or more polypeptide chains, the different polypeptide chains may encode the same or different nucleic acids. Thus, the administered nucleic acid may be a mixture of different nucleic acid molecules. When administered to a patient, the nucleic acid encoding the binding agent may be in a naked form, present in a suitable delivery vehicle, such as a liposome, nanoparticle, or viral particle, or present within a host cell. The provided nucleic acid can produce an agent that persistently alleviates the instability observed, at least in part, for a long period of time with therapeutic antibodies. The nucleic acid delivered to a patient can be produced by recombinant means. When the nucleic acid is administered to a patient without being present within the host cell, it is preferably taken up by the patient's cells for expression of the binding agent encoded by the nucleic acid. When the nucleic acid is administered to a patient while present in the host cell, it is preferably expressed by the host cell within the patient's body to produce the binding agent encoded by the nucleic acid.

[0219] The term "recombinant" in the context of the present invention means "produced by genetic engineering." Preferably, a "recombinant," e.g., a recombinant nucleic acid, in the context of the present invention, is not naturally occurring.

[0220] As used herein, the term "naturally occurring" refers to the fact that an entity can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by man in a laboratory is naturally occurring.

[0221] The term "nucleic acid," as used herein, is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0222] The nucleic acid may be contained in a vector. As used herein, the term "vector" includes any vector known to those skilled in the art, such as a plasmid vector, a cosmid vector, a phage vector, such as lambda phage, a viral vector, such as an adenovirus or baculovirus vector, or an artificial chromosome vector, such as a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a P1 artificial chromosome (PAC). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors. Expression vectors generally contain a desired coding sequence and appropriate DNA sequences required to express the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammalian) or in an in vitro expression system. Cloning vectors are generally used to engineer and amplify desired DNA fragments and may lack functional sequences required for expression of the desired DNA fragment.

[0223] In the context of the present invention, the term "RNA" refers to a molecule comprising, preferably composed entirely or substantially of, ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. This term includes double-stranded RNA, single-stranded RNA, isolated RNA, including partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from natural RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications can include the addition of non-nucleotide material, for example, to the end of the RNA, for example, at one or more nucleotides of the RNA. Nucleotides in an RNA molecule can also include non-standard nucleotides, such as unnatural nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs are sometimes referred to as analogs or analogs of natural RNA.

[0224] According to the present invention, the term "RNA" includes and preferably relates to "mRNA," which means "messenger RNA," and further relates to "transcripts" that may be produced using DNA as a template and encode peptides or proteins. mRNA typically comprises a 5'-untranslated region (5'-UTR), a protein or peptide coding region, and a 3'-untranslated region (3'-UTR). mRNA has a limited half-life in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the present invention, RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to those skilled in the art. For example, various in vitro transcription kits are commercially available.

[0225] In one embodiment of the invention, the RNA is a self-replicating RNA, such as a single-stranded self-replicating RNA. In one embodiment, the self-replicating RNA is a positive-sense single-stranded RNA. In one embodiment, the self-replicating RNA is a viral RNA or RNA derived from a viral RNA. In one embodiment, the self-replicating RNA is an alphavirus genomic RNA or is derived from an alphavirus genomic RNA. In one embodiment, the self-replicating RNA is a viral gene expression vector. In one embodiment, the virus is Semliki Forest virus. In one embodiment, the self-replicating RNA comprises one or more transgenes, at least one of which encodes a binding factor described herein. In one embodiment, when the RNA is a viral RNA or is derived from a viral RNA, the transgene may partially or completely replace viral sequences, such as viral sequences encoding structural proteins. In one embodiment, the self-replicating RNA is an in vitro transcribed RNA.

[0226] The genome of alphaviruses is a positive-sense single-stranded RNA (ssRNA(+)) encoding two open reading frames (ORFs) for a large polyprotein. The 5'-terminal ORF encodes the nonstructural proteins nSP1 to nSP4 (nsP1-4), which are translated and processed by an RNA-dependent RNA polymerase (replicase); the 3'-terminal ORF encodes the structural proteins—capsid and glycoprotein. Both ORFs are separated by a so-called subgenomic promoter (SGP), which controls the transcription of the structural ORF. When used as a gene vector, the structural proteins behind the SGP are typically replaced by a transgene. To package such vectors into viral particles, the structural proteins are typically expressed in trans from a helper construct. Alphaviruses replicate exclusively in the cytoplasm of infected cells at the RNA level. After infection, the ssRNA (+) genome serves as mRNA for translation of the nsP1234 polyprotein precursor, an early step in the viral life cycle where it is autoproteolytically processed into fragments nsP123 and nsP4. These fragments form a negative-strand replicase complex that transcribes negative-strand RNA from the genomic RNA template. Later, the nsP1234 polyprotein is cleaved completely into subgenomic transcripts or transgenes encoding structural proteins, as well as single proteins that assemble the positive-strand replicase complex that synthesizes the new positive-strand genome. The subgenomic RNA and the new genomic RNA are capped and polyadenylated, and thus recognized as mRNA after infection of the target cell. Only the new genomic RNA contains a packaging signal that ensures exclusive packaging of the genomic RNA into budding virions. The attractiveness of alphavirus replicons for vectorology is based on the positive orientation of the capped and polyadenylated RNA genome.Translatable replicon RNA can be easily synthesized in vitro, where capping may be achieved using a cap analog added to the in vitro transcription reaction, or the transcription reaction and poly(A) tail may be encoded as a poly(T) track on the plasmid template. In vitro transcribed (IVT) replicons are transfected by conventional transfection techniques, rapidly increasing even with small amounts of starting IVT RNA. Within a few hours of transcription, transgenes placed downstream of the SGP are transcribed to very high copy numbers, approximately 40,000–200,000 subgenomic RNAs per cell, and it is not surprising that recombinant proteins are therefore strongly expressed. Depending on the specific purpose, IVT replicons can be directly transfected into target cells or packaged into alphavirus particles with a helper vector that delivers the structural gene in transfection. Translocation into skin or muscle leads to high and sustained local expression, paralleled by robust induction of humoral and cellular immune responses.

[0227] Modifications may be made to increase the expression and / or stability of the RNA used in the present invention, preferably without altering the sequence of the expressed peptide or protein.

[0228] The term "modification" in the context of RNA as used according to the present invention includes any modification of RNA that does not occur naturally in said RNA.

[0229] In one embodiment of the present invention, the RNA used in accordance with the present invention does not have uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating the RNA with a phosphatase.

[0230] The RNA according to the present invention may contain modified natural or synthetic ribonucleotides to increase stability and / or reduce cytotoxicity and / or immunogenicity. For example, in one embodiment, 5-methylcytidine is partially or completely, preferably completely, replaced by cytidine in the RNA used according to the present invention. Alternatively or additionally, in one embodiment, pseudouridine is partially or completely, preferably completely, replaced by uridine in the RNA used according to the present invention.

[0231] In one embodiment, the term "modified" relates to providing RNA with a 5' cap or a 5' cap analog. The term "5' cap" refers to the cap structure found at the 5' end of an mRNA molecule, generally consisting of a guanosine nucleotide attached to the mRNA via an unconventional 5'-5' triphosphate linkage. In one embodiment, the guanosine is methylated at position 7. A "normal 5' cap" refers to the 5' cap of a natural RNA, preferably the 7-methylguanosine cap (m7G). In the context of the present invention, the term "5' cap" includes 5' cap analogs that have been modified to resemble the RNA cap structure and have the ability to stabilize the RNA, preferably in vivo and / or intracellularly, when bound to the RNA.

[0232] Providing RNA with a 5' cap or 5' cap analog can be achieved by in vitro transcription of a DNA template in the presence of the 5' cap or 5' cap analog, where the 5' cap is co-transcriptionally incorporated into the generated RNA strand, or RNA can be generated, for example by in vitro transcription, and the 5' cap added to the RNA post-transcriptionally using a capping enzyme, for example, using vaccinia virus capping enzyme.

[0233] The RNA may comprise further modifications. For example, further modifications of the RNA used in the present invention may be an extension or shortening of the naturally occurring poly(A) tail, or a modification of the 5'- or 3'-untranslated region (UTR), such as the introduction of a UTR that is not associated with the coding region of the RNA, for example the insertion of one or more, preferably two copies of a 3'-UTR from a globin gene, such as alpha2-globin, alpha1-globin, beta-globin, preferably beta-globin, more preferably human beta-globin.

[0234] Therefore, to increase the stability and / or expression of the RNA used according to the present invention, it may be modified to contain a polyA sequence, preferably having 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, and particularly 100 to 150 adenosine residues. In a particularly preferred embodiment, the polyA sequence has a length of approximately 120 adenosine residues. Furthermore, the incorporation of two or more 3'-untranslated regions (UTRs) into the 3'-untranslated region of an RNA molecule can improve translation efficiency. In a specific embodiment, the 3'-UTR is derived from the human β-globin gene.

[0235] Preferably, the delivered or transfected RNA into cells, particularly cells present in vivo, expresses the encoded protein, peptide or antigen.

[0236] The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of the present invention, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, where the cells may be present in a subject, e.g., a patient. Thus, according to the present invention, cells for transfection of nucleic acids as described herein can be present in vitro or in vivo, e.g., the cells can form part of an organ, tissue, and / or organism of a patient. According to the present invention, transfection can be transient or stable. For some applications of transfection, it is sufficient that the transfected genetic material is expressed only transiently. Because nucleic acids introduced during the transfection process are not typically integrated into the nuclear genome, the foreign nucleic acid is diluted out via mitosis or degradation. Cells that allow episomal amplification of nucleic acids have a significantly reduced dilution rate. Stable transfection must occur if it is desired that the transfected nucleic acid actually remain in the genome of the cell or daughter cells. RNA can be transfected into cells to express its encoded protein.

[0237] The term "stability" of RNA relates to the "half-life" of RNA. "Half-life" refers to the period of time required to halve the activity, amount or number of a molecule. In the context of the present invention, the half-life of an RNA is an indicator of the stability of said RNA. The half-life of an RNA may affect the "duration of expression" of the RNA. RNA with a long half-life can be expected to be expressed for a long period of time.

[0238] In the context of the present invention, the term "transcription" relates to the process by which the genetic code in a DNA sequence is transcribed into RNA. The RNA may then be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription", where the term "in vitro transcription" relates to a process by which RNA, in particular mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, cloning vectors are applied for the production of transcripts. These cloning vectors are generally designated as transcription vectors and are encompassed by the term "vector" according to the present invention.

[0239] The term "translation" according to the present invention relates to the process in the ribosomes of a cell where a chain of messenger RNA directs the assembly of a sequence of amino acids to make a peptide or protein.

[0240] The term "expression" is used in accordance with the present invention in its most general sense and relates to the production of RNA and / or peptides or proteins, for example by transcription and / or translation. With respect to RNA, the terms "expression" or "translation" particularly relate to the production of peptides or proteins. It also includes partial expression of nucleic acids. Furthermore, expression may be transient or stable. According to the present invention, the term "expression" also includes "aberrant expression" or "unusual expression".

[0241] "Aberrant expression" or "unusual expression" according to the present invention means that expression is altered, preferably increased, compared to a reference, e.g., the state of a subject without a disease associated with the abnormal or unusual expression of a particular protein, e.g., a tumor antigen. Increased expression refers to an increase of at least 10%, particularly at least 20%, at least 50%, or at least 100% or more. In one embodiment, expression is found only in diseased tissue and is suppressed in healthy tissue.

[0242] The term "specifically expressed" means that a protein is essentially expressed only in a particular tissue or organ. For example, a tumor antigen that is specifically expressed in the gastric mucosa means that the protein is primarily expressed in the gastric mucosa and not expressed in other tissues or to a significant extent in other types of tissues or organs. Thus, a protein that is exclusively expressed in the gastric mucosa and is significantly less expressed in any other tissue, such as the testis, is specifically expressed in the gastric mucosa. In some embodiments, a tumor antigen may be specifically expressed in two or more tissue types or organs, for example, two or three tissue types or organs, but preferably three or fewer different tissue or organ types, under normal conditions. In this case, the tumor antigen is specifically expressed in these organs. For example, if a tumor antigen is expressed in the lung and the stomach, preferably to approximately equal degrees, under normal conditions, the tumor antigen is specifically expressed in the lung and the stomach.

[0243] According to the present invention, the term "RNA encoding" means RNA that can be expressed to produce the encoded protein or peptide when present in an appropriate environment, preferably within a cell.

[0244] Some aspects of the invention rely on adoptive transfer of host cells that are transfected in vitro with nucleic acids, such as RNA, encoding the binding agents described herein, preferably expanded ex vivo to clinically relevant cell numbers from low precursor frequencies, and then transferred into a recipient, such as a patient. Host cells used for treatment according to the invention may be autologous, allogeneic, or syngeneic to the treated recipient.

[0245] The term "autologous" is used to describe something that is derived from the same subject. For example, "autologous transplant" refers to the transplantation of tissue or organs from the same subject. Such procedures are advantageous because they overcome immunological barriers that otherwise result in rejection.

[0246] The term "allogeneic" is used to describe something that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.

[0247] The term "syngeneic" is used to describe individuals or tissues that have the same genotype, i.e., derived from identical twins or animals of the same breed.

[0248] The term "xenogeneic" is used to describe something that is composed of multiple dissimilar elements. As an example, transferring bone marrow from one individual to a different individual constitutes a xenograft. A xenogeneic gene is a gene that comes from a source other than the subject.

[0249] The term "peptide" according to the present invention includes oligo- and polypeptides and refers to a substance comprising two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably nine or more, preferably ten or more, preferably thirteen or more, preferably more than sixteen, preferably twenty one or more, and preferably up to 8, 10, 20, 30, 40 or 50, especially 100 amino acids covalently linked by peptide bonds. The term "protein" refers to large peptides, preferably peptides having more than 100 amino acid residues, although in general the terms "peptide" and "protein" are synonymous and are used interchangeably herein.

[0250] Any teachings provided herein regarding specific amino acid sequences, e.g., those shown in the Sequence Listing, should also be construed to relate to variants of the specific sequences, leading to sequences that are functionally equivalent to the specific sequences, e.g., amino acid sequences that exhibit the same or similar properties as the specific amino acid sequences. One important property is retaining target binding or effector function. Preferably, a sequence that is variant with respect to a specific sequence, when substituted for a specific sequence in an antibody, retains the binding of the antibody to claudins and / or CD3, preferably the function of the antibody described herein, e.g., CDC-mediated lysis or ADCC-mediated lysis. Furthermore, preferably, a sequence that is variant with respect to a specific sequence, when substituted for a specific sequence in a binding agent, retains the binding of the binding agent to claudins and / or CD3, preferably the function of the binding agent described herein, e.g., cytotoxic T cell-mediated lysis.

[0251] For example, the sequences shown in the sequence listing may be modified to remove one or more, preferably all, free cysteine ​​residues, in particular by replacing the cysteine ​​residue with an amino acid other than cysteine, preferably serine, alanine, threonine, glycine, tyrosine, tryptophan, leucine or methionine.

[0252] It will be understood by those skilled in the art that the sequences of the CDRs, hypervariable, and variable regions, in particular, can be modified without losing the ability to bind to claudins and / or CD3. For example, the CDR regions can be identical or highly homologous to the regions described herein. By "highly homologous," it is intended that 1 to 5, preferably 1 to 4, e.g., 1 to 3, or 1 or 2 substitutions can be made in the CDRs. Furthermore, the hypervariable and variable regions can be modified to exhibit substantial homology with the regions specifically disclosed herein. In one embodiment, the variable region sequences can only deviate from the variable region sequences specifically disclosed herein in the framework sequences.

[0253] For the purposes of the present invention, "variants" of an amino acid sequence include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. Amino acid deletion variants, including deletions at the N-terminus and / or C-terminus of the protein, are also referred to as N-terminal and / or C-terminal truncation variants.

[0254] Amino acid insertion variants include the insertion of one or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertions with appropriate screening of the resulting products are also possible.

[0255] Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.

[0256] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be at any position in the protein.

[0257] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or the substitution of an amino acid with another amino acid with similar properties are preferred. Preferably, the amino acid changes in protein variants are conservative amino acid changes, i.e., substitutions with similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions with members of a family of amino acids related to their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are jointly classified as aromatic amino acids.

[0258] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given over the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0259] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.

[0260] The term "percent identity" is intended to refer to the percentage of amino acid residues that are identical between the two sequences being compared, obtained after best alignment, and this percentage is purely statistical, with the differences between the two sequences being randomly distributed over their entire length. Sequence comparison between two amino acid sequences is usually performed by comparing these sequences after they have been optimally aligned, said comparison being performed by segments or "comparison windows" in order to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be created manually, or by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Inc., 575 Science Drive, Madison, Wis.).

[0261] The percent identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions being compared, and multiplying the result by 100 to obtain the percent identity between the two sequences.

[0262] Binding agents of the invention can be produced intracellularly (e.g., in the cytosol, periplasm, or inclusion bodies) with subsequent isolation from the host cells and, optionally, further purification, or extracellularly (e.g., in the medium in which the host cells are cultured) with subsequent isolation from the culture medium and, optionally, further purification. The methods and reagents used for recombinant production of polypeptides, such as particularly appropriate expression vectors, transformation or transfection methods, selectable markers, methods for inducing protein expression, culture conditions, etc., are well known to those skilled in the art. Similarly, protein isolation and purification techniques are well known to those skilled in the art.

[0263] The term "cell" or "host cell" relates to an intact cell, i.e., a cell with an intact membrane that has not released normal intracellular components such as enzymes, organelles, or genetic material. An intact cell is preferably a viable cell, i.e., a living cell that is capable of performing normal metabolic functions. Preferably, the term relates, according to the present invention, to any cell that can be transfected with an exogenous nucleic acid. Preferably, the cell is capable of transfecting an exogenous nucleic acid and expressing the nucleic acid in the recipient when transferred to the recipient. The term "cell" includes bacterial cells. Other useful cells are yeast cells, fungal cells, or mammalian cells. Suitable bacterial cells include cells from gram-negative bacterial strains such as Escherichia coli, Proteus, and Pseudomonas, and gram-positive bacterial strains such as Bacillus subtilis, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cells include cells from species of Trichoderma, Neurospora, and Aspergillus. Suitable yeast cells include cells from species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, HEK293 cells, etc. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for expression of heterologous proteins may be used as well.Mammalian cells, such as human, mouse, hamster, pig, goat, and primate cells, are particularly preferred for adoptive transfer. Cells can be derived from numerous tissue types and include primary cells and cell lines, such as cells of the immune system, antigen-presenting cells, particularly dendritic cells and T cells, stem cells, such as hematopoietic stem cells and mesenchymal stem cells, as well as other cell types. Antigen-presenting cells are cells that present antigens in the context of major histocompatibility complexes on their surface. T cells can recognize this complex using their T cell receptors (TCRs).

[0264] As used herein, "reduce," "lower," or "inhibit" refers to an overall decrease or ability to cause an overall decrease, preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more, at a level, e.g., level of expression or level of cell proliferation.

[0265] Terms such as "increase" or "enhance" relate to an increase or enhancement of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or more.

[0266] Antibody-dependent cell-mediated cytotoxicity ADCC refers to the cell killing ability of effector cells, particularly lymphocytes, as described herein, which preferably requires that the target cells be marked with an antibody.

[0267] ADCC preferably occurs when an antibody binds to an antigen on a tumor cell, and the antibody Fc domain binds to an Fc receptor (FcR) on the surface of an immune effector cell. Several families of Fc receptors have been identified, and specific cell populations characteristically express defined Fc receptors. ADCC can be viewed as a mechanism that directly induces varying degrees of immediate tumor destruction, leading to antigen presentation and the induction of tumor-directed T cell responses. Preferably, in vivo induction of ADCC leads to tumor-directed T cell responses and host-derived antibody responses.

[0268] Complement-dependent cytotoxicity CDC is another cell killing method that can be induced by antibodies. IgM is the most effective isotype for complement activation. Both IgG1 and IgG3 are highly effective in directing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex is initiated by the CDC of the participating antibody molecules, such as IgG molecules. H This results in the exposure of multiple C1q binding sites adjacent to the C1 domain (C1q is one of the three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert the previously low affinity C1q-IgG interaction to one of high avidity, which triggers a cascade of events involving a series of other complement proteins, leading to the proteolytic release of the effector cell chemotactic / activating agents C3a and C5a. Preferably, the complement cascade culminates in the formation of a membrane attack complex, which forms pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell.

[0269] Antibodies described herein, e.g., antibodies for forming the VL and VH regions, can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique described by Kohler and Milstein, Nature 256:495 (1975). While somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can be utilized, e.g., viral or oncogenic transformation of B lymphocytes or phage display techniques using libraries of antibody genes.

[0270] The preferred animal system for preparing hybridomas secreting monoclonal antibodies is the murine system. Hybridoma production in mice is a very well-established procedure. Immunization protocols for fusion and techniques for isolation of immunized splenocytes are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0271] Other preferred animal systems for preparing hybridomas secreting monoclonal antibodies are the rat and rabbit systems (see, e.g., Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92:9348 (1995) and Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

[0272] In yet another preferred embodiment, human monoclonal antibodies can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice." Production of human antibodies in such transgenic mice can be performed as described in detail for CD20 in WO 2004 / 035607.

[0273] Yet another strategy for generating monoclonal antibodies is to directly isolate the antibody-encoding genes from lymphocytes that produce antibodies of defined specificities. See, e.g., Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. For details on recombinant antibody engineering, see also Welschof and Kraus, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny KC Lo, Antibody Engineering ISBN 1-58829-092-1.

[0274] To generate antibodies, mice may be immunized with carrier-conjugated peptides derived from the antigen sequence, i.e., the sequence to which the antibody is directed as described herein, recombinantly expressed antigen or fragment thereof, and / or an enriched preparation of cells expressing the antigen. Alternatively, mice may be immunized with DNA encoding the antigen or fragment thereof. If immunization with a purified or enriched preparation of antigen does not produce antibodies, mice may be immunized with cells, e.g., a cell line, expressing the antigen to boost the immune response.

[0275] The immune response may be monitored over the course of the immunization protocol with plasma and serum samples obtained by tail vein or retroorbital bleeds. Mice with sufficient titers of immunoglobulin may be used for fusions. Mice may be boosted intraperitoneally or intravenously with antigen-expressing cells 3 days before sacrifice and removal of the spleen to enrich for the proportion of specific antibody-secreting hybridomas.

[0276] To generate hybridomas that produce monoclonal antibodies, spleen cells and lymph node cells from immunized mice may be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas may then be screened for the production of antigen-specific antibodies. Individual wells may then be screened for antibody-secreting hybridomas by ELISA. Antibodies with specificity for the antigen can be identified by immunofluorescence and FACS analysis using antigen-expressing cells. Antibody-secreting hybridomas may be replated, screened again, and if still positive for monoclonal antibodies, may be subcloned by limiting dilution. Stable subclones may be cultured in vitro to produce and characterize antibodies in tissue culture medium.

[0277] Antibodies may also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods, as is well known in the art (Morrison, S. (1985) Science 229:1202).

[0278] For example, in one embodiment, a gene of interest, e.g., an antibody gene, can be ligated into an expression vector, such as a eukaryotic expression plasmid, used with the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338 841, or other expression systems known in the art. The purified plasmid carrying the cloned antibody gene can be introduced into eukaryotic host cells, such as CHO cells, NS / 0 cells, HEK293T cells, or HEK293 cells, or alternatively, other eukaryotic cells, such as plant-derived cells, fungal cells, or yeast cells. The method used to introduce these genes can be any method described in the art, such as electroporation, lipofectin, or lipofectamine. After introducing these antibody genes into host cells, cells expressing the antibody can be identified and selected. These cells represent transfectomas, which can then be scaled up to amplify the expression levels of their antibody genes and produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0279] Alternatively, cloned antibody genes can be expressed in other expression systems, including prokaryotic cells such as microorganisms, e.g., E. coli. Furthermore, antibodies can be produced in the milk of sheep and rabbits, or in hens' eggs, or in transgenic non-human animals, such as transgenic plants. See, e.g., Verma, R. et al. (1998) J. Immunol. Meth. 216:165-181; Pollock et al. (1999) J. Immunol. Meth. 231:147-157; and Fischer, R. et al. (1999) Biol. Chem. 380:825-839.

[0280] Chimerization Unlabeled mouse antibodies are highly immunogenic in humans, leading to a decrease in therapeutic efficacy when administered repeatedly. The primary immunogenicity is mediated by the heavy chain constant region. The immunogenicity of mouse antibodies in humans can be reduced or completely avoided if the respective antibodies are chimerized or humanized. Chimeric antibodies are antibodies in which different portions of the antibody are derived from different animal species, such as antibodies with a variable region derived from a mouse antibody and a human immunoglobulin constant region. Antibody chimerization is achieved by linking the variable regions of the heavy and light chains of a mouse antibody to human heavy and light chain constant regions (e.g., as described in Kraus et al., Methods in Molecular Biology series, Recombinant antibodies for cancer therapy, ISBN-0-89603-918-8). In a preferred embodiment, a chimeric antibody is generated by linking a human kappa light chain constant region to a mouse light chain variable region. In another preferred embodiment, a chimeric antibody is generated by linking a human lambda light chain constant region to a mouse light chain variable region. Preferred heavy chain constant regions for generating chimeric antibodies are IgG1, IgG3, and IgG4. Other preferred heavy chain constant regions for generating chimeric antibodies are IgG2, IgA, IgD, and IgM.

[0281] Humanization Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity-determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse among individual antibodies than sequences outside the CDRs. Because CDR sequences are involved in most antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing expression vectors containing CDR sequences from that particular naturally occurring antibody grafted onto framework sequences from different antibodies with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033). Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. These germline sequences differ from mature antibody gene sequences because they do not contain fully assembled variable genes formed by V(D)J joining during B-cell maturation, and they differ from high-affinity secondary repertoire antibody sequences, which individually span the variable regions uniformly.

[0282] The ability of antibodies and other binding agents to bind to an antigen can be determined using standard binding assays (e.g., ELISA, Western blot, immunofluorescence and flow cytometry analysis).

[0283] To purify the antibody, the selected producer cell line can be grown in a 2-liter spinner flask for recombinant antibody purification. Alternatively, the antibody can be produced in a dialysis-based bioreactor. The supernatant can be filtered, concentrated if necessary, and then subjected to affinity chromatography using protein L-Sepharose. The eluted antibody can be checked by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer is exchanged into PBS, and the concentration can be determined by OD280 using the respective extinction coefficients. The recombinant antibody can be aliquoted and stored at -65 to -85°C.

[0284] Flow cytometry may be used to demonstrate binding of monoclonal antibodies to live cells expressing the antigen. Cell lines expressing the antigen naturally or after transfection, as well as negative controls lacking antigen expression (grown under standard growth conditions), may be mixed with various concentrations of monoclonal antibodies in hybridoma supernatant or PBS containing 1% FBS and incubated for 30 minutes at 4°C. After washing, a fluorescently labeled detection reagent (e.g., fluorescently conjugated anti-IgG antibody, anti-Fab antibody, or protein L) may be combined with the antigen-bound monoclonal antibody under the same conditions as the primary antibody staining. Samples may be analyzed by flow cytometry on a FACS instrument using light and side scatter properties to gate on single live cells. Cotransfection methods can be used to distinguish antigen-specific monoclonal antibodies from nonspecific binders in a single measurement. Cells transiently transfected with plasmids encoding the antigen and a fluorescent marker can be stained as described above. Transfected cells may be detected in a different fluorescence channel than antibody-stained cells. When the majority of transfected cells express both transgenes, the antigen-specific monoclonal antibody will preferentially bind to the fluorescent marker-expressing cells, while the non-specific antibody will bind at a rate comparable to that of non-transfected cells. In addition to or instead of the flow cytometry assay, an alternative assay using a fluorescent microscope may be used. Cells can be stained exactly as described above and examined by fluorescent microscopy.

[0285] Immunofluorescence microscopy can be used to verify the binding of monoclonal antibodies to live cells expressing the antigen. For example, cell lines expressing the antigen spontaneously or after transfection, as well as negative controls lacking antigen expression, are grown in chamber slides under standard growth conditions in DMEM / F12 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin. Cells may be fixed with methanol or paraformaldehyde or left untreated. The cells can then be reacted with a monoclonal antibody against the antigen for 30 minutes at 25°C. After washing, the cells can be reacted with an Alexa555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. The cells can then be examined by fluorescence microscopy.

[0286] Cell extracts from cells expressing the antigen and appropriate negative controls can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked, and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed with ECL substrate.

[0287] Antibodies can also be tested for reactivity with the antigen by immunohistochemistry, using methods well known to those skilled in the art, for example, using paraformaldehyde- or acetone-fixed frozen sections or paraformaldehyde-fixed paraffin-embedded tissue sections from non-cancerous or cancerous tissue samples obtained from patients during routine surgery, or from mice bearing xenograft tumors inoculated with cell lines expressing the antigen, either spontaneously or after transfection. For immunostaining, antibodies reactive with the antigen can be subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies (DAKO) according to the vendor's instructions.

[0288] Preclinical trials The binding agents described herein can also be tested in in vivo models (e.g., in immunodeficient mice bearing xenograft tumors inoculated with claudin-expressing cell lines) to determine their effectiveness in controlling the growth of claudin-expressing tumor cells.

[0289] In vivo testing can be performed using the binding agents described herein after xenografting of claudin-expressing tumor cells into immunodeficient mice or other animals. The binding agents can be administered to tumor-free mice, followed by injection of tumor cells, to measure the effectiveness of the binding agents in preventing the formation of tumors or tumor-related symptoms. The binding agents can be administered to tumor-bearing mice to determine the therapeutic efficacy of each binding agent in reducing tumor growth, metastasis, or tumor-related symptoms. Application of the binding agents can be combined with the application of other agents, such as mitogenic drugs, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors, or antibodies, to determine the synergistic efficacy and potential toxicity of the combination. To analyze toxic side effects mediated by the binding agents, animals can be inoculated with the binding agents or control agents and thoroughly examined for symptoms potentially associated with claudin-binding agent therapy.

[0290] Mapping of epitopes recognized by binding factors can be performed as described in detail in "Epitope Mapping Protocols (Methods in Molecular Biology)" by Glenn E. Morris, ISBN-089603-375-9 and "Epitope Mapping: A Practical Approach" by Olwyn M. R. Westwood and Frank C. Hay, Practical Approach Series, 248.

[0291] The compounds and agents described herein can be administered in the form of any suitable pharmaceutical composition.

[0292] The pharmaceutical compositions of the present invention are preferably sterile and can contain an effective amount of a binding agent described herein and optionally an additional agent described herein to produce a desired response or a desired effect.

[0293] The pharmaceutical composition is usually provided in a certain dosage form and may be prepared in a manner known per se The pharmaceutical composition may, for example, be in the form of a solution or suspension.

[0294] Pharmaceutical compositions may include salts, buffers, preservatives, carriers, diluents and / or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to a material that is non-toxic and does not interact with the active ingredients of the pharmaceutical composition.

[0295] Pharmaceutically unacceptable salts may be used to prepare pharmaceutically acceptable salts and are included in the present invention. Such pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts may also be prepared as alkali metal salts, alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0296] Suitable buffering substances for use in pharmaceutical compositions include acetate, citrate, borate, and phosphate.

[0297] Suitable preservatives for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0298] Injectable formulations may contain pharmaceutically acceptable excipients such as Ringer's lactate solution.

[0299] The term "carrier" refers to a natural or synthetic organic or inorganic ingredient that is combined with an active ingredient to facilitate, enhance or enable application. According to the present invention, the term "carrier" includes one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration to a patient.

[0300] Carrier materials contemplated for parenteral administration are, for example, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes, and especially biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0301] The term "excipient," as used herein, is intended to include all substances that may be present in a pharmaceutical composition but which are not active ingredients, for example, carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents, or coloring agents.

[0302] The agents and compositions described herein can be administered via any conventional route, for example, parenterally, including injection or infusion. Administration is preferably parenteral, for example, intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly.

[0303] Compositions suitable for parenteral administration usually comprise a sterile aqueous or insoluble preparation of the active compound, which in certain embodiments is isotonic with the recipient's blood. Examples of suitable carriers and solvents include Ringer's solution and isotonic sodium chloride solution. Additionally, sterile fixed oils are usually used as a medium for solutions or suspensions.

[0304] The agents and compositions described herein are administered in an effective amount. An "effective amount" refers to an amount that alone or together with further doses achieves the desired response or desired effect. When treating a specific disease or a specific condition, the desired response particularly relates to inhibiting the progression of the disease. This includes slowing the progression of the disease, particularly halting or reversing the progression of the disease. In treating a disease or condition, the desired response may also be delaying the onset or preventing the onset of the disease or condition.

[0305] The effective amount of the agents or compositions described herein will depend on factors such as the condition being treated, the severity of the disease, individual parameters such as the patient's age, physical condition, size, and weight, the duration of treatment, the type of concomitant treatment (if any), and the particular route of administration. Thus, the dose administered of the agents described herein will depend on such various parameters. If the patient does not respond adequately to the initial dose, a higher dose (or a more effective dose achieved by a different, more localized route of administration) is used.

[0306] The agents and compositions described herein can be administered to a patient, e.g., in vivo, to treat or prevent various disorders, such as those described herein. Preferred patients include human patients with disorders that can be repaired or ameliorated by administering the agents and compositions described herein. This includes disorders involving cells characterized by altered expression patterns of claudins, such as CLDN18.2 and / or CLDN6.

[0307] For example, in one embodiment, the agents described herein can be used to treat a patient having a cancer disease, e.g., a cancer disease as described herein that is characterized by the presence of cancer cells that express claudins.

[0308] The pharmaceutical compositions and treatment methods described according to the present invention can also be used for immunization or vaccination to prevent the diseases described herein.

[0309] To further increase its effectiveness, preferably to achieve a synergistic immune stimulation effect, the pharmaceutical composition of the present invention may be administered together with or contain one or more supplementary immune enhancing substances, such as one or more adjuvants. The term "adjuvant" refers to a compound that prolongs, enhances, or promotes an immune response. Depending on the type of adjuvant, various mechanisms are possible in this regard. For example, compounds that enable DC maturation, such as lipopolysaccharide or CD40 ligand, form a first class of suitable adjuvants. In general, any agent that influences the immune system, such as "danger signal" species (LPS, GP96, dsRNA, etc.) or cytokines, such as GM-CSF, can be used as an adjuvant that can enhance and / or influence the immune response in a controlled manner. As mentioned above, CpG oligodeoxynucleotides can optionally be used in this context, although their side effects, which may occur under certain circumstances, should be taken into consideration. Particularly preferred adjuvants are cytokines such as monokines, lymphokines, interleukins, or chemokines, e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-α, INF-γ, GM-CSF, LT-α, or growth factors such as hGH. Further known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as Montanide®, most preferably Montanide® ISA51. Lipopeptides such as Pam3Cys are also suitable for use as adjuvants in the pharmaceutical compositions of the invention.

[0310] The agents and compositions provided herein can be used alone or in combination with conventional treatments such as surgery, radiation, chemotherapy and / or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated).

[0311] Cancer treatment is a particularly desirable area for combination strategies, since the combined action of two, three, four, or even more cancer drugs / therapies often produces significantly greater synergistic effects than the effects of monotherapy approaches. Therefore, in another embodiment of the present invention, cancer treatments utilizing immune- or vaccination-based mechanisms, such as the methods and pharmaceutical compositions of the present invention, may be effectively combined with a variety of other drugs and / or methods targeting similar or other specific mechanisms. These include, for example, combinations with conventional tumor therapies, multi-epitope strategies, additional immunotherapies, and treatment approaches targeting angiogenesis or apoptosis (for reviews, see, e.g., Andersen et al., 2008: Cancer treatment: the combination of vaccination with other therapies. Cancer Immunology Immunotherapy, 57(11):1735-1743). Sequential administration of different drugs may inhibit cancer cell growth at different checkpoints, while other drugs may inhibit, for example, angiogenesis, malignant cell survival or metastasis, or the potential for cancer to transform into a chronic disease. Some non-limiting examples of anti-cancer drugs and treatments that can be used in combination with the present invention are listed below.

[0312] 1. Chemotherapy Chemotherapy is the standard treatment for multiple types of cancer. Most common chemotherapy drugs act by killing rapidly dividing cells, which is one of the main characteristics of cancer cells. Therefore, the combination of conventional chemotherapy drugs such as alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor drugs that affect cell division or DNA synthesis can significantly improve the therapeutic effects of the present invention by eliminating suppressor cells, restarting the immune system, sensitizing tumor cells to immune-mediated killing, or further activating cells of the immune system. The synergistic anti-cancer effects of chemotherapy and vaccination-based immunotherapeutics have been demonstrated in multiple studies (see, e.g., Quoix et al., 2011: Therapeutic vaccination with TG4010 and first-line chemotherapy in advanced non-small-cell lung cancer: a controlled phase 2B trial. Lancet Oncol. 12(12):1125-33; also Liseth et al., 2010: Combination of intensive chemotherapy and anticancer vaccines in the treatment of human malignancies: the hematological experience. J Biomed Biotechnol. 2010:6920979; also Hirooka et al., 2009: A combination therapy of gemcitabine with immunotherapy for patients with inoperable locally advanced pancreatic cancer. Pancreas 38(3):e69-74). Essentially, there are hundreds of available chemotherapy drugs suitable for combination therapy.Some (non-limiting) examples of chemotherapeutic agents that can be combined with the present invention are carboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ), cyclophosphamide (Cytoxan, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (VePesid), fluorouracil (5-FU), gemcitabine (Gemzar), imatinib mesylate (Gleevec), irinotecan (Camptosar), methotrexate (FOLEX, MEXATE, amethopterin), paclitaxel (Taxol, Abraxane), sorafenib (Nexavar), sunitinib (Sutent), topotecan (Hycamtin), vincristine (Oncovin, Vincasar). PFS), and vinblastine (Velban).

[0313] 2. Surgery Cancer surgery—operation to remove tumors—remains the backbone of cancer treatment. Surgery can be combined with other cancer treatments to remove remaining tumor cells. Combining surgical methods with subsequent immunotherapeutic treatments is a promising approach that has been demonstrated countless times.

[0314] 3. Radiation Radiation therapy remains an important component of cancer treatment, with approximately 50% of cancer patients receiving radiation therapy during the course of their disease. The primary goal of radiation therapy is to deprive cancer cells of their ability to multiply (divide). The types of radiation used to treat cancer are photon radiation (X-rays and gamma rays) and particle radiation (electron, proton, and neutron beams). There are two methods for delivering radiation to the location of the cancer. External beam radiation is delivered from outside the body by directing high-energy rays (photon, proton, or particle beams) toward the tumor location. Internal radiation, or brachytherapy, is delivered from inside the body by a radioactive source encapsulated in a catheter or seeds directly to the tumor site. Radiation therapy techniques that may be used in conjunction with the present invention include, for example, fractionation (radiation therapy delivered in a fractionated regime, e.g., daily fractions of 1.5 to 3 Gy over several weeks), three-dimensional conformal radiation therapy (3DCRT; radiation delivery to the entire tumor volume), intensity-modulated radiation therapy (IMRT; computer-controlled intensity modulation of multiple radiation beams), image-guided radiation therapy (IGRT; a technique that includes pre-radiation imaging to allow correction), and stereotactic body radiotherapy (SRBT; very high doses of individual radiation delivered to a small number of treatment fractions). For a review of radiation therapy, see Baskar et al., 2012: Cancer and radiation therapy: current advances and future directions. Int. J Med Sci. 9(3):193-199.

[0315] 4. Antibodies Antibodies (preferably monoclonal antibodies) achieve their therapeutic effect on cancer cells through various mechanisms. They can have a direct effect by inducing apoptosis, or programmed cell death. For example, they can block components of signaling pathways, such as growth factor receptors, effectively inhibiting tumor cell growth. In cells expressing monoclonal antibodies, they can lead to the formation of anti-idiotypic antibodies. Indirect effects include recruiting cytotoxic cells, such as monocytes and macrophages. This type of antibody-mediated cell killing is called antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies also bind complement, resulting in direct cytotoxicity, known as complement-dependent cytotoxicity (CDC). Combining surgical methods with immunotherapeutic drugs or methods is a successful approach, as described, for example, in Gadri et al., 2009: Synergistic effect of dendritic cell vaccination and anti-CD20 antibody treatment in the therapy of murine lymphoma. J Immunother. 32(4):333-40. Some non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that can be used in combination with the present invention are listed below: abagovomab (CA-125), abciximab (CD41), adecatumumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), altumomab pentetate (CEA), amatuximab (MORAb-009), anatumomab mafenatoxin (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), bavituximab (phosphatidylserine), bectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumomab (CD19), brentuximab Vedotin (CD30 TNFRSF8), CantuzumabMertansine (mucin CanAg), cantuzumab ravtansine (MUC1), capromab pendetide (prostate cancer cells), carlumab (CNTO888), catumaxomab (EpCAM, CD3), cetuximab (EGFR), sitatuzumab bogatoxin (EpCAM), xiquistumumab (IGF-1 receptor), claudiximab (claudin), clivatuzumab Tetraxetan (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), dalotuzumab (insulin-like growth factor I receptor), denosumab (RANKL), detumomab (B-lymphoma cells), drozitumab (DR5), ecromeximab (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enavatuzumab (PDL192), ensituximab E (NPC-1C), epratuzumab (CD22), ertumaxomab (HER2 / neu, CD3), etaracizumab (integrin αvβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (SCH 900105), figitumumab (IGF-1 receptor), framvotumab (glycoprotein 75), fresolimumab (TGF-β), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gevokizumab (IL-1β), girentuximab (carbonic anhydrase 9 (CA-IX)), glembatumumab vedotin (GPNMB), ibritumomab tiuxetan (CD20), icrucumab (VEGFR-1), igovomab (CA-125), indatuximab Ravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30), labetuzumab (CEA), lexatumumab (TRAIL-R2), ribivirumab (hepatitis B surface antigen), lintuzumab (CD33), lorvotuzumab mertansine (CD56), lucatumumab (CD40), rumiliximab (CD23), mapatumumab (TRAIL-R1), matuzumab (EGFR), mepolizumab (IL-5), milatuzumab (CD74), mitumomab (GD3 ganglioside), mogamulizumab (CCR4), moxetumomab Pasudotox (CD22), nacolomabTafenatox (C242 antigen), naptumomab, estafenatox (5T4), narutumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumab (PDGF-R α), onartuzumab (human scatter factor receptor kinase), oportuzumab Monatox (EpCAM), oregovomab (CA-125), oxelumab (OX-40), panitumumab (EGFR), patritumab (HER3), pemtumomab (MUC1), pertuzumab (HER2 / neu), pintumomab (adenocarcinoma antigen), pritumumab (vimentin), racotumomab (N-glycolylneuraminic acid), radrez Mab (fibronectin extra domain-B), rafivirumab (rabies virus glycoprotein), ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), lobatumumab (IGF-1 receptor), samalizumab (CD200), sibrotuzumab (FAP), siltuximab (IL-6), tabalumab (BAFF), tacatuzumab Tetraxetan (alpha-fetoprotein), taplitumomab paptox (CD19), tenatumomab (tenascin-C), teprotumumab (CD221), ticilimumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL-13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucotuzumab celmoleukin (EpCAM), ublituximab (MS4A1), urelumab (4-1BB), volociximab (integrin α5β1), votumumab (tumor antigen CTAA16.88), zalutumumab (EGFR), and zanolimumab (CD4).

[0316] 5. Cytokines, chemokines, costimulatory molecules, and fusion proteins The combined use of pharmaceutical compositions encoding the antigens of the present invention with cytokines, chemokines, costimulatory molecules, and / or fusion proteins thereof that induce beneficial immunomodulatory or tumor-inhibitory effects is another embodiment of the present invention. Various cytokines with C, CC, CXC, and CX3C structures can be used to increase immune cell infiltration into tumors and facilitate the migration of antigen-presenting cells to tumor-draining lymph nodes. Some of the most promising chemokines are, for example, CCR7 and its ligands CCL19 and CCL21, as well as CCL2, CCL3, CCL5, and CCL16. Other examples are CXCR4, CXCR7, and CXCL12. Additionally, costimulatory or regulatory molecules, such as B7 ligands (B7.1 and B7.2), are useful. Other cytokines, such as interleukins, particularly (e.g., IL-1 through IL-17), interferons (e.g., IFN alpha 1 through IFN alpha 8, IFN alpha 10, IFN alpha 13, IFN alpha 14, IFN alpha 16, IFN alpha 17, IFN alpha 21, IFN beta 1, IFNW, IFNE1, and IFNK), hematopoietic factors, TGFs (e.g., TGF-α, TGF-β, and other members of the TGF family), terminal members of the tumor necrosis factor family of receptors and their ligands, and other stimulatory molecules, such as, but not limited to, 4-1BB, 4-1BB-L, CD137, CD Also useful are 137L, CTLA-4, GITR, GITRL, Fas, Fas-L, TNFR1, TRAIL-R1, TRAIL-R2, p75NGF-R, DR6, LT.beta.R, RANK, EDAR1, XEDAR, Fn114, Troy / Trade, TAJ, TNFRII, HVEM, CD27, CD30, CD40, 4-1BB, OX40, GITR, GITRL, TACI, BAFF-R, BCMA, RELT, and CD95 (Fas / APO-1), glucocorticoid-induced TNFR-associated protein, TNF receptor-associated apoptosis-mediating protein (TRAMP), and death receptor-6 (DR6). CD40 / CD40L and OX40 / OX40L, in particular, are important targets for combinatorial immunotherapy because of their direct impact on T cell survival and proliferation.For a review, see Lechner et al., 2011: Chemokines, costimulatory molecules and fusion proteins for the immunotherapy of solid tumors. Immunotherapy 3(11), 1317-1340.

[0317] 6. Bacterial treatment Researchers have used anaerobic bacteria, such as Clostridium novyi, to deplete the oxygen-poor interior of tumors. These bacteria die when they come into contact with the oxygenated side of tumors, meaning they are harmless to the rest of the body. Another strategy is to use anaerobic bacteria transformed with enzymes that can convert non-toxic prodrugs into toxic drugs. The bacteria grow in the necrotic and hypoxic areas of tumors, and the enzymes are expressed only in tumors. Thus, systemically applied prodrugs are metabolized into toxic drugs only in tumors. This has been demonstrated to be effective with the non-pathogenic anaerobic bacterium Clostridium sporogenes.

[0318] 7. Kinase inhibitors Another large group of promising targets for complementary cancer therapies includes kinase inhibitors, because cancer cell growth and survival are closely linked to deregulated kinase activity. A wide range of inhibitors are used to restore normal kinase activity and reduce tumor growth. The group of target kinases includes receptor tyrosine kinases such as BCR-ABL, B-Raf, EGFR, HER-2 / ErbB2, IGF-IR, PDGFR-α, PDGFR-β, c-Kit, Flt-4, Flt3, FGFR1, FGFR3, FGFR4, CSF1R, c-Met, RON, c-Ret, ALK, cytoplasmic tyrosine kinases such as c-SRC, c-YES, Abl, JAK-2, serine / threonine kinases such as ATM, Aurora A&B, CDKs, mTOR, PKCi, PLKs, b-Raf, S6K, STK11 / LKB1, and lipid kinases such as PI3K, SK1. Examples of small molecule kinase inhibitors include PHA-739358, nilotinib, dasatinib, and PD166326, NSC 743411, lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sutent (SU11248), sorafenib (BAY 43-9006), and leflunomide (SU101). For further information, see, for example, Zhang et al., 2009: Targeting cancer with small molecule kinase inhibitors. Nature Reviews Cancer 9, 28-39.

[0319] 8. Toll-like receptors Members of the Toll-like receptor (TLR) family are important for linking innate and adaptive immunity, and the effectiveness of many adjuvants depends on TLR activation. The majority of established cancer vaccines incorporate ligands for TLRs to enhance vaccine responses. In addition to TLR2, TLR3, TLR4, and particularly TLR7 and TLR8, are being considered for cancer treatment in passive immunotherapy approaches. The closely related TLR7 and TLR8 contribute to antitumor responses by influencing immune cells, tumor cells, and the tumor microenvironment and can be activated by nucleoside analog structures. All TLRs have been used as standalone immunotherapies or cancer vaccine adjuvants and can be synergistically combined with the formulations and methods of the present invention. For more information, see van Duin et al., 2005: Triggering TLR signaling in vaccination. Trends in Immunology, 27(1):49-55.

[0320] 9. Angiogenesis inhibitors In addition to therapies targeting immunomodulatory receptors and immunosuppression affected by tumor-mediated escape mechanisms, there are also therapies targeting the tumor environment. Angiogenesis inhibitors prevent the extensive growth of blood vessels (angiogenesis) that tumors need to survive. Angiogenesis, which is promoted by tumor cells to meet their increased nutrient and oxygen demands, can be blocked, for example, by targeting different molecules. Non-limiting examples of angiogenesis mediating molecules or angiogenesis inhibitors that can be combined with the present invention include soluble VEGF (VEGF isoforms VEGF121 and VEGF165, receptors VEGFR1, VEGFR2 and co-receptors neuropilin-1 and neuropilin-2) 1 and NRP-1, angiopoietin 2, TSP-1 and TSP-2, angiostatin and related molecules, endostatin, vasostatin, calreticulin, platelet factor-4, TIMPs and CDAI, Meth-1 and Meth-2, IFN-α, -β and -γ, CXCL10, IL-4, -12 and -18, prothrombin (kringle domain-2), antithrombin III fragment, prolactin, erythromycin ... These include vasculitis, VEGI, SPARC, osteopontin, maspin, canstatin, proliferin-related protein, restin, and drugs such as bevacizumab, itraconazole carboxyamidotriazole, TNP-470, CM101, IFN-α, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, angiogenic steroids plus heparin, cartilage-derived angiogenesis inhibitory factor, matrix metalloproteinase inhibitors, 2-methoxyestradiol, tecogalan, tetrathiomolybdic acid, thalidomide, thrombospondin, prolactin Vβ3 inhibitors, linomide, and tasquinimod. For a review, see, e.g., Schoenfeld and Dranoff 2011: Anti-angiogenesis immunotherapy. Hum Vaccin. (9):976-81.

[0321] 10. Small molecule targeted therapeutics Small molecule targeted therapeutics are generally inhibitors of the enzymatic domains of mutated, overexpressed, or other important proteins in cancer cells. Notable, non-limiting examples are the tyrosine kinase inhibitors imatinib (Gleevec) and gefitinib (Iressa). The use of small molecules targeting certain kinases, such as sunitinib malate and / or sorafenib tosylate, in combination with vaccines for cancer treatment has also been described in the prior patent application U.S. Patent Application Publication No. 2009004213.

[0322] 11. Virus-based vaccines There are several virus-based cancer vaccines available or under development that can be used in combination therapeutic approaches with the formulations of the present invention. One advantage of using such viral vectors is their inherent ability to initiate an immune response, along with the inflammatory response that occurs as a result of viral infection, generating the danger signals necessary for immune activation. An ideal viral vector should be safe and not induce an anti-vector immune response, allowing for the boosting of anti-tumor-specific responses. Recombinant viruses such as vaccinia virus, herpes simplex virus, adenovirus, adeno-associated virus, retrovirus, and avipox virus have been used in animal tumor models, and based on their promising results, human clinical trials have begun. A particularly important virus-based vaccine is the virus-like particle (VLP), a small particle containing specific proteins derived from the viral envelope. Virus-like particles do not contain any genetic material from viruses and cannot cause infection, but can be constructed to present tumor antigens on their envelope. VLPs can be derived from a variety of viruses, such as hepatitis B virus, or other viral families including parvoviruses (e.g., adeno-associated virus), retroviruses (e.g., HIV), and flaviviridae (e.g., hepatitis C virus). For a general review, see Sorensen and Thompsen 2007: Virus-based immunotherapy of cancer: what do we know and where are we going? APMIS 115(11):1177-93.Virus-like particles against cancer are reviewed in Buonaguro et al., 2011: Developments in virus-like particle-based vaccines for infectious diseases and cancer. Expert Rev Vaccines 10(11): 1569-83; and Guillen et al., 2010: Virus-like particles as vaccine antigens and adjuvants: application to chronic disease, cancer immunotherapy and infectious disease preventive strategies. Procedia in Vaccinology 2(2), 128-133.

[0323] 12. Multi-epitope strategy The use of multiple epitopes shows promising results for vaccination. Rapid sequencing technology combined with intelligent algorithmic systems allows for the utilization of tumor mutanomes, providing multiple epitopes for personalized vaccines that can be combined with the present invention. For further information, see 2007: Vaccination of metastatic colorectal cancer patients with mature dendritic cells loaded with multiple major histocompatibility complex class I peptides. J Immunother 30:762-772; and Castle et al., 2012: Exploiting the mutanome for tumor vaccination. Cancer Res 72(5):1081-91.

[0324] 13. Adoptive T cell transfer For example, the combination of tumor antigen vaccination and T cell transfer is described in Rapoport et al., 2011: Combination immunotherapy using adoptive T-cell transfer and tumor antigen vaccination on the basis of hTERT and survivin after ASCT for myeloma. Blood 117(3):788-97.

[0325] 14. Peptide-based targeted therapy Peptides can bind to cell surface receptors or to the affected extracellular matrix surrounding tumors. Radionuclides (e.g., RGD) attached to peptides ultimately kill cancer cells when the nuclide decays near the cells. In particular, oligomers or multimers of these binding motifs are of great interest because they can lead to enhanced tumor specificity and avidity. For a non-limiting example, see Yamada 2011: Peptide-based cancer vaccine therapy for prostate cancer, bladder cancer, and malignant glioma. Nihon Rinsho 69(9):1657-61.

[0326] 15. Other Treatments There are many other cancer therapies that can be combined with the formulations and methods of the present invention to produce synergistic effects. Non-limiting examples include apoptosis-targeting treatments, hyperthermia, hormone therapy, telomerase therapy, insulin-augmenting therapy, gene therapy, and photodynamic therapy.

[0327] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0328] Example 1: Generation, purification, and analysis of bispecific antibody derivatives targeting CLDN6 and CD3 (CLDN6 x CD3) a. Sequence origin, bstb construct design, and cloning into expression vectors The bispecific chimeric TriMAB (bstb) construct was designed as an antigen-binding fragment (Fab) with two single-chain variable fragments (scFv) at the C-terminus of the constant region. The Fab binding domain is specific for the human T-cell receptor component CD3ε. The anti-CD3 heavy chain variable region (V H ) and the corresponding light chain variable region (V L The heavy chain constant region (C) domain is derived from mouse IgG TR66 (Lanzavecchia and Scheidegger 1987). The cysteine ​​at position 114 (according to the IMGT nomenclature) is replaced by serine. H 1) and light chain constant region (C L ) are of human origin. A kappa-type light chain constant region was selected for our construct. The two scFv binding moieties are specific for the human tumor-associated antigen (TAA) CLDN6. The corresponding V H and V L The region is derived from the chimeric IgG1 IMAB206-SUBW (Ganymed Pharmaceuticals AG, Mainz, Germany). H and the amino acid sequences of the VL domains.

[0329] C H Two bstb molecules were generated that differ in one region. bstb_369 / 367 is a C molecule derived from human IgG1. H 1 region, and bstb_371 / 367 is a C region derived from human IgG2. H It has one region. Different subclasses of C H 1 and C L Due to the different disulfide bond formation by kappa C, it was reasonable to compare heterodimer formation (Rothlisberger et al., 2005). L Since the sequence of is conserved in IgG1 and IgG2, the same C L The constructs were used for the formation of two constructs. For the formation of the bstb molecules, the following molecules were obtained at the protein level: bstb_369 / 367: NV H αCD3 -C H 1(IgG1)-V H αCLDN6 -V L αCLDN6 -6xHis tag-C (Fd bstb_369 / 367) NV L αCD3 -C L -V H αCLDN6 -V L αCLDN6 -Strep tag-C (L bstb_369 / 367) bstb_371 / 367: NV H αCD3 -C H 1(IgG2)-V H αCLDN6 -V L αCLDN6 -6xHis tag-C (Fd bstb_371 / 367) NV L αCD3 -C L -V H αCLDN6 -V L αCLDN6 -Strep tag-C (L bstb_371 / 367)

[0330] C is the C-terminus, C H is the heavy chain constant region, C L indicates the light chain constant region, Fd indicates the digestible fragment (heavy chain portion of Fab), L indicates the light chain portion of Fab, N indicates the N-terminus, V H is the heavy chain variable domain, V L is the light chain variable domain.

[0331] Table 1 summarizes information about the BSTB constructs specific for TAA CLDN6 generated in the course of the present invention. The bstb construct fragments are identified as GeneArt Strings and the V of the corresponding IgG antibody. H and V L Sequence and conserved human C H and C LThe sequences were used to generate the target cell binding moieties by gene synthesis by GeneArt AG (GeneArt / Thermo Fisher Scientific, Regensburg, Germany). CHO codon optimization was performed using GeneArt's GeneOptimizer® software. Information on specificity, origin of the sequence from a monoclonal antibody (mAB), parameters for codon usage optimization, and additional sequence features is listed in Table 1. The sequences encoding the variable domains of the target cell binding moieties were originally obtained from Ganymed Pharmaceuticals AG.

[0332] DNA cloning and expression vector construction were performed using the Seamless PLUS Cloning and Assembly Kit (GeneArt / Thermo Fisher Scientific), which is well known to those skilled in the art. A human IgG secretory signal sequence encoding MGWSCIILFLVATATGVHS was inserted at the 5' end upstream of the Fd- and L-bstb V-region sequences for protein secretion into the culture medium. For the construction of anti-TAA scFv, a sequence encoding a 20-amino acid (AA) flexible glycine-serine (GS) peptide linker ((GGGGS)4) was inserted to form the V region. H and V L The domains were linked. H and V L One cysteine ​​substitution was introduced in each domain to form an scFv-stable disulfide bridge. One scFv domain sequence was connected to the Fd portion (C) by a sequence encoding an 18-AA linker (SGPGGGRS(GGGGS)2). H The other scFv is linked to the L portion (C terminus) via a sequence encoding a six AA linker (DVPGGS). LThe Fd and L portions were linked to the C-terminus of the Fd-scFv portion. Different linker lengths allowed the Fd and L portions to be distinguished by size. A 6xHis tag sequence was added to the 3' end of the Fd-scFv portion and a Strep tag to the 3' end of the L-scFv portion to facilitate the purification of the first-generation pure heterodimeric bstb. Table 2 shows the full-length DNA sequence encoding the bstb protein. Table 3 shows the DNA sequence encoding the double (scFv)2 used as a reference.

[0333] The bstb antibody construct was cloned into a standard mammalian pCEP4-based expression vector (Invitrogen / Thermo Fisher Scientific, Darmstadt, Germany) and digested with NaeI and PmlI to obtain a blunt-ended linearized plasmid. C-terminal 6xHis and Strep tags were used for affinity chromatography purification of the protein and for detection purposes. An untagged construct was generated as well. All constructs were verified by an external sequencing service. The construct and protein schema are shown in Figure 1.

[0334] [Table 1]

[0335] [Table 2-1]

[0336] [Table 2-2]

[0337] [Table 2-3]

[0338] [Table 2-4]

[0339] [Table 2-5]

[0340] [Table 2-6]

[0341] [Table 3]

[0342] B. Transient transfection and production For transient production of CLDN6-specific bstb proteins, the Expi293™ Expression System (Thermo Fisher Scientific, Darmstadt, Germany)—a derivative of the human embryonic kidney cell line HEK293—was used according to the manufacturer's instructions. For this purpose, Expi293F™ cells were cultured at 2.0 × 10 per ml of Expi293™ Expression Medium in cell culture suspension flasks. 6 On the day of transfection, cells were pre-cultured at a density of 2.5 x 10 cells per ml of fresh Expi293™ Expression Medium for 2 days. 6Expi293F™ cells were transferred to a sterile Erlenmeyer glass flask with a capacity for 5x the volume. The Fd fragment and L fragment-containing pCEP4 plasmid were mixed with Opti-MEM (Gibco / Thermo Fisher Scientific) at a 1:1 ratio. 1 μg of DNA mixture per ml of cell suspension was prediluted in 50 μl of Opti-MEM in a tube, and 2.7 μl of ExpiFectamine™ 293 reagent per ml of cell suspension was prediluted in 50 μl of Opti-MEM in a separate tube and incubated at room temperature for 5 minutes. The prediluted DNA mixture was then added to the prediluted ExpiFectamine™ 293 reagent, mixed, and incubated at room temperature for 20 minutes before being added to the cell suspension. The cell culture flask was agitated at 125 rpm in a humidified Multitron Cell shaker (Infors HT, Bottmingen, Switzerland) at 37°C in 8% CO2 for 16–18 hours. Next, 3.9 μl of Enhancer 1 and 39 μl of Enhancer 2 were added per ml of cell suspension, and the cell culture flasks were further incubated. Protein-containing supernatants were collected 7 days after transfection. The supernatants were filtered through 0.22 μm Nalgene Rapid Flow 90 mm filter units, low protein binding (Thermo Fisher Scientific). Secreted protein content was assessed by BLItz quantification using Protein L Biosensors (Pall / ForteBio, Dreieich, Germany) according to the manufacturer's protocol. The supernatants were stored at 2–8°C until purification.

[0343] c. Purification of tagged proteins bstb_369 / 367 and bstb_371 / 367 Cell culture supernatants from transiently transfected Expi293F™ cells containing proteins bstb_369 / 367 or bstb_371 / 367 (described in Example 1b) were subjected to immobilized metal affinity chromatography (IMAC) using standard procedures (Coligan et al., 2001b). Briefly, cell culture supernatants supplemented with 10 mM imidazole were loaded onto a HisTrap HP 1 ml column (both from GE Healthcare Life Sciences, Freiburg, Germany) coupled to an AKTA pure 25 FPLC system and equilibrated with wash buffer (20 mM NaH2PO4, 500 mM NaCl, 10 mM imidazole, pH 7.5). Samples were loaded at a rate of 1 ml / min. The elution buffer differed from the wash buffer by a concentration of 500 mM imidazole. After washing with 10 column volumes (CV), the protein was eluted using a linear gradient of 0 to 100% elution buffer. The eluate was collected in 1 ml fractions at a rate of 1 ml / min. High molecular weight (HMW) and monomeric species (main peak) were collected (Figure 2A). The eluted bstb proteins were pooled and immediately dialyzed against Strep-Tactin® binding buffer DPBS (Dulbecco's Phosphate Buffered Saline, Gibco / Thermo Fisher Scientific) containing 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 2 mM KHPO, pH 7.4, using a Slide-A-Lyzer G2 Dialysis Cassette 10K MWCO (Pierce / Thermo Fisher Scientific, Rockford, IL, USA). The dialyzed bstb protein from the main IMAC peak was further purified by Strep-Tactin® affinity chromatography (GE Healthcare Life Sciences). All following Strep-Tactin® purification steps were performed at a flow rate of 1 ml / min.

[0344] The Strep-Tactin® column was equilibrated with binding buffer before application of bstb protein. After a five-CV binding buffer wash step, bstb protein was eluted with a 0-100% linear gradient of binding buffer containing 2.5 mM desthiobiotin (Sigma-Aldrich, Taufkirchen, Germany) (Figure 2B). The bstb protein eluted in 1 ml fractions was pooled and sub-injected onto an equilibrated size-exclusion chromatography (SEC) HiLoad 16 / 600 Superdex 200 pg column (GE Healthcare Life Sciences), followed by isocratic elution with one CV DPBS. The eluted HMW and monomeric bstb species were collected in 1 ml fractions (Figure 2C) and pooled.

[0345] The bstb concentration was determined by measuring at 280 nm using a NanoDrop2000c, taking into account the extinction coefficient and the theoretical molecular weight determined via the ProtParam tool (http: / / web.expasy.org / protparam / ). The purified protein was aliquoted and stored at 2–8 °C.

[0346] d. Purification of untagged proteins bstb_5726 / 5725 and bstb_5727 / 5725 Cell culture supernatants from transiently transfected Expi293F™ cells containing proteins bstb_5726 / 5725 and bstb_5727 / 5725 (described in Example 1b), both untagged, were filtered using Supracap™ 50-depth filter capsules (Pall Corporation, Crailsheim, Germany) and subsequently subjected to protein purification. Purified bstb proteins were analyzed by size-exclusion high-performance liquid chromatography (SE-HPLC) (Figure 2D).

[0347] SE-HPLC was performed using a Dionex Ultimate 3000 (Thermo Scientific) equipped with a size-exclusion TSK gel G3000SWxl column (300 × 7.8 mm, Tosoh Bioscience, Griesheim, Germany). A maximum of 100 μl was injected. The column was rinsed with SE-HPLC buffer (0.3 M NaHPO, pH 7.2), and bstb proteins were separated by isocratic elution.

[0348] Both bstb_5726 / 5725 and bstb_5727 / 5725 (data not shown) could be efficiently purified using the established process, and the monomer content increased to >93% after the purification process (Figure 2D). To further concentrate the monomer fraction to approximately 100%, the sample dialyzed against PBS was finally subjected to preparative SEC using a HiLoad 26 / 600 Superdex 200 pg column (GE Healthcare Life Sciences).

[0349] This allows us to demonstrate a purification strategy for untagged bstb.

[0350] e. Analysis of tagged proteins bstb_369 / 367 and bstb_371 / 367 The quality and purity of the tagged bstb protein were examined by polyacrylamide gel electrophoresis using 4-15% Criterion™ TGX Stain-Free™ Gels (Bio-Rad Laboratories, Dreieich, Germany), followed by stain-free gel analysis (Figure 3A) and Western blot analysis (Figure 3B) according to standard procedures known to those skilled in the art. Antibodies used for Western blot analysis were anti-6xHis Tag™ antibody (HRP) (1:10,000, Abcam, Cambridge, MA, USA) and StrepMAB-Classic, HRP conjugate (1:10,000, IBA GmbH, Göttingen, Germany). Gels and Western blots were documented using a Bio-Rad Chemidoc MP Imaging System. The signal for the bstb protein was detected at 50-55 kD under reducing conditions (with DTT) and at approximately 100 kD under non-reducing conditions, relative to an internal molecular weight standard (Figure 3). The heterodimeric composition of bstb was revealed by separation of the Fd and L fragments on a stain-free gel under reducing conditions.

[0351] The aggregation state of the purified protein was analyzed over time by analytical SE-HPLC. SE-HPLC was performed using an Agilent 1260 Infinity system (Agilent Technologies, Waldbronn, Germany) equipped with a size-exclusion TSKgel G3000SWxl column (300 × 7.8 mm, Tosoh Bioscience). A maximum of 100 μl was injected. The column was rinsed with SE-HPLC buffer (0.3 M NaHPO, pH 7.2), and the bstb proteins were separated by isocratic elution. As summarized in Table 4, the purified bstb_369 / 367 proteins were tested by analytical SE-HPLC over the course of more than 6 months and showed high stability at 2–8°C in DPBS with no signs of aggregation or degradation.

[0352] [Table 4]

[0353] This demonstrated the high stability of the bstb molecule, and subsequent purification yielded 100% monomeric bstb_369 / 367 protein.

[0354] Example 2: Determination of the specific lytic activity of bstb_369 / 367 and bstb_371 / 367 in an in vitro cytotoxicity assay To determine the specific lytic effect, an in vitro luciferase cytotoxicity assay was used. The target cell lines used were the human ovarian cancer cell line OV-90 (ATCC CRL-11732) and the teratocarcinoma cell line PA-1 (ATCC CRL-1572), which stably express luciferase and endogenously express CLDN6. The human CLDN6-negative breast cancer cell line MDA-MB-231 (ATCC HTB-26), which also stably expresses luciferase, was used as a specificity (off-target) control.

[0355] Human peripheral blood mononuclear cells (PBMCs) isolated from human blood of healthy donors (University Medicine of the JGU Mainz, Blood Transfusion Centre, Mainz, Germany) according to standard procedures ( Coligan et al., 2001a ) were selected as effector cells.

[0356] a. Determination of EC50 To determine the half-maximal effective concentration (EC50) of highly monomeric bstb_369 / 367, a titration series of the protein was tested in an in vitro luciferase cytotoxicity assay.

[0357] In the described examples, effector (E) and target (T) cells were mixed at an E:T ratio of 5:1 and seeded into white 96-well cell culture plates (Nunclon® Delta Surface, Thermo Scientific, Braunschweig, Germany), with a final cell number per well of 1 × 10 4 5 x 10 target cells and 5 x 10 4 The cells were incubated with a 10-point, 10-fold serial dilution series of bstb_369 / 367 or bstb_371 / 367 protein at concentrations ranging from 4.85 aM to 48.50 nM. min and L max Wells representing values ​​(see below) were supplemented with DPBS instead of bstb protein.

[0358] The cell culture microplates were incubated at 37°C and 5% CO2 for 48 hours. For analysis, 50 μl of an aqueous solution containing 1 mg / ml luciferin (BD Monolight, BD Biosciences, Heidelberg, Germany) and 50 mM HEPES was added per well, and the plates were then incubated at 37°C in the dark for 30 minutes. Luminescence resulting from the oxidation of luciferin by live cells expressing luciferase was measured using an Infinite M200 Tecan microplate reader (Tecan, Mannedorf, Switzerland). The percentage of specific target cell lysis was calculated by the following formula: % specific lysis = [1-(luminescence 試験試料 -L max ) / (L min -L max )] x 100 In the formula, "L" indicates dissolution. min indicates the emission at minimum dissolution in the absence of bstb, and L max refers to the luminescence (equal to spontaneous luminescence counts) at maximum lysis achieved by adding Triton X-100 (2% final concentration) in the absence of bstb.

[0359] EC50 values ​​were calculated using a sigmoidal dose-response algorithm (log(agonist) vs. response - variable slope (3 parameters)) integrated into PRISM6 software (GraphPad Software, San Diego, California, USA).

[0360] The determined EC50 of bstb_369 / 367 against CLDN6-positive OV-90 cells was 39.71 pM, thus three-fold lower than the EC50 of bstb_371 / 367, which was 117.00 pM (Figure 4A). No lysis was observed in the CLDN6-negative control cell line, MDA-MB-231. The results of this assay are highly dependent on the potency of human PBMCs and, as reported by others, vary according to the donor's immune status (see, e.g., Lutterbuese et al., 2010). Therefore, using eight different PBMC donors, the EC50 titer range of bstb_369 / 367 proteins was determined to be 0.98 fM–39.71 pM (data not shown).

[0361] b. Comparison of bstb_369 / 367 and CLDN6×CD3 double (scFv)2 The bivalent CLDN6-binding bstb_369 / 367 was compared with a highly monomeric (99.5%) in-house generated CDLN6×CD3 bi(scFv)2 (BioNTech AG, Stadler et al., 2015) to investigate the beneficial avidity effect of bivalent antitumor targeting on lytic efficacy.

[0362] To this end, bstb and double(scFv)2 were used at equimolar concentrations, and their lytic effects were tested against PA-1, OV-90, and MDA-MB-231 in a cytotoxicity assay essentially as described in Example 2a (Figure 4B). PA-1 cells were co-incubated for only 24 h. Surprisingly, bstb_369 / 367-mediated PA-1 cell killing was 58% at 0.24 pM, the lowest concentration used against PA-1, while the double(scFv)2 control showed a lysis curve starting at 0% killing. In the case of OV-90, the higher potency of bstb_369 / 367 compared to double(scFv)2 was evident by an EC50 55-fold lower than that of bstb. Both molecules showed no off-target lytic effects. Thus, bivalent CLDN6 binding by bstb molecules strongly enhances target cell killing potential compared to monovalent CLDN6 binding by dual (scFv)2 molecules.

[0363] C. Investigating the effect of bstb_369 / 367 high molecular weight species on lytic activity HMW species, such as dimers or multimers, may exhibit different activities compared to monomeric species due to their multivalency towards the corresponding antigen. Although the molecule has been shown to be stable in its monomeric state, the effect on the activity of HMW species was examined for additional safety aspects.

[0364] For this purpose, HMW species were collected during the purification procedure, concentrated by SEC, and spiked into a monomer preparation to a 5% fixed concentration. Subsequently, 99.3% and 94.3% monomers supplemented with 5% HMW species were compared in a cytotoxicity assay as described in Example 2a, but using a 15-point, 20-fold serial dilution series. PA-1 and OV-90 were used as target cells. Co-incubation with bstb_369 / 367 was performed for 16 hours with the moderately sensitive cell line PA-1 and for 48 hours with the more robust cell line OV-90. EC50 values ​​were calculated using a sigmoidal dose-response algorithm (log(agonist) vs. response—variable slope (four parameters)) integrated into PRISM6 software (GraphPad Software, Inc.), as shown in Figure 4C. The calculated EC50 for the 5% HMW bstb_369 / 367 sample was 1.5-fold higher for PA-1 and 2.3-fold higher for OV-90. Thus, a slight decrease in activity was observed for PA-1 and a slight increase in activity for OV-90. Overall, the effect of the 5% HMW species on activity was small and may depend on the target cell line and / or incubation time. The stability of the bstb protein and the low impact of the somewhat higher proportion of HMW species make the bstb molecule suitable for therapeutic product development.

[0365] d. Comparison of bstb_369 / 367 with untagged variants bstb_5726 / 5725 and bstb_5727 / 5725 The untagged bstb variants were evaluated in a cytotoxicity assay and compared to bstb_369 / 367. Additionally, the effect of HMW species on activity was examined. The assay setup was as described in Example 2c, except that the test items were 10-point 5-fold serial dilutions of CLDN6. + Only OV-90 cells were used as target cells.

[0366] bstb_369 / 367 and its untagged analogue bstb_5726 / 5725 (99.9% monomeric) showed equal lysis efficiency with an EC50 value of approximately 9.3 pM (Figure 4D, left plot). 5% HMW led to a 1.4-fold lower EC50 value.H The EC50 of the 1 (IgG2)-bearing variant bstb_5727 / 5725 (100% monomer) was approximately 1.6-fold higher than that of the reference bstb_369 / 367, confirming the slightly lower potency of this variant, as shown for the untagged variant in Example 2a. For bstb_5727 / 5725, 5% HMW had little effect on its potency (Figure 4D, right plot). In this exemplary study, the EC50 with spiked HMW was only 1.1-fold lower than that with the monomer.

[0367] In conclusion, tagged and untagged C H 1 (IgG1)-bearing bstb is equivalent in function and H 1 (IgG2)-bearing bstb. The effect of HMW species is low for both variants.

[0368] Example 3: T cell regulation mediated by bstb_369 / 367 and bstb_371 / 367 The regulation of T cells mediated by bstb_369 / 367 and bstb_371 / 367, specifically T cell activation and proliferation, was investigated in the same target cell line with a similar setup as described in Example 2. Briefly, CLDN6 + Target cells OV-90 and PA-1 and CLDN6 - MDA-MB-231 control cells were seeded in 24-well plates with human PBMCs at a 5:1 E:T ratio in 0.5 ml of complete medium. To further confirm target dependency, PBMCs were co-incubated with bstb proteins without target cells. The bstb proteins bstb_369 / 367 and bstb_371 / 367 were added in a four-point 100-fold serial dilution series at concentrations ranging from 0.005 to 5,000 ng / ml. As a positive control, 100 ng / ml of human anti-CD3 IgG2a clone OKT3 (BioXCell, West Lebanon, NH, USA), a target-independent T cell activating mouse IgG, was applied. DPBS, bstb buffer, was added as a negative control.

[0369] a. T cell activation Effector and target cells were co-incubated with bstb for 48 hours. PBMCs were harvested, transferred to round-bottom 96-well plates (Fisher Scientific, Schwerte, Germany), centrifuged, and washed. Cells were stained with anti-human fluorescently labeled antibodies anti-CD5-PE-Cy7 (Abcam), CD69-APC, and CD25-PE (BD Biosciences), and the live / dead dye eFluor506 (eBioscience, Frankfurt am Main, Germany). Samples were measured on a BD FACSCanto II, and 10,000 CD5 + Single live lymphocytes were recorded. Data were analyzed using FlowJo software V10 (Tree Star, San Carlos, CA, USA) and Microsoft Excel 2010 (Microsoft, Deutschland GmbH, Unterschleißheim, Germany). Background signals from mock samples were subtracted.

[0370] As shown in Figure 5A, both bstbs mediate potent CLDN6-dependent T cell activation. After 48 hours, T cells are at an intermediate activation stage, as determined by a high percentage of double positive T cells for the early activation marker CD69 and the late activation marker CD25, and the absence of T cells positive for CD25 alone. The data further indicate that lower bstb concentrations result in earlier activation states. Only at a high concentration of 5,000 ng / ml did bstb protein induce measurable target-independent T cell activation, and this activation was less than 5.5%. bstb_369 / 367 demonstrates slightly higher, but not significant, potency in mediating target-dependent T cell activation than bstb_371 / 367 (see also Table 5).

[0371] [Table 5]

[0372] b. T cell proliferation PBMCs were labeled using the CellTrace™ CFSE Cell Proliferation Kit (Thermo Fisher Scientific, Darmstadt, Germany) according to the manufacturer's protocol before assay setup. Effector and target cells were co-incubated with bstb for 72 hours. PBMCs were harvested as described in Example 3.a. Cells were stained with anti-human fluorescently labeled antibody anti-CD5-APC (BD Biosciences) and the live / dead dye eFluor506 (eBioscience). Samples were run on a BD FACSCantoII and 10,000 CD5 + Singlet viable lymphocyte counts were recorded, and data were analyzed using Tree Star FlowJo software V10 and Microsoft Excel 2010.

[0373] As shown in Figure 5B, both bstb molecules mediate strong CLDN6-dependent T cell proliferation as determined by CFSE-positive bleaching, indicative of cell division. In the presence of PA-1, no clear difference in the potency of the two bstb molecules in terms of robust proliferation could be determined. Also, as seen in T cell activation in the presence of OV-90, the bstb_369 / 367-mediated effect was more potent than bstb_371 / 367. No nonspecific or target-independent T cell proliferation was detected.

[0374] Taken together, both bstb molecules mediate potent T cell regulation in a strictly target-dependent manner.

[0375] (Example 4) Binding of bstb_369 / 367 and bstb_5726 / 5725 to CLDN6 The relative binding affinities of the dual (scFv)2 reference protein, bstb_369 / 367, and its untagged analog, bstb_5726 / 5725, were determined using flow cytometry analysis. PA-1 cells, which endogenously express CLDN6, were harvested with 0.05% trypsin / EDTA, washed with PBS, and plated at 2 × 10 in FACS buffer (PBS containing 2% FCS and 0.1% sodium azide). 6 Cells were resuspended at a concentration of 100 μl / ml. 100 μl of the cell suspension was incubated with a concentration series of different antibodies diluted in FACS buffer (11-point, 2-fold serial dilutions from 9.77 to 10,000 ng / ml) for 45 min at 4°C. The cells were then washed three times with FACS buffer and incubated with FITC-conjugated protein L (Pierce / Thermo Scientific) at a concentration of 4 μg / ml by Squarix (Squarix Biotechnology, Marl, Germany) for 45 min at 4°C. The cells were then washed twice and resuspended in 100 μl of FACS buffer. Binding was analyzed by flow cytometry using a BD FACSArray (BD Biosciences). EC50 values ​​for half-maximal binding (specific binding) were determined using GraphPad Prism 6 One Site.

[0376] The dual (scFv)2 protein, which has a monovalent CLDN6-binding site, exhibited low relative binding affinity (EC50: approximately 8.3 μg / ml, corresponding to approximately 150 nM). In comparison, bstb_369 / 367 and bstb_5726 / 5725 exhibited half-maximal binding at a concentration of approximately 1.9 μg / ml, corresponding to approximately 19 nM, and thus their relative binding affinity was approximately 8-fold higher compared to the dual (scFv)2 reference protein (Figure 6A). This indicates that binding to the target CLDN6 is strongly improved by the use of a bivalent binding site, thus increasing binding affinity by implementing the avidity effect. Notably, the binding curves of tagged and untagged bstb are identical.

[0377] We further analyzed the effect of HMW species on the binding properties of bstb_5726 / 5725 monomers by flow cytometry using Protein L-FITC at a concentration of 4 μg / ml or a PE-conjugated mouse anti-human IgG (Fab region) antibody (antikoerper-online, Aachen, Germany) at a concentration of 6 μg / ml as a secondary detection reagent. The EC50 for half-maximal binding was similar among 100% monomer, 97% monomer / 3% HMW, and 95% monomer / 5% HMW (varying between 28 nM and 31-34 nM depending on the secondary detection reagent). Figure 6B shows only Protein L-FITC detection as an example. These data are consistent with the data from the cytotoxicity assay (see Example 2c), which clearly demonstrates that 5% HMW has no relevant effect on the activity of the bstb_369 / 367 molecule, making this format advantageous for product development.

[0378] Example 5: Efficacy in a mouse xenograft model To investigate the therapeutic efficacy of the protein bstb_369 / 367 in vivo, we transfected the immunodeficient mouse strain NOD.Cg-Prkd scid IL2rg tm1Wjl We selected mice with either the / SzJ or short NSG strains (Jackson Laboratory, Bar Harbor, ME, USA). All mice were used in accordance with guidelines from the Institutional Animal Care Committee of Johannes Gutenberg University (Mainz, Germany).

[0379] a. Treatment of highly CLDN6-expressing tumors in mice with protein bstb_369 / 367 In a typical study, male and female NSG mice aged 8 weeks and weighing 21 to 36 g were used. CLDN6-positive OV-90 cells were used as tumor cells, and PBMCs were used as effector cells.

[0380] 5×10 6 Tumor cells were inoculated subcutaneously (sc), and 8 days later, 1 × 107 PBMCs were administered intraperitoneally (ip). Only PBMC-engrafted mice - analyzed in peripheral blood 5 days after PBMC injection - were stratified into groups according to tumor volume and sex (both sexes in all groups). Treatments were administered in approximately 30 mm per group. 3 Mice were treated with vehicle (DPBS), 31 μg / kg bstb (low dose), or 308 μg / kg bstb (high dose) by ip injection three times a week (Monday-Wednesday-Friday). Group "G1-Vehicle" consisted of four mice (n=4), "G2-bstb_369 / 367 (low dose)" consisted of eight mice (n=8), and "G3-bstb_369 / 367 (high dose)" consisted of nine mice (n=9). Treatment groups are summarized in Table 6. Treatment was intraperitoneal for 5 consecutive weeks (G1), 6 weeks (G2), or 4 weeks (G3), depending on tumor volume readout (Figure 7A). Tumor size was measured twice a week with a digitally calibrated caliper, and tumor volume was calculated using the formula: tumor volume [mm 3 ] = length [mm] x (width [mm]) 2 The tumor volume was calculated by the formula: 3 Mice were sacrificed by cervical dislocation when they reached 0.05 or in case of severe morbidity (mainly symptoms of graft-versus-host disease (GVHD)).

[0381] Figure 7B shows the inhibition of tumor growth in all mice in the bstb-treated groups G2 and G3. Antitumor efficacy was measured as tumor growth inhibition / elimination and survival compared to the vehicle control G1. Treatment with test item bstb_369 / 367 resulted in tumor elimination in both treatment groups, in contrast to the control group. All nine mice in the bstb_369 / 367 high-dose group (G3) were tumor-free after seven injections. All eight mice in the bstb_369 / 367 low-dose group (G2) were tumor-free after 15 injections. Tumor-free mice were observed for tumor recurrence until GVHD symptoms developed. No tumor recurrences were recorded during this period.

[0382] For the purposes of constructing the Kaplan-Meier survival plot (Figure 7C), the day of treatment initiation was considered day 0. As determined by Kaplan-Meier survival statistics combined with the Mantel-Cox log-rank test for pairwise comparisons, survival in both bstb_369 / 367-treated groups was significantly prolonged compared with the control group G1. Comparisons of G2 to G1 yielded p-values ​​of p≤0.0001, and comparisons of G3 to G1 yielded p=0.0043. Median survival was 31 days in the control group G1, and 52 and 42 days in the treatment groups G2 and G3, respectively. In summary, bstb_369 / 367 was highly efficient in terms of tumor elimination and further demonstrated beneficial effects in terms of survival in this GVHD-restricted mouse model.

[0383] [Table 6]

[0384] b. Determining the effect of treatment on body weight The weight of each mouse was checked twice a week using a laboratory scale. None of the mice in either group showed significant weight loss over the course of treatment (data not shown).

[0385] c. Splenocyte isolation After euthanasia, mice were euthanized and their spleens were dissected to detect human cell engraftment by flow cytometry analysis. Splenocyte isolation was performed by mashing the spleen through a 70 µm cell strainer into a 50 ml reaction tube using the sterile plunger of a 3-5 ml syringe immediately after dissection, followed by repeated flushing of the cell strainer with RT DPBS. The isolated splenocytes were centrifuged, the DPBS was decanted, and the splenocyte pellet was resuspended in 1 ml of heat-inactivated fetal bovine serum supplemented with 10% DMSO. The samples were immediately frozen at -65 to -85 °C and stored until splenocyte samples from all mice were completed.

[0386] d. Analysis of human T lymphocyte engraftment in mouse spleens Completely harvested splenocyte samples were thawed once, and all cells were washed twice with warmed DPBS, with 1 × 10 cells per sample. 6 Splenocytes were incubated with fluorescently labeled antibodies (BD Biosciences) for 20 min at 4°C in the dark. Human cell engraftment was detected by anti-hCD45-APC staining, and the percentage of human T cells was detected by anti-hCD3-FITC staining. Flow cytometry analysis was performed using a FACSCant II (BD Biosciences). Human T cell engraftment was confirmed by the percentage of hCD45 / hCD3 double-positive singlet splenocytes, ranging from 50 to 95% (data not shown).

[0387] (Example 6) Estimation of pharmacokinetic behavior in immunodeficient mice To determine the approximate half-life and clearance of bstb_369 / 367 protein in mice, female NSG mice aged 9 to 49 weeks were used. Each mouse was injected i.p. with 5 mg / kg of highly monomeric (100%) bstb_369 / 367 in DPBS. Group sizes corresponding to one time point for blood collection consisted of three mice per group. One group was injected with vehicle buffer (DPBS) alone as the baseline (post-injection time = 0 h). Additional time points for blood collection were set at 15 min, 1 h, 2 h, 3 h, 6 h, and 8 h. Blood was collected directly into Li-heparin tubes (Microvette 300 LH, Sarstedt, Nurmbrecht, Germany), and plasma was separated by centrifugation as known to those skilled in the art. Plasma was collected, immediately flash-frozen in liquid nitrogen, and stored at -65 to -85°C until use.

[0388] ELISA was performed to quantify bstb_369 / 367 in plasma. To this end, plasma aliquots from each group were thawed at room temperature and diluted with PBS / 0.2% BSA. The diluted bstb_369 / 367-containing plasma and bstb_369 / 367 protein were added as a standard series (0.39 ng / ml to 3.41 μg / ml) to a MaxiSorp™ plate (Thermo Scientific) coated with goat anti-human IgG F(ab')2 antibody (Abd Serotec, Oxford, UK) and blocked with 3% milk. After a washing step, mouse IgG specific for the anti-CLDN6 binding site of bstb_369 / 367 (Ganymed Pharmaceuticals Ag) was incubated at a concentration of 3.5 μg / ml, followed by a washing step and incubation with a 1:500 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (Fc) antibody (Dianova, Hamburg, Germany). Finally, 4-nitrophenyl phosphate disodium salt hexahydrate, short PNPP (PanReac AppliChem, Darmstadt, Germany) was added as a substrate for alkaline phosphatase, and the reaction was stopped after 30 min with 3 M potassium hydroxide. ELISA was measured at 405 / 492 nm using a microplate reader M200Pro (Tecan). The measured absorbance values ​​were converted to concentration values ​​based on the bstb_369 / 367 standard curve.

[0389] As shown in Figure 8, a maximum bstb_369 / 367 plasma concentration of 34 μg / ml was reached within 1 hour after ip injection. At the analytical endpoint of 8 hours after ip injection, bstb_369 / 367 protein was still detectable in serum (8 ng / ml).

[0390] Considering binding to tumor targets and T cells in patients where long-term circulation is assured, a cycle of injection of bstb molecules once or twice per week is contemplated.

[0391] (Example 7) In vivo dose-finding test of untagged bstb_5726 / 5725 A xenograft model was established as described in Example 5, according to the schedule shown in Figure 9A. Briefly, 44 male and female NSG mice aged 10 to 36 weeks were engrafted with OV-90 (sc) and human PBMCs (ip). Mice were randomized into seven groups: six treatment groups of six mice and one PBS control group of eight mice. To determine the optimal bstb_5726 / 5725 dose, concentrations above and below the effective dose of bstb_369 / 367 used in Example 5 (approximately 300 and 30 μg / kg) were selected, specifically, 1,000 μg / kg, 300 μg / kg, 100 μg / kg, 30 μg / kg, 10 μg / kg, and 3 μg / kg. On the day of treatment initiation, the mean tumor volume was approximately 150 mm in the randomized groups. 3 Treatment was administered i.p. three times per week starting 28 days after tumor cell inoculation. A total of 11 injections were administered per group. Tumors were measured twice per week with digital calipers.

[0392] As shown in Figure 9B, tumor shrinkage correlated with dose. 3 Tumor volume appears to represent a critical size that is only eliminated by bstb doses >30 μg / kg. Interestingly, even 3 μg / kg led to a long-term reduction in tumor growth and size, suggesting a cumulative effect of bstb in the tumor mass.

[0393] In this setting, 100 μg / kg was determined to be a highly effective and safe dose. Adverse effects such as yellowing of the skin and weight loss were observed in the highest dose group of 1,000 μg / kg.

[0394] Example 8: Generation, purification, and analysis of bispecific antibody derivatives targeting CLDN18.2 and CD3 a. Sequence origin, bstb construct design, and cloning into expression vectors The bispecific chimeric TriMAB (bstb) construct was designed as an antigen-binding fragment (Fab) with two single-chain variable fragments (scFv) at the C-terminus of the constant region. The Fab binding domain is specific for the human T-cell receptor component CD3ε. The anti-CD3 heavy chain variable region (V H ) and the corresponding light chain variable region (V L The heavy chain constant (C) domain is derived from IgG TR66 (Lanzavecchia and Scheidegger 1987). The cysteine ​​at position 114 is replaced by a serine. H 1) and light chain constant region (C L ) are of human origin. A kappa-type light chain constant region was selected for our construct. The two scFv binding moieties are specific for the human tumor-associated antigen (TAA) CLDN18.2. The corresponding V H and V L The region is derived from the chimeric IgG1 IMAB362 (Ganymed Pharmaceuticals AG, Mainz, Germany), which is currently undergoing clinical trials such as NCT01630083 and NCT01671774 (see, e.g., Woll et al., 2014). H and V L The orientation of the domains and the choice of linker in the scFv portion correlate with the dual (scFv)2 molecules 5506 and 5538 described in patent application PCT / EP2013 / 003399. H and the amino acid sequences of the VL domains.

[0395] The four bstb molecules are C H bstb_5730 / 5728 and bstb_5731 / 5729 were generated as first-generation antibodies with different C domains and IMAB362 scFv portion. H bstb_5732 / 5728 and bstb_5733 / 5729 contain C domains derived from human IgG2. H 1 region. Furthermore, bstb_5730 / 5728 and bstb_5732 / 5728 contain V H -V L, while bstb_5731 / 5729 and bstb_5733 / 5728 possess the IMAB362 scFv portion in the V orientation. L -V H For the formation of bstb molecules, the following molecules were obtained at the protein level: bstb_5730 / 5728: NV H αCD3 -C H 1(IgG1)-V H αCLDN18.2 -V L αCLDN18.2 -6xHis tag-C (Fd bstb_5730 / 5728) NV L αCD3 -C L -V H αCLDN18.2 -V L αCLDN18.2 -Strep tag-C (L bstb_5730 / 5728) bstb_5732 / 5728: NV H αCD3 -C H 1(IgG2)-V H αCLDN18.2 -V L αCLDN18.2 -6xHis tag-C (Fd bstb_5732 / 5728) NV L αCD3 -C L -V H αCLDN18.2 -V L αCLDN18.2 -Strep tag-C (L bstb_5732 / 5728) bstb_5731 / 5729: NV H αCD3 -C H 1(IgG1)-V L αCLDN18.2 -V H αCLDN18.2 -6xHis tag-C (Fd bstb_5731 / 5729) NV L αCD3 -C L -V L αCLDN18.2 -V H αCLDN18.2 -Strep tag-C (L bstb_5731 / 5729) bstb_5733 / 5729: NV H αCD3 -C H 1(IgG2)-V L αCLDN18.2 -V H αCLDN18.2 -6xHis tag-C (Fd bstb_5733 / 5729) NV L αCD3 -C L -V L αCLDN18.2 -V H αCLDN18.2 -Strep tag-C (L bstb_5733 / 5729)

[0396] C is the C-terminus, C H is the heavy chain constant region, C L indicates the light chain constant region, Fd indicates the digestible fragment (heavy chain portion of Fab), L indicates the light chain portion of Fab, N indicates the N-terminus, V H is the heavy chain variable domain, V L indicates the light chain variable domain.

[0397] Table 7 summarizes information about the bstb constructs specific for the TAA CLDN18.2 generated during the course of this invention. Information regarding specificity, origin of the sequence from a monoclonal antibody (mAB), codon usage, and additional sequence features is listed. The sequences encoding the variable domains of the target cell binding moieties were originally obtained from Ganymed Pharmaceuticals AG.

[0398] DNA cloning and construction of the pCEP4 expression vector were performed by GeneArt (GeneArt / Thermo Fisher Scientific). A human IgG secretory signal sequence encoding MGWSCIILFLVATATGVHS was inserted 5' upstream of the Fd- and L-bstb V-region sequences for protein secretion into the culture medium. H -V L For the construction of an anti-TAA scFv in the V orientation, a sequence encoding a 20 amino acid (AA) flexible glycine-serine (GS) peptide linker ((GGGGS)4) was inserted to form the V H and V L domains, whereas V L -V H A 25 AA flexible (GGGGS)5-peptide linker was inserted to construct an anti-TAA scFv with the following orientation: One scFv domain sequence was connected to the Fd portion (C) by a sequence encoding an 18 AA linker (SGPGGGRS(GGGGS)2). H The other scFv is linked to the L portion (C terminus) via a sequence encoding a six AA linker (DVPGGS). L The Fd and L portions were linked to the C-terminus of the Fd-scFv and L-scFv fragments, respectively. Different linker lengths allowed the Fd and L portions to be distinguished by size. A 6xHis tag sequence was added to the 3' end of the Fd-scFv portion and a Strep tag to the 3' end of the L-scFv portion to facilitate the purification of the first-generation pure heterodimeric bstb. Table 8 shows the full-length DNA sequence encoding the bstb protein. Table 9 shows the protein-encoding DNA sequences of the parental duplex (scFv)2 and the reference duplex (scFv)2 used in in vitro studies.

[0399] The C-terminal 6xHis tag and Strep tag were used for affinity purification of the protein and for detection analysis. All constructs were verified by an external sequencing service. The construction schema is shown in Figure 10A. The second generation bstb was designed without tags and had a disulfide bond. The cross-linking was introduced into the scFv portion.

[0400] Table 7

[0401] Table 8-1

[0402] Table 8-2

[0403] Table 8-3

[0404] Table 8-4

[0405] Table 8-5

[0406] Table 8-6

[0407] Table 8-7

[0408] Table 8-8

[0409] Table 8-9

[0410] [Table 8-10]

[0411] [Table 8-11]

[0412] [Table 8-12]

[0413] [Table 8-13]

[0414] [Table 8-14]

[0415] [Table 9-1]

[0416] [Table 9-2]

[0417] [Table 9-3]

[0418] Example 9 Comparison of the specific lytic activity of four CLDN18.2×CD3-bstb in an in vitro cytotoxicity assay Proteins bstb_5730 / 5728, bstb_5731 / 5729, bstb_5732 / 5728, and bstb_5733 / 5729 (all containing tags in the scFv portion but without sulfide bridges) were produced and purified as described in Examples 1b and 1c. Prior to isolation of highly monomeric species by SEC, the functionality of the proteins (50-77% monomeric species) was examined in an in vitro luciferase cytotoxicity assay essentially as described in Example 2a. Deviations are noted below.

[0419] In a typical study, the human gastric cancer cell line NUGC-4_hCLDN18.2, which stably expresses luciferase and CLDN18.2, was used as the target cell line. The human CLDN18.2-negative breast cancer cell line MDA-MB-231 was used as a specificity control. PBMC effector and target cells were incubated with a 10-point, 10-fold serial dilution series of bstb protein at concentrations ranging from 47.44 aM to 47.44 nM.

[0420] As shown in Figure 11, all four CLDN18.2 x CD3-bstb molecules (Figure 10B) resulted in efficient lysis of NUGC-4_hCLDN18.2 cells after 48 hours of co-incubation in a typical study. Up to 93% of target cells were lysed. No lysis was observed in the CLDN18.2 negative control cell line, MDA-MB-231. Regarding monomer content and efficiency in the cytotoxicity assay before SEC, the C of IgG1 H Two bstb variants with one moiety, bstb_5730 / 5728 and bstb_5731 / 5729, were selected for further testing without tags and with or without disulfide bridging of the scFv portion.

[0421] Example 10 Comparison of Physicochemical Properties of Purified, Untagged CLDN18.2×CD3-bstb Cell culture supernatants of transiently transfected Expi293F™ cells containing proteins bstb_5745 / 5747, bstb_5746 / 5748, bstb_5749 / 5751, or bstb_5750 / 5752 were filtered using Supracap™ 50 depth filter capsules (Pall Corporation, Port Washington, NY, USA) and subsequently subjected to the protein purification process.

[0422] To analyze the aggregation state of various purified CLDN18.2×CD3-bstb proteins, size-exclusion high-performance liquid chromatography (SE-HPLC) was performed using a Dionex Ultimate 3000 (Thermo Scientific) equipped with a size-exclusion TSK gel G3000SWxl column (300 × 7.8 mm, Tosoh Bioscience). The column was rinsed with SE-HPLC buffer (0.3 M NaHPO, pH 7.2), and bstb proteins were separated by isocratic elution. bstb_5745 / 5747 and bstb_5746 / 5748 were shown to be relatively low amounts of monomeric proteins (approximately 60% and 40%, respectively), whereas bstb_5749 / 5751 and bstb_5750 / 5752 were shown to be highly monomeric after purification (approximately 96% and 89%, respectively) (Figures 12A and 12B). Furthermore, short-term storage of bstb_5745 / 5747 and bstb_5746 / 5748 at room temperature resulted in the formation of visible aggregates.

[0423] bstb_5745 / 5747 and bstb_5749 / 5751, and bstb_5746 / 5748 and bstb_5750 / 5752, respectively, differ in only two amino acids in each anti-CLDN18.2 scFv portion, which allows the formation of disulfide bridges within the anti-CLDN18.2 scFv portion of bstb_5749 / 5751 and bstb_5750 / 5752. Introduction of additional disulfide bridges in the anti-CLDN18.2 scFv portion favors the formation of monomeric proteins, thus improving the physicochemical properties of each bstb.

[0424] All bstb variants after the initial purification were dialyzed into PBS and finally subjected to preparative SEC using a HiLoad26 / 600 Superdex 200 pg column (GE Healthcare, Life Sciences) to enrich the monomeric fraction to approximately 100% for further characterization of activity and binding.

[0425] Example 11: Direct comparison of the activity of CLDN18.2xCD3 bstb and dual (scFv)2 proteins The monomeric proteins bstb_5749 / 5751 and bstb_5750 / 5752 (all containing disulfide bridges in the scFv portion) and their dual(scFv)2 analogs, dual(scFv)_5506 and dual(scFv)_5538 (without disulfide bridges), were tested in an in vitro luciferase cytotoxicity assay essentially as described in Example 9. Except as previously described, a 10-point, 5-fold serial dilution series was applied over a concentration range of 24.64 fM to 48.11 nM. Additionally, a reference was included on each plate for normalization. The bstb and dual(scFv)2 proteins were used at equimolar concentrations.

[0426] bstb_5750 / 5752 showed an EC50 (40.63 pM) 1.8-fold lower than that of bstb_5749 / 5751 (70.94 pM) (Figure 13A, left plot), thus identifying a slightly higher activity than the first bstb. Both bstb proteins led to efficient lysis of approximately 95% of target cells at the highest concentration. No lysis was observed in the CLDN18.2-negative control cell line, MDA-MB-231 (data not shown). Interestingly, the respective double(scFv)2-double(scFv)2_5506 and 5538-analogue (Figure 13A, right plot) showed significant differences with EC50 values ​​of 9.99 pM and 777.20 pM, respectively. Subsequently, EC50 values ​​were normalized to the plate reference (double(scFv)2_5376), and the double(scFv)2 EC50 was set to 1 to calculate the fold difference. Strikingly, the EC50 of bstb_5749 / 5751 was 6-fold higher compared to the dual scFv_5506, pointing to an initial loss of approximately 80% activity (Figure 13B, left plot), whereas the EC50 of bstb_5750 / 5752 was 23-fold lower compared to the dual scFv_5538, suggesting a >2000% increase in activity with the use of the bstb format (Figure 13B, right plot).

[0427] In summary, switching from monovalent to bivalent depending on the binding domains used and their orientation can improve the activity of bispecific antibodies, as demonstrated by the dramatic increase in activity of bstb_5750 / 5752 compared to the bispecific scFv_5538. Furthermore, the protein properties of the bstb format appear to be superior to the bispecific (scFv)2 format in terms of stability.

[0428] Example 12: Generation of IVT-mRNA-based bispecific antibody-derivatives targeting CLDN6 and CD3 a. Cloning of bstb IVT-mRNA template vector and IVT-mRNA synthesis For the generation of CLDN6 x CD3 bispecific chimeric TriMAB (bstb) as in vitro transcribed messenger RNA (IVT-mRNA), the DNA sequences of bstb_369 / 367 and bstb_371 / 367 (described in Example 1) were subcloned into the IVT-mRNA template vector pST1-TEV-MCS-FI (BioNTech AG, Mainz, Germany) using standard techniques. The TEV leader sequence has been described elsewhere (Zeenko und Gallie 2005; Weingarten-Gabbay et al., 2016; Gallie et al., 1995), and the FI sequence has been described in the parent application "3'UTR Sequences for RNA Stabilization" (PCT / EP2015 / 073180). The following constructs were obtained for the generation of bstb molecules: bstb_435 / 434: pST1-5'TEV-Sec-V H αCD3 -C H 1(IgG1)-V H αCLDN6 -V L αCLDN6 -6xHis tag-FI-A30 linker A70 (Fd) pST1-5'TEV-Sec-V L αCD3 -C L -V H αCLDN6 -V L αCLDN6 -Strep tag-FI-A30 linker A70 (L) bstb_436 / 434: pST1-5'TEV-Sec-V H αCD3 -C H 1(IgG2)-V H αCLDN6 -V L αCLDN6 -6xHis tag-FI-A30 linker A70 (Fd) pST1-5'TEV-Sec-V L αCD3 -C L -V HαCLDN6 -V L αCLDN6 -Strep tag-FI-A30 linker A70 (L)

[0429] A is adenine, Fd is a digestible fragment (heavy chain portion of Fab), FI is the 3'UTR sequence, L is the light chain portion of Fab, Sec is a secretion signal, and 5'TEV is 5'UTR derived from tobacco etch virus.

[0430] Table 10 summarizes information about the bstb constructs specific for the TAA CLDN6 generated during the course of this invention. Information on specificity, origin of the sequence from a monoclonal antibody (mAB), codon usage, and additional sequence characteristics is listed. The sequences encoding the variable domains of the target cell binding moieties were originally obtained from Ganymed Pharmaceuticals AG.

[0431] [Table 10]

[0432] b. IVT-mRNA synthesis To purify templates for in vitro transcription, plasmid DNA was linearized downstream of the poly(A) tail-encoding region using a class II restriction endonuclease, thereby generating a template for transcribing RNA with no additional nucleotides beyond the poly(A) tail (Holtkamp et al., 2006). The linearized template DNA was purified, quantified spectrophotometrically, and then transcribed in vitro using T7 RNA polymerase essentially as previously described (Grudzien-Nogalska et al., 2013) in the presence of 7.5 mM each of ATP, CTP, UTP, 1.5 mM GTP, and 6 mM beta-S-ARCA (D2) cap analog (Kuhn et al., 2010). RNA was purified using magnetic particles (Berensmeier 2006). For in vivo studies, non-immunogenic RNA was used. For this purpose, N1-methylpseudouridine-5'-triphosphate (TriLink Biotechnologies, San Diego, CA, USA) was incorporated in place of UTP to remove double-stranded RNA. RNA was enzymatically capped with the ScriptCap m7G Capping System and 2'O-methyltransferase (CellScript, Madison, WI, USA). RNA concentration and quality were assessed spectrophotometrically and analyzed on a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). A sketch of the two IVT-mRNAs required for the formation of the complete bstb molecule is shown in Figure 14.

[0433] Example 13: Determination of the specific lytic activity of bstb_435 / 434 and bstb_436 / 434 in an in vitro cytotoxicity assay a. Electroporation of producer cells To produce bstb protein from IVT-mRNA, 4 × 10 cells were used per ml of log-phase growth. 6K-562 cells (ATCC CCL-243, LGC Standards GmbH, Wesel, Germany) were electroporated with HO (mock) or 25 μg / ml IVT-mRNA per bstb strand in a 0.4 mm cuvette using a GenePulser MXCell (Bio-Rad Laboratories, Dreieich, Germany) at 200 V, 2 pulses, 8 ms. The cells were then plated at 5 × 10 in 6-well tissue culture plates. 5 After 48 hours of incubation, the supernatant was collected by centrifugation and filtered through 0.2 μm Minisart NML Syringe Filters (Sartorius). The solution was sterile filtered using a filter (Göttingen, Germany).

[0434] b. Quantification of bstb in producer cell supernatants via ELISA Supernatants from electroporated K-562 cells were subjected to an in-house designed ELISA for quantification. Briefly, 96-well MaxiSorp™ plates (Thermo Scientific, Braunschweig, Germany) were coated with 4.5 μg / ml goat anti-human IgG F(ab')2 antibody (Abd Serotec, Puchheim, Germany) in DPBS for 1 h at 37°C, washed three times (wash buffer: PBS / 0.05% Tween-20), and blocked overnight at 2-8°C with DPBS containing 3% milk powder. After the washing step, K-562 supernatants containing serial dilutions of bstb_369 / 367 and bstb_371 / 367 proteins as references and serial dilutions of bstb_435 / 434 and bstb_436 / 434 proteins (diluent: 0.2% BSA) were added to the coated plates in triplicate. After 2 hours at room temperature, the plates were washed three times. For detection of bstb bound to anti-F(ab')2, 3.5 μg / ml mouse anti-IMAB206 (Ganymed Pharmaceuticals AG, Mainz, Germany) was added and incubated for 1 hour at room temperature. The plates were again washed three times. A 1:500 dilution of AP-conjugated goat anti-mouse IgG Fc antibody (Jackson ImmunoResearch, Newmarket, UK) was added and incubated for 1 hour at room temperature. For visualization, the plates were incubated with a homemade pNPP substrate solution for 30 minutes at room temperature in the dark, and the reaction was finally stopped with 3 M KOH. Analysis was performed at 405 / 492 nm on a Tecan M200 microplate reader (Tecan, Mannedorf, Switzerland). Data were analyzed using GraphPad Prism software 6 and Microsoft Excel 2010 (data not shown).

[0435] Concentrations of 253 ng / ml of bstb_435 / 434 and 427 ng / ml of bstb_436 / 434 were measured in the K-562 supernatant. Thus, both bstbs were translated and secreted in different amounts. The amount of protein obtained was highly dependent on electroporation and varied over time. Over the course of various experiments, 1.0- to 1.7-fold higher amounts of bstb_436 / 434 were produced compared to bstb_435 / 434.

[0436] c. Western blot analysis of producer cell lysates and supernatants The supernatant and cell lysate of K-562 cells (Example 13a.) were used for the analysis of bstb protein translation and secretion. The cell lysate was diluted to 1 x 10 in 200 μl of 4x Laemmli buffer (Bio-Rad Laboratories) containing 20 units of benzonase (Merck Millipore, Darmstadt, Germany). 6The bstb proteins were obtained by incubation of 1000 washed cells in 1000 ml of PBS. Samples were then heated to 95°C for 10 minutes with (reducing) or without (non-reducing) 1 M dithiothreitol (DTT, final concentration 0.1 M). The supernatants were similarly treated with Laemmli buffer. The prepared cell lysates and supernatants and the corresponding purified bstb proteins were separated by polyacrylamide gel electrophoresis using 4-15% Criterion™ TGX Stain-Free™ Gels (Bio-Rad Laboratories), followed by Western blot analysis (Figure 15) according to standard procedures known to those skilled in the art. For Western blot analysis, horseradish peroxidase (HRP)-conjugated antibodies anti-6xHis tag (1:10,000, Abcam, Cambridge, MA, USA), StrepMAB-Classic (1:10,000, IBA GmbH, Göttingen, Germany), and anti-β-actin (1:25,000, Abcam) were used. Western blots were recorded using a Bio-Rad Chemidoc MP Imaging System. bstb protein signals were detected at 50-55 kD under reducing conditions and approximately 100 kD under non-reducing conditions, relative to internal molecular weight standards. bstb_435 / 436 (Figure 15A) and bstb_436 / 434 (Figure 15B) were detected primarily in the supernatant but also in the cell lysate, indicating incomplete secretion at the time of collection. The heterodimeric composition of the bstb protein is evident under reducing conditions and detection using a mixture of anti-6xHis tag HRP and StrepMAB-Classic HRP (Figure 15C). The Fd and L fragments differ in size by approximately 2 kD (approximately 51 kD and 49 kD, respectively). Quantitation of the protein bands corresponding to HMW and monomeric bstb detected under nonreducing conditions determined 3% HMW and 88% monomeric bstb_435 / 434 species and 1.4% HMW and 92% monomeric bstb_436 / 434 species. A few minor bands (approximately 8% and 6% total, respectively) were detected, indicating slight degradation during cell culture.

[0437] In summary, both bstb molecules can be efficiently translated from IVT-mRNA and are secreted into the cell culture supernatant. bstb_436 / 434 exhibits a somewhat favorable protein pattern with respect to HMW species and degradation products, although the percentage of HMW species is low in both bstb molecules.

[0438] d. Luciferase cytotoxicity assay and EC50 determination Cytotoxicity assays were performed essentially as described in Example 2a. bstb protein was used as a reference. K-562 supernatant samples from Example 13a were diluted with K-562 mock supernatant (electroporation without IVT-mRNA). min and L max The K-562 supernatant samples were supplemented with K-562 mock supernatant.

[0439] 20 μl of the supernatant containing bstb was added to OV-90 and MDA-MB-231 cancer cells (stably transfected with luciferase) plated with PBMCs as effector cells (E:T 5:1) in a 7-point 5-fold serial dilution series ranging from 33.1 fM to 516.5 pM. After 48 hours of co-incubation, luciferase solution was added and the plates were analyzed.

[0440] As shown in Figure 16, both bstb + mediated efficient lysis of OV-90, but CLDN6 - The effect was not mediated by MDA-MB-231. Approximately 77-80% maximal lysis was achieved with bstb_436 / 434 and bstb_435 / 434 at 516.5 pM, respectively. The EC50 values ​​indicate 1.3-fold higher activity than bstb_435 / 434. The EC50 values ​​of the RNA-translated bstb were 4-fold and 7.5-fold lower than the corresponding protein fragments bstb_369 / 367 and bstb_371 / 367, respectively. This effect could arise from higher protein activity in the absence of any further protein manipulation, from the influence of HMW species, quantitation deviations, or a combination thereof.

[0441] In summary, the RNA approach is feasible, highly efficient, and time-saving since protein expression, purification, stabilization, and analysis are omitted.

[0442] Example 14: Efficacy of IVT-RNA encoding a bispecific antibody compared to a protein reference in a mouse xenograft model To investigate the therapeutic efficacy of IVT-mRNA encoding the bispecific bstb and dual (scFv)2 antibodies compared to protein references in vivo, the immunodeficient mouse strain NOD.Cg-Prkd scid IL2rg tm1Wjl We again selected mice with either SzJ or short NSG (Jackson Laboratory, Bar Harbor, ME, USA). All mice were used in accordance with guidelines from the Institutional Animal Care Committee of Johannes Gutenberg University (Mainz, Germany).

[0443] a. Treatment of highly CLDN6-expressing tumors in mice with bstb and dual (scFv)2-encoding IVT-RNA compared to protein In a typical study, male and female NSG mice aged 6–25 weeks and weighing 18–38 g were used. CLDN6-positive OV-90 cells were used as tumor cells, and PBMCs were used as effector cells.

[0444] 5×10 6 Tumor cells were inoculated subcutaneously (sc), and 19 days later, 1 × 10 7 PBMCs were administered intraperitoneally (ip). PBMC-engrafted mice, analyzed in peripheral blood 6 days after PBMC injection, were stratified into groups according to tumor volume and sex (both sexes in all groups). Approximately 250 mm per group 3Treatment began 8 days after PBMC administration, when the tumors were at a fairly advanced stage with a mean tumor volume of 100 μg. Mice were treated once weekly by intravenous injection (iv) into the retro-orbital venous plexus (Figure 17A). Groups "G1-RNA control," "G2-bstb RNA," and "G3-dual-scFv RNA" received 200 μl of IVT-mRNA per mouse, each containing 3 μg of IVT-mRNA complexed with the TransIT®-mRNA Transfection Kit (Mirus Bio, Madison, WI, USA). Luciferase IVT-mRNA was used as the RNA control; the bstb-encoding RNAs were CDLN6×CD3 bstb Fd and LC chain IVT-mRNAs 435 and 434 (mixed 1:1); and the dual (scFv)2-encoding RNA was CDLN6×CD3 dual (scFv)2 IVT-RNA 123r. All IVT-RNAs were synthesized in vivo according to Example 12b. "G4-vehicle control" included six mice treated with DPBS and six mice treated with 10 mM NaOAc buffer, pH 5.5 (bstb and double (scFv)2 formulation buffers, respectively). "G5-bstb protein" and "G6-double scFv protein" were treated with protein analogs to the IVT-mRNA items. The bstb protein was bstb_369 / 367 (100 μg / kg, 5 μl / g body weight dose) and the double (scFv)2 protein was double scFv_123 (200 μg / kg, 5 μl / g body weight dose). The treatment groups are summarized in Table 11. Treatment was for four consecutive weeks. Tumor size was measured twice a week with a digital calibrated caliper, and tumor volume was calculated using the formula: tumor volume [mm 3 ] = length [mm] x (width [mm]) 2 The tumor volume was calculated by the formula: 3 Mice were sacrificed by cervical dislocation when they reached 0.05 or in case of severe morbidity (mainly symptoms of graft-versus-host disease (GVHD)).

[0445] Figure 17B shows the effect of treatment on tumor growth. Antitumor efficacy was measured as tumor growth inhibition / reduction and survival compared to the RNA control group G1 or the vehicle control G4. Compared to the control group G1, in which tumor growth reduction was 0 / 10 mice, treatment with test item bstb_435 / 434 (G2) led to tumor reduction in 8 / 9 mice, and treatment with test item bi-scFv_123r (G3) led to tumor reduction in 9 / 9 mice. Treatment with bstb protein analogs (G5, G6) was similar, but the efficacy of bi(scFv)2 was lower. Importantly, for direct comparison, the protein was administered according to an RNA injection scheme (once weekly, intravenously via the retroorbital route) rather than the established protein treatment schedule of three times weekly, i.p. While prolonged protein translation and secretion over several days likely resulted in more efficient RNA, injected protein was rapidly cleared. In vivo protein blood levels after RNA injection are unknown. Three mice from G1 to G3 were sacrificed early and tumor-infiltrating lymphocytes (TILs) were examined by flow cytometry analysis. To this end, single tumor cell suspensions were stained with anti-human CD45 and CD3 fluorescently labeled antibodies (BD Biosciences, Heidelberg, Germany), and whole tumor singlet cells were analyzed using a BD FACS Canto II flow cytometer. In the RNA control group, ≤3% infiltrating human T cells were detected, whereas >7% and >12% were detected in the bstb- and double(scFv)2-RNA groups, respectively (Figure 17C). These data demonstrate the directional infiltration of human T cells bound by antibodies encoded by bispecific RNA.

[0446] b. Determining the effect of treatment on body weight The weight of each mouse was checked weekly on a laboratory scale, and no treatment-related weight loss was observed (data not shown).

[0447] References [Table 11]

Claims

1. A binding agent comprising at least three binding domains, The first binding domain binds to CD3, the second binding domain and the third binding domain bind to claudin 6 (CLDN6), the first binding domain has the format of a Fab molecule, and the second and third binding domains have the format of an scFv molecule; The binding factor is (a) a first polypeptide comprising a VH domain having specificity for CD3 (VH(CD3)), a VH domain having specificity for CLDN6 (VH(CLDN6)), and a VL domain having specificity for CLDN6 (VL(CLDN6)); (b) a second polypeptide comprising a VL domain having specificity for CD3 (VL(CD3)), a VH domain having specificity for CLDN6 (VH(CLDN6)), and a VL domain having specificity for CLDN6 (VL(CLDN6)); Including, the first polypeptide and the second polypeptide are associated to form a binding agent; (i) the CD3-binding domain comprises VH(CD3) and VL(CD3); VH(CD3) HCDR1 with the amino acid sequence GYTFTRYT, HCDR2 having the amino acid sequence INPSRGYT, and an HCDR3 having the amino acid sequence ARYYDDHYSLDY; VL(CD3) LCDR1, which has the amino acid sequence SSVSY; LCDR2 having the amino acid sequence DTS, and comprising an LCDR3 having the amino acid sequence QQWSSNPLT, (ii) a combination of VH(CLDN6) and VL(CLDN6), in which the CLDN6-binding domain is selected from the group consisting of: (a) VH(CLDN6) HCDR1 comprising the sequence set forth in SEQ ID NO: 44, HCDR2 comprising the sequence set forth in SEQ ID NO: 45, and comprising an HCDR3 comprising the sequence set forth in SEQ ID NO: 46, VL(CLDN6) LCDR1 comprising the sequence shown in SEQ ID NO: 50; LCDR2 comprising the sequence set forth in SEQ ID NO: 51, and comprising an LCDR3 comprising the sequence set forth in SEQ ID NO: 52; (b) VH(CLDN6) HCDR1 comprising the sequence set forth in SEQ ID NO: 44, HCDR2 comprising the sequence set forth in SEQ ID NO: 45, and comprising an HCDR3 comprising the sequence set forth in SEQ ID NO: 47, VL(CLDN6) LCDR1 comprising the sequence shown in SEQ ID NO: 50; LCDR2 comprising the sequence set forth in SEQ ID NO: 51, and comprising an LCDR3 comprising the sequence set forth in SEQ ID NO: 53; and (c) VH(CLDN6) HCDR1 comprising the sequence set forth in SEQ ID NO: 44, HCDR2 comprising the sequence set forth in SEQ ID NO: 45, and comprising an HCDR3 comprising the sequence set forth in SEQ ID NO: 46, VL(CLDN6) LCDR1 comprising the sequence shown in SEQ ID NO: 50; LCDR2 comprising the sequence set forth in SEQ ID NO: 51, and comprising an LCDR3 comprising the sequence set forth in SEQ ID NO: 53; Including, binding factor.

2. 2. The binding agent of claim 1, wherein the first polypeptide further comprises a constant domain 1 (CH1) of an immunoglobulin heavy chain and the second polypeptide further comprises a constant domain (CL) of an immunoglobulin light chain, and both domains are capable of associating.

3. 3. The binding agent of claim 2, wherein the first and second polypeptides are covalently linked via a disulfide bridge between the CH1 and CL domains.

4. In the first polypeptide and the second polypeptide, the VH domain, the VL domain, the CH1 domain, and the CL domain are arranged from the N-terminus to the C-terminus as follows: (i) VH(CD3)-CH1-VH(CLDN6)-VL(CLDN6) and VL(CD3)-CL-VH(CLDN6)-VL(CLDN6); or (ii) VH(CD3)-CH1-VL(CLDN6)-VH(CLDN6) and VL(CD3)-CL-VL(CLDN6)-VH(CLDN6) The binding agent according to claim 2 or 3, wherein the binding agent is arranged in the order:

5. 5. The binding agent of claim 4, wherein the VH-VL or VL-VH domain is linked to the CH1 domain or CL domain via a peptide linker.

6. The peptide linker has the amino acid sequence DVPG 2 S or SGPG 3 RS(G 4 S) 2 6. The binding agent of claim 5, comprising:

7. The VH and VL domains have the amino acid sequence (G 4 S) x (wherein x is 3, 4, 5 or 6) to form a VH-VL or VL-VH domain.

8. 8. The binding agent of claim 1, wherein CD3 is expressed on the surface of T cells.

9. 9. The binding agent of claim 1, wherein the first binding domain binds to the epsilon chain of CD3.

10. The binding agent of any one of claims 1 to 9, wherein CLDN6 is expressed on the surface of a cancer cell.

11. A binding agent according to any one of claims 1 to 10, which binds to the extracellular domain of CLDN6.

12. 12. The binding agent according to claim 1, wherein the binding to CD3 and / or the binding to CLDN6 is specific binding.

13. 13. The binding agent of any one of claims 1 to 12, which induces T cell-mediated cytotoxicity against cancer cells that express CLDN6.

14. The binding agent of claim 13, which induces T cell-mediated cytotoxicity against CLDN6-expressing cancer cells with an EC50 of ≦10 nM.

15. VH(CD3) (a) the amino acid sequence represented by SEQ ID NO: 5 or a fragment thereof; or (b) an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 5 or a fragment thereof and / or VL(CD3) (a) the amino acid sequence represented by SEQ ID NO: 6 or a fragment thereof, or (b) an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 6 or a fragment thereof comprising or consisting of 15. A binding agent according to any one of claims 1 to 14.

16. VH(CLDN6) (a) the amino acid sequence represented by SEQ ID NO: 8 or a fragment thereof; or (b) an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 8 or a fragment thereof and / or VL(CLDN6) (a) the amino acid sequence represented by SEQ ID NO: 10 or a fragment thereof, or (b) an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 10 or a fragment thereof comprising or consisting of 16. A binding agent according to any one of claims 1 to 15.

17. (i) the first polypeptide is (a) the amino acid sequence represented by SEQ ID NO: 14 or a fragment thereof, or (b) an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 14 or a fragment thereof comprising or consisting of The second polypeptide is (a) the amino acid sequence represented by SEQ ID NO: 16 or a fragment thereof, or (b) an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 16 or a fragment thereof comprising or consisting of; (ii) the first polypeptide is (a) the amino acid sequence represented by SEQ ID NO: 15 or a fragment thereof, or (b) an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 15 or a fragment thereof comprising or consisting of The second polypeptide is (a) the amino acid sequence represented by SEQ ID NO: 16 or a fragment thereof, or (b) an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 16 or a fragment thereof comprising or consisting of; (iii) the first polypeptide is (a) the amino acid sequence represented by SEQ ID NO: 17 or a fragment thereof; or (b) an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 17 or a fragment thereof comprising or consisting of The second polypeptide is (a) the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof; or (b) an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof comprising or consisting of; or (iv) the first polypeptide is (a) the amino acid sequence represented by SEQ ID NO: 18 or a fragment thereof; or (b) an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 18 or a fragment thereof comprising or consisting of The second polypeptide is (a) the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof; or (b) an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof comprising or consisting of 17. A binding agent according to any one of claims 1 to 16.

18. - an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 5, which contains no more than two amino acid substitutions relative to SEQ ID NO: 5; - an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 6, and contains no more than two amino acid substitutions relative to SEQ ID NO: 6; - an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 8, which contains no more than two amino acid substitutions relative to SEQ ID NO: 8; - an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 10, which contains no more than two amino acid substitutions relative to SEQ ID NO: 10; - an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 14, which contains no more than two amino acid substitutions relative to SEQ ID NO: 14; - an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 15, which contains no more than two amino acid substitutions relative to SEQ ID NO: 15; - an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 16, which contains no more than two amino acid substitutions relative to SEQ ID NO: 16; - an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 17, which contains no more than two amino acid substitutions relative to SEQ ID NO: 17; - an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 18, which contains no more than two amino acid substitutions relative to SEQ ID NO: 18, or - an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 19, which contains no more than two amino acid substitutions relative to SEQ ID NO: 19; 18. A binding agent according to any one of claims 15 to 17.

19. 19. The binding agent of claim 18, wherein the amino acid substitutions are made by modifying one or more free cysteine ​​residues to replace them with an amino acid residue other than cysteine, wherein the amino acid residue other than cysteine ​​is selected from the group consisting of serine, alanine, threonine, glycine, tyrosine, tryptophan, leucine, or methionine.

20. 20. The binding agent of any one of claims 1 to 19, wherein the first polypeptide and the second polypeptide of the binding agent are encoded on the same nucleic acid or on different nucleic acids.

21. 21. A nucleic acid encoding a binding agent according to any one of claims 1 to 20.

22. 22. The nucleic acid of claim 21, wherein the binding agent comprises two or more polypeptide chains, and the different polypeptide chains are encoded on the same nucleic acid or on different nucleic acids.

23. A composition comprising a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide, or a nucleic acid encoding a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are as defined in any one of claims 1 to 20, and the nucleic acid is in the form of a vector or in the form of RNA.

24. 23. A host cell comprising the nucleic acid of claim 21 or 22.

25. 25. A medicament comprising a binding agent according to any one of claims 1 to 20, a nucleic acid according to claim 21 or 22, a composition according to claim 23, or a host cell according to claim 24.

26. 26. The pharmaceutical composition according to claim 25, for use in the treatment or prevention of cancer.

27. 25. A pharmaceutical composition comprising a binding agent according to any one of claims 1 to 20, a nucleic acid according to claim 21 or 22, a composition according to claim 23, or a host cell according to claim 24.

28. 28. A binding agent according to any one of claims 1 to 20, a nucleic acid according to claim 21 or 22, a composition according to claim 23, a host cell according to claim 24, or a pharmaceutical composition according to claim 27 for use in the treatment or prevention of cancer.

29. The pharmaceutical of claim 26, the binding factor of claim 28, the nucleic acid of claim 28, the composition of claim 28, the pharmaceutical composition of claim 28, or the host cell of claim 28, wherein the cancer is characterized by cancer cells that express claudin 6 and the cancer is selected from the group consisting of bladder cancer, ovarian cancer, lung cancer including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, cancer of the bladder, kidney cancer, colon cancer, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, testicular teratoma and embryonic testicular cancer, uterine cancer, germ cell tumors, and metastatic forms thereof.

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