Drug conjugates comprising antibodies against claudin 18.2

Abstract

To provide, generally, a therapy for effectively treating and / or preventing cancer associated with cells expressing CLDN18.2 such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, hepatic cancer, head-neck cancer, and cancer of the gallbladder and metastases thereof, particularly gastric cancer metastasis such as Krukenberg tumors, peritoneal metastasis and lymph node metastasis; where particularly preferred cancer diseases are adenocarcinomas of the stomach, the esophagus, the pancreatic duct, the bile ducts, the lung and the ovary.SOLUTION: The present invention provides anti-CLDN18.2 antibody-drug conjugates which are effective for treating and / or preventing cancer diseases associated with cells expressing CLDN18.2, including gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, hepatic cancer, head-neck cancer, and cancer of the gallbladder and metastases thereof.SELECTED DRAWING: None

Description

[Background technology]

[0001] Monoclonal antibodies (mABs) have revolutionized cancer treatment over the past two decades (Sliwkowski, MX et al. (2013), Science 341(6151), 1192-1198). A crucial feature of mABs is their high specificity and their ability to target tumor cells, mark them for immune effector-mediated cell killing (complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC)), and / or result in reduced proliferation and apoptosis (Kubota, T. et al. (2009) Cancer Sci. 100(9), 1566-1572). Conjugation to cytotoxic drugs can extend the utility of mABs and improve their potency and efficacy (Goldmacher, VS et al. (2011) Ther. Deliv. 2(3), 397-416; Sievers, EL (2013) Annu. Rev. Med. 64, 15-29).

[0002] Historically, the use of cytotoxic drugs to treat cancer has focused on chemotherapy, which targets dividing cancer cells. These compounds target not only cancer cells but also other dividing healthy cells in the body, and patients undergoing treatment experience severe side effects that are dose-limiting. The therapeutic index (maximum tolerated dose / minimum effective dose) of these drugs is low, resulting in a narrow therapeutic window (Ismael, GFV et al. (2008) Cancer Treat Rev. 34(1), 81-91). To circumvent this obstacle in drug development and improve the therapeutic index, antibodies can be used to deliver cytotoxic drugs specifically to tumors. By combining the unique targeting capabilities of antibodies with the cancer-killing capabilities of cytotoxic drugs, antibody-drug conjugates (ADCs) exhibit lower side effects and result in a wider therapeutic window compared to traditional chemotherapy agents (Gerber, H.-P. et al. (2013) Nat. Prod. Rep., 30(5), 625-639).

[0003] ADCs are designed to kill cancer cells in a target-dependent manner. The first step in this process is the binding of an antibody to its antigen. Upon ADC binding, the entire antigen-ADC complex is internalized and the cytotoxic payload is released into the tumor cell, resulting in cell death. Factors that influence the therapeutic index of an ADC include the antibody, the tumor targeting antigen, the cytotoxic drug, and the linker (Panowksi, S. et al. (2014) MAbs, 6(1), 34-45).

[0004] As a basic requirement for developing ADCs, tumor target antigens must be localized on the cell surface and accessible to circulating antibodies. Furthermore, tumor selectivity and expression level of target antigens are crucial parameters for designing safe and effective ADCs. Currently, various tumor-associated cell surface antigens are being evaluated as ADC targets for cancer therapy (Trail, PA(2013) Antibodies, 2(1), 113-129; Teicher, BA(2009) Curr. Cancer Drug Targets 9(8), 982-1004).

[0005] The efficiency of ADCs also depends on the cytotoxic drug. The amount of antibody that localizes to the tumor is very small compared to the dose administered, so toxic compounds with subnanomolar potency are required. Auristatins and maytansinoids are two classes of highly potent cytotoxins currently used in ADC development (Trail, PA (2013) Antibodies, 2(1), 113-129). Both are antimitotic agents that block tubulin polymerization and cause cell death by G2 / M phase cell cycle arrest (Lopus, M. et al. (2010) Mol. Cancer Ther. 9(10), 2689-2699; Francisco, J. A et al. (2003) Blood, 102(4), 1458-1465). In addition to mAB specificity and drug potency, the linker is a critical element in ADC development. Linkers should be stable to exploit the pharmacokinetic half-life of the mAB and should not release the cytotoxic drug until antigen-mediated internalization. Linkers can be classified according to their mechanism of drug release: cleavable linkers release the drug by hydrolysis or enzymatic cleavage after antigen-specific internalization, whereas non-cleavable linkers release the drug via degradation of the mAB in lysosomes after internalization (Dosio, F. et al. (2011) Toxins (Basel) 3(7), S. 848-883).

[0006] Depending on the linker design, membrane-permeable (lipophilic) toxins released inside target-positive cells can cross the cell membrane and kill other cells in close proximity, including adjacent cancer cells that lack antigen expression (bystander effect) (Kovtun, YV et al. (2006) Cancer Res., 66(6), 3214-3221). The ability of these cytotoxic drugs to mediate local bystander killing is an important selection criterion for ADCs directed against antigens that are heterogeneously expressed in tumors.

[0007] The tight junction molecule claudin 18 isotype 2 (CLDN18.2) is a cancer-associated splice variant of claudin 18. CLDN18.2 is a 27.8 kDa transmembrane protein that contains four membrane-spanning domains with two small extracellular loops (loop 1 encompassed by hydrophobic regions 1 and 2; loop 2 encompassed by hydrophobic regions 3 and 4). CLDN18.2 is a highly selective gastric lineage antigen that is exclusively expressed on short-lived differentiated gastric epithelial cells and is not detectable in any other normal human tissues. The antigen is ectopically expressed at significant levels in a variety of human cancers, including gastroesophageal and pancreatic cancers (Sahin, U. et al., Clin Cancer Res, 2008.14(23):7624-34). CLDN18.2 protein is frequently detected in lymph node metastases of gastric cancer and also in distant metastases. CLDN18.2 appears to be involved in the proliferation of CLDN18.2-positive tumor cells, because downregulation of the target by siRNA technology inhibits the proliferation of gastric cancer cells.

[0008] IMAB362 is a chimeric monoclonal antibody of IgG1 subtype directed against CLDN18.2. IMAB362 recognizes the first extracellular domain of CLDN18.2 with high affinity and specificity and does not bind to any other claudin family members, including the closely related splice variant 1 of claudin 18 (CLDN18.1). In human xenografts expressing CLDN18.2, survival benefit and tumor regression were observed in mice after administration of IMAB362. When administered intravenously in relevant animal species, toxicity in stomach tissue is not observed because the target epitope is not accessible. However, the tumor target becomes accessible to IMAB362 during malignant transformation. IMAB362 bundles four independent and highly potent mechanisms of action: (i) antibody-dependent cellular cytotoxicity (ADCC), (ii) complement-dependent cytotoxicity (CDC), (iii) induction of apoptosis induced by crosslinking targets at the tumor surface, and (iv) direct inhibition of proliferation. A previous Phase I trial evaluated IMAB362 as monotherapy at a single dose in patients with late-stage gastroesophageal cancer. The study failed to observe any relevant differences in the AE profile and other safety parameters between dose arms, and therefore single doses of the antibody were administered at doses up to 1000 mg / m 2 The best results regarding antitumor activity were obtained at a dose of 300 mg / m 2 and 600 mg / m 2 A Phase IIa clinical trial was conducted to determine the safety, tolerability, and antitumor activity of repeated doses of IMAB362 in patients with metastatic, refractory, or recurrent disease from advanced adenocarcinoma of the stomach or lower esophagus, as proven by histology.

[0009] As mentioned above, CLDN18.2 has a restricted expression pattern in normal cells, and therefore appears to be an ideal target for antibody-mediated therapy of CLDN18.2-expressing cancers. Thus, there is a need for a therapy specifically directed to CLDN18.2-expressing cancer cells that can exert clinically useful cytotoxic or cytostatic effects on CLDN18.2-expressing cells without exerting undesirable effects on non-CLDN18.2-expressing cells. Preferably, the therapy should not be associated with the disadvantages and undesirable side effects commonly associated with approaches that have been used to increase the therapeutic efficacy of antibodies, such as radiolabeling and combination with chemotherapy. For example, isotope therapy is associated with myelosuppression, and combination therapy with antibodies and chemotherapeutic agents is associated with immunosuppression. Furthermore, isotope-labeled substances are difficult to produce, and patients often experience relapsed disease after initial treatment with isotope-labeled substances.

[0010] The present invention demonstrates the existence of anti-CLDN18.2 monoclonal antibodies that can be highly efficiently internalized upon binding to CLND18.2 on CLND18.2-expressing cells, and thus are suitable for ADC development. Furthermore, the successful conjugation of such antibodies to the drugs DM4 and MMAE using cleavable SPDB or Val-Cit (vc) linkers, respectively, is also disclosed. In vitro, the antibody conjugates reduce the viability of gastric and pancreatic cancer cells expressing CLDN18.2. IMAB362-vcMMAE and IMAB362-DM4 do not bind to or affect the viability of CLDN18.2-negative cells. Both DM4 and vcMMAE conjugates exert bystander killing effects on CLDN18.2-negative cancer cells co-cultured with CLDN18.2-positive cancer cells in vitro. Furthermore, intravenous administration of the antibody conjugates in vivo in nude mice bearing CLDN18.2-positive gastric or pancreatic xenograft tumors results in dose-dependent tumor growth inhibition, survival benefit, and even complete regression of early and advanced tumors. Significant therapeutic effects are observed at single-dose intravenous application of approximately 4-8 mg / kg, with optimal therapeutic effects being achieved at 15-16 mg / kg. The maximum tolerated single dose of both conjugates could not be determined, as the highest possible tested doses of 15.2 and 16 mg / kg did not result in hepatotoxicity or other toxic effects.

[0011] From the data presented herein, it can be concluded that anti-CLDN18.2 antibody-drug conjugates such as those described herein are highly potent drugs for treating CLDN18.2-positive human cancers, such as gastric and pancreatic cancer. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] U.S. Patent No. 4,151,042 [Patent Document 2] U.S. Patent No. 4,137,230 [Patent Document 3] U.S. Patent No. 4,248,870 [Patent Document 4] U.S. Patent No. 4,256,746 [Patent Document 5] U.S. Patent No. 4,260,608 [Patent Document 6] U.S. Patent No. 4,265,814 [Patent Document 7] U.S. Patent No. 4,294,757 [Patent Document 8] U.S. Patent No. 4,307,016 [Patent Document 9] U.S. Patent No. 4,308,268 [Patent Document 10] U.S. Patent No. 4,308,269 [Patent Document 11] U.S. Patent No. 4,309,428 [Patent Document 12] U.S. Patent No. 4,313,946 [Patent Document 13] U.S. Patent No. 4,315,929 [Patent Document 14] U.S. Patent No. 4,317,821 [Patent Document 15] U.S. Patent No. 4,322,348 [Patent Document 16] U.S. Patent No. 4,331,598 [Patent Document 17] U.S. Patent No. 4,361,650 [Patent Document 18] U.S. Patent No. 4,364,866 [Patent Document 19] U.S. Patent No. 4,424,219 [Patent Document 20] U.S. Patent No. 4,362,663 [Patent Document 21] U.S. Patent No. 4,371,533 [Patent Document 22] US Patent Application Publication No. 2003 / 0118592 [Patent Document 23] U.S. Patent Application No. 2003 / 0133939

Patent document 24

Patent document 25

Patent document 26

Patent document 27

Patent document 28

Patent document 29

Patent document 30

Non-licensed literature

[0013]

Non-licensed literature 1

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-licensed Document 38

Non-licensed Document 39

Non-licensed Document 40

Non-licensed Document 41

Non-licensed Document 42

Non-licensed Document 43

Non-licensed Document 44

Non-licensed Document 45

Non-licensed Document 46

Non-licensed Document 47

[0014] The present invention generally provides a therapy for effectively treating and / or preventing cancers associated with cells expressing CLDN18.2, including, for example, 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, and metastases thereof, particularly gastric cancer metastases, e.g., Krukenberg tumors, peritoneal metastases, and lymph node metastases. Particularly preferred cancer diseases are gastric, esophageal, pancreatic duct, bile duct, lung, and ovarian adenocarcinomas. [Means for solving the problem]

[0015] In one aspect, the present invention provides a method of treating or preventing a CLDN18.2-expressing cancer, comprising administering to a cancer patient an antibody-drug conjugate comprising an antibody capable of binding to CLDN18.2 covalently attached to at least one toxin drug moiety.

[0016] In one embodiment, the antibody-drug conjugate is internalized within the cell after binding to CLDN18.2 expressed by the cell.

[0017] In one embodiment, the antibody having the ability of binding to CLDN18.2 specifically binds to CLDN18.2. In one embodiment, the antibody-drug conjugate specifically binds to CLDN18.2.

[0018] In one embodiment, the antibody capable of binding to CLDN18.2 is a monoclonal, chimeric, or humanized antibody, or a fragment of an antibody. In one embodiment, the antibody capable of binding to CLDN18.2 is a monoclonal antibody.

[0019] In one embodiment, the antibody capable of binding to CLDN18.2 binds to the natural epitope of CLDN18.2 present on the surface of living cells.In one embodiment, the antibody capable of binding to CLDN18.2 binds to the extracellular domain of CLDN18.2.In one embodiment, the antibody capable of binding to CLDN18.2 binds to the first extracellular loop of CLDN18.2.

[0020] In one embodiment the antibody having the ability of binding to CLDN18.2 is an antibody selected from the group consisting of antibodies produced by and / or obtained from the clone deposited under accession numbers DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809 or DSM ACC2810, (ii) antibodies which are chimerized or humanized forms of antibodies belonging to (i), (iii) antibodies which have the specificity of antibodies belonging to (i), and (iv) antibodies which comprise an antigen-binding portion or antigen-binding site, in particular the variable region, of an antibody belonging to (i) and preferably having the specificity of an antibody belonging to (i). In one embodiment, the antibody capable of binding to CLDN18.2 comprises a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 32 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 39 or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the antibody capable of binding to CLDN18.2 comprises a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 17 or 51 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 24 or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the antibody capable of binding to CLDN18.2 comprises a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 30 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 35 or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the antibody capable of binding to CLDN18.2 comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 15 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof, or a variant of said amino acid sequence or fragment.In one embodiment, the antibody capable of binding to CLDN18.2 recognizes the same or essentially the same epitope as a CLDN18.2-binding antibody comprising a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 32 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 39 or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or competes with said CLDN18.2-binding antibody for binding to CLDN18.2. In one embodiment, the antibody capable of binding to CLDN18.2 recognizes the same or essentially the same epitope as a CLDN18.2-binding antibody comprising a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 17 or 51, or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 24, or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or competes with said CLDN18.2-binding antibody for binding to CLDN18.2. In one embodiment, the antibody capable of binding to CLDN18.2 recognizes the same or essentially the same epitope as a CLDN18.2 binding antibody comprising a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 30 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 35 or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or competes with said CLDN18.2 binding antibody for binding to CLDN18.2. In one embodiment, the antibody capable of binding to CLDN18.2 recognizes the same or essentially the same epitope as a CLDN18.2 binding antibody comprising a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 15 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or competes with said CLDN18.2 binding antibody for binding to CLDN18.2. An antibody that competes with a second antibody for binding to a target is preferably antagonistic to said second antibody.

[0021] In one embodiment, the toxin drug moiety is cell membrane permeable. In one embodiment, the toxin drug moiety is a cytotoxic or cytostatic agent. In one embodiment, the toxin drug moiety is a maytansinoid or an auristatin. In one embodiment, the maytansinoid is selected from the group consisting of DM1 and DM4. In one embodiment, the auristatin is selected from the group consisting of monomethylauristatin E (MMAE) and monomethylauristatin F (MMAF).

[0022] In one embodiment, the antibody capable of binding to CLDN18.2 is covalently attached to the toxin drug moiety by a linker. In one embodiment, the linker is a cleavable linker. In one embodiment, the linker is cleavable under intracellular conditions. In one embodiment, the linker is hydrolyzable at a pH of less than 5.5. In one embodiment, the linker is cleavable by intracellular proteases. In one embodiment, the linker is a cathepsin-cleavable linker. In one embodiment, the linker comprises a dipeptide. In one embodiment, the dipeptide is val-cit or phe-lys. In one embodiment, the antibody is attached to the linker via a cysteine ​​thiol of the antibody. In one embodiment, the antibody is attached to the linker via an amine group, in particular an amine group of a lysine residue of the antibody.

[0023] In one embodiment, the antibody-drug conjugate is administered in an amount effective for treating or preventing a CLDN18.2-expressing cancer. In one embodiment, the antibody-drug conjugate is administered at a dose of between 3-30 mg / kg body weight, e.g., between 4-25, 5-20, 10-18, or 15-16 mg / kg body weight. In one embodiment, the antibody-drug conjugate is administered at a dose of 8-150, 9-100, or 9-90 mg / m of body surface of a human patient. 2 For example, between 12-75, 15-60, 30-54, or 45-48 mg / m of the body surface of a human patient. 2In one embodiment, a single dose of the antibody-drug conjugate is administered, or two or more doses of the antibody-drug conjugate are administered. In one embodiment, the antibody-drug conjugate is administered by intravenous injection.

[0024] In one embodiment, the method of the invention further comprises the step of administering surgery, chemotherapy, and / or radiation therapy.

[0025] In one embodiment, the expression of CLDN18.2 is on the cell surface of the cancer cells. In one embodiment, the cancer is an adenocarcinoma, particularly an advanced adenocarcinoma. In one embodiment, 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, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof, Krukenberg tumors, peritoneal metastases, and / or lymph node metastases. In one embodiment, the cancer is selected from the group consisting of gastric cancer, esophageal cancer, particularly cancer of the lower esophagus, cancer of the esophagogastric junction, and gastroesophageal cancer. In one embodiment, the patient is a HER2 / neu negative patient, or a patient with HER2 / neu positive status but ineligible for trastuzumab therapy.

[0026] In one embodiment, CLDN18.2 has an amino acid sequence according to SEQ ID NO:1.

[0027] In a further aspect, the invention provides an antibody-drug conjugate comprising an antibody capable of binding to CLDN18.2 covalently attached to at least one toxin drug moiety.

[0028] In one embodiment, the antibody-drug conjugate is internalized within the cell after binding to CLDN18.2 expressed by the cell.

[0029] In one embodiment, the antibody having the ability of binding to CLDN18.2 specifically binds to CLDN18.2. In one embodiment, the antibody-drug conjugate specifically binds to CLDN18.2.

[0030] In one embodiment, the antibody capable of binding to CLDN18.2 is a monoclonal, chimeric, or humanized antibody, or a fragment of an antibody. In one embodiment, the antibody capable of binding to CLDN18.2 is a monoclonal antibody.

[0031] In one embodiment, the antibody capable of binding to CLDN18.2 binds to the natural epitope of CLDN18.2 present on the surface of living cells.In one embodiment, the antibody capable of binding to CLDN18.2 binds to the extracellular domain of CLDN18.2.In one embodiment, the antibody capable of binding to CLDN18.2 binds to the first extracellular loop of CLDN18.2.

[0032] In one embodiment the antibody having the ability of binding to CLDN18.2 is an antibody selected from the group consisting of antibodies produced by and / or obtained from the clone deposited under accession numbers DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809 or DSM ACC2810, (ii) antibodies which are chimerized or humanized forms of antibodies belonging to (i), (iii) antibodies which have the specificity of antibodies belonging to (i), and (iv) antibodies which comprise an antigen-binding portion or antigen-binding site, in particular the variable region, of an antibody belonging to (i) and preferably having the specificity of an antibody belonging to (i). In one embodiment, the antibody capable of binding to CLDN18.2 comprises a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 32 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 39 or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the antibody capable of binding to CLDN18.2 comprises a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 17 or 51 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 24 or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the antibody capable of binding to CLDN18.2 comprises a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 30 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 35 or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the antibody capable of binding to CLDN18.2 comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 15 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof, or a variant of said amino acid sequence or fragment.In one embodiment, the antibody capable of binding to CLDN18.2 recognizes the same or essentially the same epitope as a CLDN18.2-binding antibody comprising a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 32 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 39 or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or competes with said CLDN18.2-binding antibody for binding to CLDN18.2. In one embodiment, the antibody capable of binding to CLDN18.2 recognizes the same or essentially the same epitope as a CLDN18.2-binding antibody comprising a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 17 or 51, or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 24, or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or competes with said CLDN18.2-binding antibody for binding to CLDN18.2. In one embodiment, the antibody capable of binding to CLDN18.2 recognizes the same or essentially the same epitope as a CLDN18.2 binding antibody comprising a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 30 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 35 or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or competes with said CLDN18.2 binding antibody for binding to CLDN18.2. In one embodiment, the antibody capable of binding to CLDN18.2 recognizes the same or essentially the same epitope as a CLDN18.2 binding antibody comprising a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 15 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or competes with said CLDN18.2 binding antibody for binding to CLDN18.2. An antibody that competes with a second antibody for binding to a target is preferably antagonistic to said second antibody.

[0033] In one embodiment, the toxin drug moiety is cell membrane permeable. In one embodiment, the toxin drug moiety is a cytotoxic or cytostatic agent. In one embodiment, the toxin drug moiety is a maytansinoid or an auristatin. In one embodiment, the maytansinoid is selected from the group consisting of DM1 and DM4. In one embodiment, the auristatin is selected from the group consisting of monomethylauristatin E (MMAE) and monomethylauristatin F (MMAF).

[0034] In one embodiment, the antibody capable of binding to CLDN18.2 is covalently attached to the toxin drug moiety by a linker. In one embodiment, the linker is a cleavable linker. In one embodiment, the linker is cleavable under intracellular conditions. In one embodiment, the linker is hydrolyzable at a pH of less than 5.5. In one embodiment, the linker is cleavable by intracellular proteases. In one embodiment, the linker is a cathepsin-cleavable linker. In one embodiment, the linker comprises a dipeptide. In one embodiment, the dipeptide is val-cit or phe-lys. In one embodiment, the antibody is attached to the linker via a cysteine ​​thiol of the antibody. In one embodiment, the antibody is attached to the linker via an amine group, in particular an amine group of a lysine residue of the antibody.

[0035] In one embodiment, CLDN18.2 has an amino acid sequence according to SEQ ID NO:1.

[0036] In a further aspect, the invention provides a pharmaceutical formulation comprising an antibody-drug conjugate of the invention and a pharma- ceutically acceptable diluent, carrier, or excipient.

[0037] In a further aspect, the present invention provides a pharmaceutical formulation comprising the antibody-drug conjugate of the present invention. In one embodiment, the pharmaceutical formulation is in the form of a kit comprising a container comprising the antibody-drug conjugate. In one embodiment, the pharmaceutical formulation further comprises printed instructions for use of the formulation in a method of treating or preventing cancer, in particular a CLDN18.2-expressing cancer.

[0038] In a further aspect, the present invention provides an antibody-drug conjugate of the invention, a pharmaceutical composition of the invention, or a pharmaceutical formulation of the invention for use in therapy, in particular for use in a method of treating or preventing cancer, in particular a CLDN18.2-expressing cancer. In one embodiment, the method of treating or preventing cancer is a method of treating or preventing a CLDN18.2-expressing cancer of the invention.

[0039] Other features and advantages of the invention will become apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 shows antibody drug conjugation. [Diagram 2] FIG. 1 is a graph showing reduction in viability of HEK293-CLDN18.2 cells after co-incubation with chimeric anti-CLDN18.2 mAb and Fab-ZAP (indirect assessment of internalization). HEK293-CLDN18.2 cells were incubated with anti-CLDN18.2 specific antibody and saporin-conjugated anti-human IgG Fab fragment (Fab-ZAP human) for 72 hours. Endocytosis of IMAB362, chim mAB294, chim mAB308, and chim mAB359 was determined indirectly by measuring cell viability. Data points (n=3 replicates) are presented as mean ± SD. [Diagram 3] Figure 1 shows the reduction in viability of HEK293-CLDN18.2 cells after co-incubation with mouse anti-CLDN18.2 antibodies and Fab-ZAP (indirect assessment of internalization). HEK293-CLDN18.2 cells were incubated with anti-CLDN18.2 reactive mouse antibodies and saporin-conjugated anti-mouse IgG Fab fragments (Fab-ZAP mouse) for 72 hours. Endocytosis of different anti-CLDN18.2 reactive mouse antibodies was indirectly determined by measuring cell viability. [Figure 4]1 is a graph showing the relative binding affinity of IMAB362-DM4 and IMAB362-vcMMAE to CLDN18.2 positive cells. The relative binding affinity of IMAB362-toxin conjugates compared to unconjugated IMAB362 was determined by flow cytometry at antibody concentrations up to 20 μg / ml for (A) NUGC-4 10cF7-5 sort3a, and (B) DAN-G 1C5F2 cells that endogenously express CLDN18.2, (C) NCI-N87-CLDN18.2, and (D) BxPC-3-CLDN18.2 cells that ectopically overexpress CLDN18.2. Data points (n=2 replicates) are presented as mean ± SD. [Diagram 5] 1 is a graph showing CLDN18.2-mediated binding of IMAB362-DM4 and IMAB362-vcMMAE. CLDN18.2-mediated binding of IMAB362-toxin conjugates was analyzed by flow cytometry at antibody concentrations up to 20 μg / ml in (A) NCI-N87-CLDN18.2 cells that ectopically overexpress CLDN18.2 and (B) the corresponding CLDN18.2-negative human tumor cell lines. Data points (n=2 replicates) are presented as mean ± SD. [Figure 6] Graph showing binding specificity of IMAB362-DM4 and IMAB362-vcMMAE. Binding specificity of IMAB362-toxin conjugates was determined on (A) HEK293-CLDN18.2, (B) HEK293-CLDN18.1, or (C) HEK293-mock cells as a negative control. Binding was analyzed by flow cytometry at antibody concentrations up to 20 μg / ml. Data points (n=2 replicates) are presented as mean ± SD. [Figure 7]Graph showing the effect of IMAB362-DM4 and IMAB362-vcMMAE on the viability of CLDN18.2 expressing human cancer cell lines. Dose response curves for IMAB362-DM4 and IMAB362-vcMMAE mediated reduction of (A) NUGC-4 10cF7-5 sort 3a, (B) NCI-N87-CLDN18.2, and (C) BxPC-3-CLDN18.2 cell viability. IMAB362 was used as a negative control (no effect in the viability assay under these conditions). Cells were incubated for 72 hours in the presence of antibodies at concentrations up to 16875 ng / ml. Reduction in cell viability was measured using an XTT-based viability assay. Data points (n=3 replicates) are presented as mean ± SD. [Figure 8] 1 is a graph showing the CLDN18.2-dependence of IMAB362-vcMMAE-mediated reduction of tumor cell viability. The target dependence of IMAB362-vcMMAE-mediated reduction of cell viability was determined for NCI-N87 cells (CLDN18.2 negative) and NCI-N87-CLDN18.2 cells ectopically expressing the target. Cells were incubated with IMAB362-vcMMAE or unconjugated IMAB362 at concentrations up to 16875 ng / ml for 72 hours. IMAB362 is known to have no activity under the experimental conditions used here. Reduction of cell viability was measured using an XTT-based viability assay. Data points (n=3 replicates) are presented as mean ± SD. [Figure 9] 1 is a graph showing the specificity of IMAB362-vcMMAE-mediated reduction of cell viability. Target specificity of IMAB362-vcMMAE-mediated reduction of cell viability was tested using stably transfected HEK293-CLDN18.2, HEK293-CLDN18.1, and HEK293-Mock cells. Cells were incubated for 72 hours in the presence of IMAB362-vcMMAE at concentrations up to 16875 ng / ml. Reduction of cell viability was measured using an XTT-based viability assay. Data points (n=3 replicates) are presented as mean ± SD. [Figure 10]1 is a graph showing the bystander activity of IMAB362-DM4 and IMAB362-vcMMAE. IMAB362-DM4- and IMAB362-vcMMAE-mediated induction of bystander effect was determined in co-culture experiments using PA-1(Luc) cells (CLDN18.2 negative / luciferase positive) and NUGC-4 10cE8 cells (CLDN18.2 positive / luciferase negative). As background control, PA-1(Luc) cells were incubated with IMAB362-DM4- or IMAB362-vcMMAE. For treatment, cells were cultured for 4 days in the presence of 200ng / ml IMAB362-DM4, 800ng / ml IMAB362-vcMMAE, or 800ng / ml IMAB362. Luciferase activity was measured. [Figure 11] FIG. 13 is a graph showing tumor growth inhibition of advanced BxPC-3 to CLDN18.2 xenograft tumors by IMAB362-DM4. CLDN18.2 positive BxPC-3 to CLDN18.2 cells were implanted subcutaneously into the flank of female athymic nude mice. On day 14, mice were organized into 4 groups and injected intravenously with a single dose of vehicle, 7.5 mg / kg, 15 mg / kg IMAB362-DM4, or a repeat dose of 15 mg / kg IMAB362-DM4 (days 14 and 21). Subcutaneous tumor size was measured twice weekly (mean+SEM). Group size n=5. SD: single dose, RD: repeat dose. [Figure 12] 1 is a graph showing the average body weight of mice treated with IMAB362-DM4. Body weights of BxPC-3 to CLDN18.2 tumor-bearing nude mice treated with a single dose of vehicle control, 7.5 mg / kg, or 15 mg / kg, or a repeated dose of 15 mg / kg, IMAB362-DM4, respectively, were monitored twice weekly. Body weights of the four groups are presented as the mean. Group size n=5. [Figure 13]Graph showing clinical chemistry parameters from single and repeated dose administration of IMAB362-DM4 in xenograft nude mice. Clinical chemistry of BxPC-3 to CLDN18.2 tumor-bearing female nude mice treated intravenously with a single dose of vehicle, 7.5 mg / kg, 15 mg / kg IMAB362-DM4, or a repeated dose of 15 mg / kg IMAB362-DM4 was analyzed 49 days after implantation. A) Alanine transaminase (GPT), B) Aspartate transaminase (GOT), C) Glutamate dehydrogenase, D) Alkaline phosphatase, E) α-amylase, F) Cholinesterase, G) Creatine kinase (CK), H) Lactate dehydrogenase (LDH), I) Lipase, J) Urea, K) Glucose, L) Total protein, and M) Albumin. [Figure 14] Photographs showing histological analysis of stomach sections from IMAB362-DM4 and vehicle treated mice. Mice bearing BxPC-3 to CLDN18.2 xenograft tumors were treated with IMAB362-DM4. On day 49 post-grafting, mice were sacrificed and selected organs were dissected and fixed in formalin. Sections of these FFPE tissues were stained with hematoxylin-eosin and examined microscopically for morphological changes. (A, C) Representative mouse stomach tissues from the treatment group with the highest IMAB362-DM4 exposure (15 mg / kg IMAB362-DM4 on days 14 and 21 post-grafting). (B, D) Gastric tissues from control mice treated with vehicle only. Magnification: see scale bar. [Figure 15] FIG. 1 is a graph showing tumor growth inhibition of advanced BxPC-3 to CLDN18.2 xenograft tumor IMAB362-vcMMAE. CLDN18.2 positive BxPC-3 to CLDN18.2 cells were implanted subcutaneously into the flank of female nude mice. On day 14, mice were organized into 4 groups and injected intravenously with a single dose of vehicle, 8 mg / kg, 16 mg / kg IMAB362-vcMMAE, or a repeat dose of 16 mg / kg IMAB362-vcMMAE (days 14 and 21). Subcutaneous tumor size was measured twice weekly (mean + SEM). Group size n=5. [Figure 16]Graph showing the average body weight of mice treated with IMAB362-vcMMAE. Body weights of tumor-bearing female nude mice treated with a single dose of vehicle control, 8 mg / kg, or 16 mg / kg, or a repeat dose of 16 mg / kg IMAB362-vcMMAE were monitored twice weekly. Body weights of the four groups are presented as the mean. Group size n=5. [Figure 17] Graphs showing clinical chemistry parameters from single and repeat dose administration of IMAB362-vcMMAE in xenograft nude mice. Clinical chemistry of BxPC-3 to CLDN18.2 tumor-bearing female nude mice treated intravenously with a single dose of vehicle, 8 mg / kg, 16 mg / kg IMAB362-vcMMAE, or a repeat dose of 16 mg / kg IMAB362-vcMMAE was analyzed 37 days after implantation. A) Alanine transaminase (GPT), B) Aspartate transaminase (GOT), C) Glutamate dehydrogenase, D) Alkaline phosphatase, E) α-amylase, F) Cholinesterase, G) Creatine kinase (CK), H) Lactate dehydrogenase (LDH), I) Lipase, J) Urea, K) Glucose, L) Total protein, and M) Albumin. [Figure 18]FIG. 1 shows the dose-dependent antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE in an advanced human NCI-N87-CLDN18.2 gastric xenograft tumor model. NCI-N87-CLDN18.2 cells ectopically expressing human CLDN18.2 were implanted subcutaneously into the flank of female nude mice. Ten days after implantation, mice were organized into groups and injected intravenously on day 13 with a single dose of vehicle, 3.8, 7.6, or 15.2 mg / kg IMAB362-DM4, or 4, 8, or 16 mg / kg IMAB362-vcMMAE. Another control group received repeated doses of approximately 8 mg / kg IMAB362 twice weekly by alternating IV and ip injections. Tumor volumes were measured twice weekly. Animals were sacrificed when tumor volumes exceeded 1400 mm3 or when tumors ulcerated. Statistical analysis of tumor growth was performed using Kruskal-Wallis and post-hoc Dunn tests. Survival was analyzed using the Mantel-Cox test comparing vehicle control groups with IMAB362-DM4 and IMAB362-vcMMAE, respectively. (A-H) Tumor growth curves, (I,K) mean tumor growth (±SEM), and (J,L) survival plots of mice treated with vehicle control, IMAB362, or IMAB362-DM4, or IMAB362-vcMMAE. Group size: n=11; *: p<0.05; ***p<0.001. Arrows indicate start of treatment. [Figure 19]1 is a graph showing the antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE in an early stage human NUGC-4 10cF7-5 sort3a gastric xenograft tumor model. NUGC-4 10cF7-5 sort3a cells endogenously expressing CLDN18.2 were implanted subcutaneously into the flank of female nude mice. On day 3, mice received vehicle, 15.2 mg / kg IMAB362-DM4, or 16 mg / kg IMAB362-vcMMAE by a single IV injection. Tumor volumes were measured twice weekly. Animals were sacrificed when tumor volume exceeded 1400 mm3, when tumors ulcerated, or after a predefined observation period of 120 days. Statistical analysis of tumor growth was performed using Kruskal-Wallis and post-hoc Dunn tests. Survival was analyzed using the Mantel-Cox test. (A-C) Tumor growth curves, (D) mean tumor growth (±SEM), and (E,F) survival plots of mice treated with vehicle control, IMAB362-DM4, or IMAB362-vcMMAE. Group size: n=10; ***: p<0.001; ****: p<0.0001. Arrows indicate time points of treatment. [Figure 20]FIG. 1 is a graph showing dose-dependent antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE in an advanced human BxPC-3-CLDN18.2 pancreatic xenograft tumor model. BxPC-3-CLDN18.2 cells ectopically expressing human CLDN18.2 were implanted subcutaneously into the flank of female nude mice. On day 13, mice were organized into groups and injected intravenously with a single dose of vehicle, 3.8, 7.6, or 15.2 mg / kg IMAB362-DM4, or 4, 8, or 16 mg / kg IMAB362-vcMMAE on day 14. Mice from the antibody control group received approximately 8 mg / kg unconjugated IMAB362 twice weekly by alternating IV and ip injections. Tumor size was measured twice weekly. Animals were sacrificed when tumor volume exceeded 1400 mm3 or tumors ulcerated. Statistical analysis of tumor growth was performed using Kruskal-Wallis and post-hoc Dunn tests. Survival was analyzed using the Mantel-Cox test comparing vehicle control groups with IMAB362-DM4 and IMAB362-vcMMAE, respectively. (A-H) Tumor growth curves, (I, K) mean tumor growth (± SEM), and (J, L) survival plots of mice treated with vehicle control, IMAB362, IMAB362-DM4, or IMAB362-vcMMAE. Group size: n=11; p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. Arrows indicate time points of treatment. [Figure 21]1 is a graph showing the antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE in an early stage human DAN-G 1C5F2 pancreatic xenograft tumor model. DAN-G 1C5F2 cells endogenously expressing CLDN18.2 were implanted subcutaneously into the flank of female nude mice. Three days after implantation, mice were treated with a single IV injection of vehicle control, 15.2 mg / kg IMAB362-DM4, or 16 mg / kg IMAB362-vcMMAE. Tumor volumes were measured twice weekly. Animals were sacrificed when mice lost more than 10% body weight due to cancer cachexia, when tumors ulcerated, or after a predefined observation period of 120 days. Statistical analysis of tumor growth was performed using Kruskal-Wallis and post-hoc Dunn tests. Survival time was analyzed using the Mantel-Cox test. (A-C) Tumor growth curves, (D) mean tumor growth (±SEM), and (E,F) survival plots of mice treated with vehicle control, IMAB362-DM4, or IMAB362-vcMMAE. Group size: n=10; **: p<0.01; ***: p<0.001. Arrows indicate time points of treatment. [Figure 22] Photographs showing histological analysis of stomach sections from IMAB362-vcMMAE and vehicle treated mice. Mice bearing BxPC-3 to CLDN18.2 xenograft tumors were treated with IMAB362-vcMMAE. On day 37 post-grafting, mice were sacrificed and selected organs were dissected and fixed in formalin. Sections of these FFPE tissues were stained with hematoxylin-eosin and examined microscopically for morphological changes. (A, C) Representative mouse stomach tissues from the treatment group with the highest IMAB362-vcMMAE exposure (16 mg / kg IMAB362-vcMMAE on days 14 and 21 post-grafting). (B, D) Gastric tissues from control mice treated with vehicle only. Magnification: see scale bar. [Figure 23]Graph showing induction of apoptosis by IMAB362-DM4 and IMAB362-vcMMAE. IMAB362-DM4 and IMAB362-vcMMAE mediated induction of apoptosis was determined by measuring caspase 3 / 7 activity using target positive NUGC-4 10cE8 cells and staining with Annexin V. A) Caspase 3 / 7 activity was analyzed after incubating cells in the presence of 2.5 μg / ml IMAB362 antibody for 3 days (n=3 replicates, mean±SD). B) Flow cytometry analysis of cells co-stained with Annexin V and propidium iodide (PI) was performed 4 days after treatment with 2.5 μg / ml IMAB362 antibody (n=3 replicates). Untreated cells served as control. [Figure 24] 1 is a graph showing the antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE in an advanced human NUGC-4 10cF7-5 sort3a gastric xenograft tumor model. NUGC-4 10cF7-5 sort3a cells, which endogenously express CLDN18.2, were implanted subcutaneously into the flank of female nude mice. On day 10, mice received vehicle, 15.2 mg / kg IMAB362-DM4, or 16 mg / kg IMAB362-vcMMAE by a single IV injection. Tumor volumes were measured twice weekly. Animals were sacrificed when tumor volume exceeded 1400 mm3, when tumors ulcerated, or after a predefined observation period of 120 days. Statistical analysis of tumor growth was performed using Kruskal-Wallis and post-hoc Dunn tests. Survival was analyzed using the Mantel-Cox test. (A-C) Tumor growth curves, (D) mean tumor growth (±SEM), and (E,F) survival plots of mice treated with vehicle control, IMAB362-DM4, or IMAB362-vcMMAE. Group size: n=10; *: p<0.05; ***: p<0.001. Arrows indicate time points of treatment. [Diagram 25]Graphs showing IMAB362-DM4 and IMAB362-vcMMAE mediated ADCC against CLDN18.2 expressing human cancer cells. A) Dose response curves for IMAB362-DM4 (solid black circles), IMAB362-vcMMAE (solid black triangles), and IMAB362 (open black squares) mediated ADCC against NUGC-4 10cF7_5 sort3a p3151#10 human gastric cancer cells that endogenously express CLDN18.2. Experiments were performed using an effector to target ratio of approximately 40:1. Data points (n=4 replicates) are presented as mean±SD. B) Flow cytometry analysis of CLDN18.2 expression on NUGC-4 10cF7_5 sort3a p3151#10 cells. Grey filled histograms: anti-CLDN18.2 (IMAB362, 50 μg / ml). Black dotted line: isotype control. [Figure 26] Graphs showing IMAB362-DM4- and IMAB362-vcMMAE-mediated CDC on CLDN18.2-expressing human cancer cells. A) Dose-response curves of IMAB362-DM4 (solid black circles), IMAB362-vcMMAE (solid black triangles), and IMAB362 (open black squares)-mediated CDC on KATO-III FGF BP#12 adM p3151#25 (left) and NUGC-4 10cF7_5 sort3a p3151#10 human gastric cancer cells (right), which endogenously express CLDN18.2. Luciferase-expressing target cells were incubated for 90 min with 20% human serum (pooled from healthy human donors) and the indicated concentrations of the respective antibodies. Data points (n=3 replicates) are presented as mean ± SD. B) Flow cytometry analysis of CLDN18.2 expression on KATO-III FGF BP#12 adM p3151#25 (left) and NUGC-4 10cF7_5 sort3a p3151#10 cells (right). Grey filled histograms: anti-CLDN18.2 (IMAB362, 50 μg / ml). Black dotted line: isotype control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described in the present invention, as they may vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to limit the scope of the present invention, which will be 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.

[0042] In the following, elements of the present invention are described. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed to limit the present invention to only the embodiments explicitly described. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all described elements in this application should be considered to be disclosed by the description of this application, unless otherwise indicated by the context.

[0043] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", edited by HGW Leuenberger, B. Nagel, and H. Kolbl, Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0044] 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 Edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0045] Throughout this specification and the claims that follow, unless otherwise required by context, the word "comprise", and variations such as "comprises" and "comprising" imply 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, although it is understood that 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 to include a stated member, integer, or step, or group of members, integers, or steps. The terms "a" and "an" and "the" and similar references as used in the context of describing the invention (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the invention and does not limit the scope of the invention as otherwise claimed. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0046] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0047] Claudins are a family of proteins that are the most important components of tight junctions, where they establish a paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. Claudins are transmembrane proteins that span the membrane four times, with both N- and C-termini located in the cytoplasm. The first extracellular loop or domain consists of an average of 53 amino acids, and the second extracellular loop or domain consists of about 24 amino acids. Cell surface proteins of the claudin family, such as CLDN18.2, are expressed in tumors of various origins and, due to their selective expression (without expression in toxicity-associated normal tissues) and localization to the plasma membrane, are particularly suitable as target structures in the context of antibody-mediated cancer immunotherapy.

[0048] The term "CLDN" as used herein means claudin, including CLDN18.2. Preferably, the claudin is a human claudin.

[0049] The term "CLDN18" refers to claudin 18 and includes any variants, including claudin 18 splice variant 1 (claudin 18.1 (CLDN18.1)) and claudin 18 splice variant 2 (claudin 18.2 (CLDN18.2)).

[0050] The term "CLDN18.2" preferably refers to a protein comprising, and preferably consisting of, human CLDN18.2, in particular the amino acid sequence according to SEQ ID NO: 1 of the Sequence Listing, or a variant of said amino acid sequence. The first extracellular loop or domain 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 or domain of CLDN18.2 preferably comprises amino acids 140 to 180 of the amino acid sequence shown in SEQ ID NO: 1. The first and second extracellular loops or domains preferably form the extracellular portion or domain of CLDN18.2.

[0051] CLDN18.2 is selectively expressed in normal tissues in differentiated epithelial cells of 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, and their metastases, especially gastric cancer metastases such as Krukenberg tumors, peritoneal metastases, and lymph node metastases.

[0052] The term "CLDN18.1" preferably refers to a protein comprising, preferably consisting of, human CLDN18.1, in particular the amino acid sequence according to SEQ ID NO: 2 of the Sequence Listing, or a variant of said amino acid sequence.

[0053] The term "variant" according to the present invention refers in particular to mutants, splice variants, conformations, isoforms, allelic variants, species variants, and species homologs, especially those that occur naturally. Allelic variants refer to changes in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies multiple allelic variants for a given gene. Species homologs are nucleic acid or amino acid sequences for a species of origin that differ from that of a given nucleic acid or amino acid sequence. The term "variant" is intended to encompass any post-translationally modified variants and conformational variants.

[0054] According to the present invention, the term "CLDN18.2-expressing cancer" or "CLDN18.2-positive cancer" refers to a cancer involving cancer cells which express CLDN18.2, preferably on the surface of said cancer cells.

[0055] "Cell surface" is used according to its ordinary meaning in the art, and thus includes the outside of a cell that is accessible to binding by proteins and other molecules.

[0056] CLDN18.2, when it is located at the surface of a cell, is expressed on the surface of said cell and is accessible for binding by a CLDN18.2-specific antibody that is added to the cell.

[0057] The term "extracellular portion" or "extracellular domain" in the context of the present invention refers to a part of a molecule, such as a protein, which 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, located on the outside of said cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0058] According to the present invention, CLDN18.2 is not substantially expressed in cells when the level of expression is lower compared to expression in gastric cells or gastric tissue. Preferably, the level of expression is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05%, or even lower, of expression in gastric cells or gastric tissue. Preferably, CLDN18.2 is not substantially expressed in cells when the level of expression exceeds the level of expression in non-cancerous tissue other than the stomach by no more than 2-fold, preferably no more than 1.5-fold, preferably not exceeding the level of expression in said non-cancerous tissue. Preferably, CLDN18.2 is not substantially expressed in cells when the level of expression is below detection limits and / or the level of expression is too low to allow binding by a CLDN18.2-specific antibody added to the cells.

[0059] According to the present invention, CLDN18.2 is expressed in cells when the level of expression exceeds the level of expression in non-cancerous tissues other than the stomach by more than 2-fold, preferably more than 10-fold, more than 100-fold, more than 1000-fold, or more than 10000-fold. Preferably, CLDN18.2 is expressed in cells when the level of expression exceeds the detection limit and / or when the level of expression is high enough to allow binding by a CLDN18.2-specific antibody added to the cells. Preferably, CLDN18.2 expressed in cells is expressed or exposed on the surface of the cells.

[0060] The term "disease" refers to an abnormal condition that affects an individual's body. A disease is often interpreted as a medical condition associated with specific symptoms and signs. A disease may be caused by factors that are originally from an external source, such as an infectious disease, or a disease may be caused by a malfunction within the body, such as an autoimmune disease. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems, or death to the individual who suffers from it, or similar problems to those who are in contact with the individual. In this broader sense, disease sometimes includes injuries, physical disabilities, disorders, syndromes, infections, isolated symptoms, deviant behavior, and atypical deformations of structure and function, while in other contexts and for other purposes, these may be considered as distinct categories. Diseases usually affect individuals not only physically but also emotionally, since suffering from and living with many diseases can change one's outlook on life and personality. According to the present invention, the term "disease" includes any pathological condition, including cancer, particularly the forms of cancer described herein. Any reference herein to cancer, or to a particular form of cancer, also includes cancer metastases thereof. In a preferred embodiment, the disease treated by the present application involves cells that express CLDN18.2.

[0061] "Disease involving cells expressing CLDN18.2" or "Disease associated with cells expressing CLDN18.2" or similar expressions means, according to the present invention, that CLDN18.2 is expressed in cells of diseased tissue or organ. In one embodiment, the expression of CLDN18.2 in cells of diseased tissue or diseased organ is increased compared to the situation in the corresponding healthy tissue or organ. Increase refers to an increase of at least 10%, in particular 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 the corresponding healthy tissue is suppressed. According to the present invention, diseases associated with cells expressing CLDN18.2 include cancer diseases. Furthermore, according to the present invention, cancer diseases are preferably those in which cancer cells express CLDN18.2.

[0062] The term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is typically characterized by unregulated cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specifically, examples of such cancer include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, genital and reproductive organ cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) neoplasm, neuroectodermal cancer, spinal axis tumor, glioma, meningioma, and pituitary adenoma. The term "cancer", according to the present invention, also includes cancer metastasis. Preferably, the "cancer disease" is characterized by cells expressing CLDN18.2, and the cancer cells express CLDN18.2. The cells expressing CLDN18.2 are preferably cancer cells, preferably cancer cells of a cancer described herein.

[0063] According to the present invention, the term "tumor" or "tumor disease" refers to the abnormal proliferation of cells (called neoplastic cells, tumorigenous cells, or tumor cells) that preferably form swellings or lesions. By "tumor cells" is meant abnormal cells that grow by rapid, uncontrolled cellular proliferation and continue to grow after the stimulus that initiated the new growth has ceased. Tumors exhibit a partial or complete lack of structural organization and functional coordination with normal tissues and usually form a separate mass of tissue that may be benign, premalignant, or malignant. According to the present invention, the "cancer disease" is preferably a "tumor disease". In general, however, the terms "cancer" and "tumor" are used interchangeably herein.

[0064] In one embodiment, the cancer according to the invention involves cancer cells expressing CLDN18.2. In one embodiment, the cancer is CLDN18.2 positive. In one embodiment, the expression of CLDN18.2 is on the surface of the cells. In one embodiment, at least 50%, preferably 60%, 70%, 80%, or 90% of the cancer cells are CLDN18.2 positive and / or at least 40%, preferably at least 50% of the cancer cells are positive for surface expression of CLDN18.2. In one embodiment, at least 95% or at least 98% of the cancer cells are CLDN18.2 positive. In one embodiment, at least 60%, at least 70%, at least 80%, or at least 90% of the cancer cells are positive for surface expression of CLDN18.2.

[0065] In one embodiment, the CLDN18.2-expressing cancer, cancer with cancer cells expressing CLDN18.2, or CLDN18.2-positive cancer is selected from the group consisting of 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, and metastases thereof, particularly gastric cancer metastases such as Krukenberg tumors, peritoneal metastases, and lymph node metastases. In one embodiment, the cancer is an adenocarcinoma, particularly an advanced adenocarcinoma. Particularly preferred cancer diseases are adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung, and ovary. In one embodiment, the cancer is selected from the group consisting of gastric cancer, esophagus, particularly cancer of the lower esophagus, cancer of the esophagogastric junction, and gastroesophageal cancer. In a particularly preferred embodiment, the cancer is gastroesophageal cancer, such as metastatic, refractory, or recurrent advanced gastroesophageal cancer.

[0066] 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.

[0067] "Adenocarcinoma" is a cancer that originates from glandular tissue. This tissue is also 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, a cell does not necessarily have to be part of a gland, as long as it has secretory properties. This form of cancer can occur in several higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while poorly differentiated adenocarcinomas may not. By staining the cells from the biopsy, the pathologist determines whether the tumor is an adenocarcinoma or some other type of cancer. Adenocarcinoma can occur in many tissues of the body due to the ubiquitous nature of glands in the body. Although each gland may not secrete the same substances, as long as there is an exocrine function to the cell, it is considered a gland, and therefore its malignant form is called adenocarcinoma. Malignant adenocarcinomas often metastasize if given enough time to invade and spread to other tissues. Ovarian adenocarcinoma is the most common type of ovarian cancer. It includes serous and mucinous adenocarcinoma, clear cell adenocarcinoma, and endometrioid adenocarcinoma.

[0068] "Metastasis" refers to the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process, depending on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter the body cavities and blood vessels, and then infiltration of the target organ after being transported by the blood. Finally, the growth of new tumors at the target site depends on angiogenesis. Tumor metastasis often occurs even after the primary tumor is removed, since tumor cells or tumor components may remain and 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 therapy of the present invention is metastasis originating from gastric cancer as the primary site. In a preferred embodiment, such gastric cancer metastasis is Krukenberg tumor, peritoneal metastasis, and / or lymph node metastasis.

[0069] Krukenberg tumor is a rare metastatic tumor of the ovary that accounts for 1%-2% of all ovarian tumors. The prognosis of Krukenberg tumor remains very poor and there is no established treatment for Krukenberg tumor. Krukenberg tumor is a metastatic signet ring cell adenocarcinoma of the ovary. The stomach is the primary site in most Krukenberg tumor cases (70%). Cancer of the colon, appendix, and breast (mainly invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg tumor originating from cancer of the gallbladder, biliary tract, pancreas, small intestine, ampulla of Vater, cervix, and bladder / urachus have been reported. The interval between diagnosis of the primary cancer and subsequent discovery of ovarian involvement is usually 6 months or less, although longer periods have been reported. In many cases, the primary tumor is very small and may escape detection. A history of prior cancer of the stomach or another organ can be obtained in only 20%-30% of cases.

[0070] Patients with Krukenberg tumors have a significantly higher overall mortality rate. Most patients die within 2 years (median survival time, 14 months). Several studies have shown that the prognosis is poorer when the primary tumor is identified after ovarian metastasis is discovered, and the prognosis is worse if the primary tumor remains occult.

[0071] The optimal treatment strategy for Krukenberg tumors has not been clearly established in the literature. Whether surgical resection should be performed has not been fully addressed. Chemotherapy or radiation therapy has no significant effect on the prognosis of patients with Krukenberg tumors.

[0072] In particular, the term "(therapeutic) treatment" in relation to cancer treatment, as used herein, refers to any treatment aimed at improving the health and / or extending (increasing) the life span of a patient. The treatment may eliminate cancer, reduce the size or number of tumors in a patient, stop or slow the development of cancer in a patient, inhibit or slow the development of new cancers in a patient, reduce the frequency or severity of symptoms in a patient, and / or reduce recurrence in a patient who currently has cancer or who has previously had cancer. The (therapeutic) treatment for cancer may be selected from the group consisting of surgery, chemotherapy, radiation therapy, and targeted therapy.

[0073] The term "surgery" as used herein includes the removal of a tumor in an operation, which is a common treatment for cancer. A surgeon may remove a tumor using local excision.

[0074] The term "chemotherapy" as used herein preferably refers to the use of a chemotherapy agent or combination of chemotherapy agents to stop the growth of cancer cells by killing the cells or stopping them from dividing. When chemotherapy is taken by mouth or injected into a vein or muscle, the drug can enter the bloodstream and reach cancer cells throughout the body (systemic chemotherapy). When chemotherapy is placed directly into the cerebrospinal fluid, organs, or body cavities such as the abdomen, the drug mainly affects cancer cells in these areas (local chemotherapy).

[0075] Chemotherapeutic agents according to the present invention include cytostatic and cytotoxic compounds. Conventional chemotherapeutic agents act by killing rapidly dividing cells, which is one of the main properties of most cancer cells. This means that chemotherapy also harms cells that divide rapidly under normal circumstances, such as cells in the bone marrow, digestive tract, and hair follicles. This results in the most common side effects of chemotherapy. According to the present invention, the term "chemotherapy" does not include antibodies that act by targeting abnormally expressed proteins (tumor antigens such as CLDN18.2) in cancer cells and recruiting the patient's immune system to destroy tumor cells. However, antibodies that target abnormally expressed proteins (tumor antigens such as CLDN18.2) in cancer cells and act by a therapeutic moiety or agent conjugated to the antibody can be considered as a form of chemotherapy. However, in the strict sense, the term "chemotherapy" does not include targeted therapy according to the present invention.

[0076] According to the present invention, the term "targeted therapy" refers to any therapy that can be used to preferentially target diseased cells, such as cancer cells, while non-diseased cells are not targeted or are targeted to a lesser extent. Targeting of diseased cells preferably results in killing of diseased cells and / or impairing their proliferation or viability. Such therapies include i) antibodies, antibody fragments, and proteins (e.g., antibodies or antibody conjugates against CLDN18.2 described herein), either naked or conjugated to a therapeutic moiety that targets a certain cell surface target on diseased cells, such as a tumor antigen, e.g., CLDN18.2, or ii) small molecules that impair the proliferation or viability of diseased cells. In a specific embodiment, the agent binds to an antigen that is expressed at a greater level on diseased cells than on normal stem cells. In a specific embodiment, the agent specifically binds to a tumor antigen. Conventional chemotherapy or radiation therapy is not considered a "targeted therapy" even though it is often directed to tumors. Furthermore, the term "antibody therapy", according to the present invention, does not include therapy with antibodies, fragments or derivatives thereof that are conjugated to a therapeutic moiety, but simply relates to therapy with antibodies, fragments or derivatives thereof that act by recruiting the patient's immune system to destroy tumor cells.

[0077] In the context of the present invention, terms such as "protect", "prevent" or "prophylactic" refer to the prevention of the appearance and / or spread of disease in a subject, in particular minimizing the likelihood that the subject will develop a disease or delaying the onset of the disease. For example, subjects at risk for cancer should be candidates for a therapy that prevents the cancer.

[0078] "At risk" refers to a subject who is identified as having a higher than normal probability of developing a disease, particularly cancer, compared to the general population.Furthermore, a subject who has had or currently has a disease, particularly cancer, is a subject who is at increased risk of developing the disease, because such a subject may continue to develop the disease.A subject who currently has or has had cancer is also at increased risk of cancer metastasis.

[0079] The terms "individual" and "subject" are used interchangeably herein. They refer to a human, non-human primate, or other mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may suffer from or be susceptible to a disease or disorder (e.g., cancer), but may or may not have a disease or disorder. In many embodiments, the individual is a human. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus include adults, elderly people, children, and newborns. In a preferred embodiment of the present invention, the "individual" or "subject" is a "patient". The term "patient" refers to the subject of treatment, particularly a diseased subject, according to the present invention.

[0080] The term "antigen" relates to an agent such as a protein or peptide that contains an epitope against which and / or an immune response is directed. In a preferred embodiment, the antigen is a tumor-associated antigen such as CLDN18.2, i.e. a component of a cancer cell that may originate from the cytoplasm, the cell surface and the cell nucleus, in particular an antigen that is preferably produced in large amounts intracellularly or as a surface antigen of the cancer cell.

[0081] In the context of the present invention, the term "tumor-associated antigen" or "tumor antigen" preferably relates to a protein that is specifically expressed in a limited number of tissues and / or organs or at a particular developmental stage under normal conditions and 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.

[0082] The term "epitope" refers to an antigenic determinant within a molecule, i.e., a part of a molecule that is recognized by the immune system, e.g., by an antibody. For example, an epitope is a distinct three-dimensional site on an antigen that is recognized by the immune system. Epitopes usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural features and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope of a protein such as CLDN18.2 preferably comprises a continuous or discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, and most preferably between 10 and 25 amino acids in length, for example, an epitope may preferably be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0083] The term "antibody" includes glycoproteins comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as any molecule comprising an antigen-binding portion of such a glycoprotein. The term "antibody" includes molecules including, without limitation, monoclonal antibodies, including single-chain antibodies, e.g., scFv, and antigen-binding antibody fragments, e.g., Fab and Fab' fragments, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, binding fragments or derivatives of antibodies, and also includes all recombinant forms of antibodies, e.g., antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives described herein. 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 VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0084] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody exhibits 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.

[0085] The term "recombinant antibody," as used herein, includes all antibodies prepared, expressed, created, or isolated by recombinant means, such as, for example, (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for autologous prepared immunoglobulin genes or hybridomas, (b) antibodies isolated from host cells that have been transformed to express the antibody, e.g., from transfectomas, (c) antibodies isolated from recombinant combinatorial antibody libraries, and (d) antibodies prepared, expressed, created, or isolated by any other means of splicing immunoglobulin gene sequences to other DNA sequences.

[0086] 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 include 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).

[0087] 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 being based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise a complete variable domain fused onto a constant domain, or may comprise only the complementarity determining regions (CDRs) grafted onto appropriate framework regions in the variable domain. The antigen-binding site may be wild-type or 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 mouse antibodies). Other forms have one or more CDRs altered relative to the original antibody.

[0088] The term "chimeric antibody" refers to an antibody in which one portion of each of the amino acid sequences of the heavy and light chains is homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular class, while the remaining segments of the chains are homologous to the corresponding sequence in another. In general, the variable regions of both the light and heavy chains mimic the variable regions of an antibody derived from one species of mammal, while the constant portions are homologous to the sequences of an antibody derived from another species. One obvious advantage of such chimeric forms is that the variable regions can be derived from currently known sources, for example, using readily available B cells or hybridomas from non-human host organisms, conveniently in combination with constant regions derived from human cell samples. The variable regions have the advantage of ease of preparation and the specificity is not affected by the source, but the constant regions that are human are less likely to elicit an immune response from a human subject when the antibody is injected than do constant regions from non-human sources. However, the definition is not limited to this particular example.

[0089] The term "antigen-binding portion" (or simply "binding portion") of an antibody, or "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 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) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH domains; (iv) an 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 joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined using recombinant methods by a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as 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 within the term "antigen-binding fragment" of an antibody. Further examples are binding domain immunoglobulin fusion proteins 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 of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0090] Naturally occurring antibodies are generally monospecific, i.e., they bind to a single antigen. The present invention includes antibodies that bind to a target cell (by engaging a tumor antigen) and to a second entity, such as a cytotoxic cell (e.g., by engaging a CD3 receptor). The antibodies of the present invention may be bispecific or multispecific, e.g., trispecific, tetraspecific, etc.

[0091] The term "bispecific molecule" is intended to include an agent having two different binding specificities. For example, the molecule can bind to or interact with (a) a cell surface antigen, such as CLDN18.2, and (b) a receptor, such as an Fc receptor, on the surface of an effector cell. The term "multispecific molecule" is intended to include an agent having more than two different binding specificities. For example, the molecule can bind to or interact with (a) a cell surface antigen, such as CLDN18.2, (b) a receptor, such as an Fc receptor, on the surface of an effector cell, and (c) at least one other component. Thus, the term "antibody" includes bispecific, trispecific, tetraspecific, and other multispecific molecules directed against other targets, such as, but not limited to, tumor antigens and Fc receptors on effector cells. The term "bispecific antibody" also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are on a single polypeptide chain, but are expressed using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (see, e.g., Holliger, P. et al., (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-6448; Poljak, RJ et al., (1994) Structure, 2: 1121-1123).

[0092] Antibodies may be derived from various species, including, but not limited to, mouse, rat, rabbit, guinea pig, and human.

[0093] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0094] 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.

[0095] 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 specifically 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).

[0096] As used herein, a "heterologous antibody" is defined with respect to the transgenic organism producing such an antibody. The term refers to an antibody that has an amino acid sequence or a coding nucleic acid sequence that corresponds to one found in an organism other than the transgenic organism, and generally originates from a species other than the transgenic organism.

[0097] 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.

[0098] The antibodies described herein are preferably isolated. An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to a tumor antigen is substantially free of antibodies that specifically bind to antigens other than the tumor antigen). However, an isolated antibody that specifically binds to an epitope, isoform, or variant of a human tumor antigen may have cross-reactivity with other related antigens, for example, from other species (e.g., tumor antigen species homologs). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. In one embodiment of the present invention, an "isolated" monoclonal antibody combination relates to antibodies with different specificities and combined in a particular composition or mixture.

[0099] In the context of the present invention, if an antibody elicits immune effector functions as described herein, particularly when bound to its target, such as a tumor antigen on diseased cells, the antibody can act by recruiting the patient's immune system to destroy tumor cells. Preferably, said immune effector functions are directed against cells, such as cancer cells, carrying a tumor antigen, such as CLDN18.2, on their surface.

[0100] The term "immune effector function" in the context of the present invention includes any function mediated by components of the immune system that results in the inhibition of tumor growth and / or the inhibition of tumor development, including, for example, the inhibition of tumor spread and metastasis. Preferably, the immune effector function results in the killing of cancer cells. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis in cells bearing tumor antigens, cytolysis of cells bearing tumor antigens, and / or inhibition of proliferation of cells bearing tumor antigens.

[0101] Antibody-dependent cell-mediated cytotoxicity ADCC describes the cell-killing capacity of effector cells, particularly lymphocytes, which preferably involve target cells that have been marked by an antibody.

[0102] ADCC preferably occurs when an antibody binds to an antigen on a tumor cell and the antibody Fc domain engages with an Fc receptor (FcR) on the surface of an immune effector cell. Several families of Fc receptors have been identified, and certain cell populations characteristically express defined Fc receptors. ADCC can be viewed as a mechanism that directly induces immediate tumor destruction to various degrees through antigen presentation and induction of tumor-directed T-cell responses. Preferably, induction of ADCC in vivo leads to tumor-directed T-cell responses and host-derived antibody responses.

[0103] Complement-dependent cytotoxicity CDC is another cell killing method that can be directed by antibodies. IgM is the most effective isotype for complement activation. Both IgG1 and IgG3 are also very effective in directing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex leads to the CDC of participating antibody molecules, such as IgG molecules. H This results in the uncloaking of multiple Clq binding sites that are in close proximity on the 2 domain (C1q is one of three subcomponents of complement C1). Preferably, these uncloaked 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, resulting in the proteolytic release of the effector chemoattractants / activators C3a and C5a. Preferably, the complement cascade culminates in the formation of a cell membrane attack complex, which creates pores in the cell membrane that facilitate the free passage of water and solutes in and out of the cell.

[0104] It has been surprisingly found that the antibody-drug conjugates described herein can mediate the killing of cells, particularly cells expressing CLDN18.2, such as cancer cells, by inducing complement-dependent cytotoxicity (CDC)- and / or antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis. Thus, in one embodiment, the antibody-drug conjugates of the invention mediate the killing of cells by inducing complement-dependent cytotoxicity (CDC)- and / or antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, preferably by inducing CDC- and ADCC-mediated lysis.

[0105] As used herein, an antibody is "derived from" a particular germline sequence if it is obtained from one line by immunizing an animal or by screening an immunoglobulin gene library, and the selected antibody is at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Generally, an antibody derived from a particular germline sequence will display no more than 10 amino acid differences, more preferably no more than 5, or even more preferably no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0106] As used herein, the term "heteroantibody" refers to two or more antibodies, derivatives thereof, or antigen-binding regions linked together, at least two of which have different specificities, including a binding specificity for an Fc receptor on an effector cell and a binding specificity for an antigen or epitope on a target cell, e.g., a tumor cell.

[0107] The term "transfectoma" as used herein includes recombinant eukaryotic host cells expressing an antibody, such as CHO cells, NS / 0 cells, HEK293 cells, HEK293T cells, plant cells, or fungal cells, including yeast cells.

[0108] The present invention includes all antibodies and antibody derivatives described herein that are encompassed by the term "antibody" for purposes of the present invention. The term "antibody derivative" refers to any modification of an antibody, for example, a conjugate of an antibody with another agent or antibody, or an antibody fragment.

[0109] The term "antibody against a tumor antigen" or similar terms refers to an antibody that is directed against or capable of binding to a tumor antigen. The term "binding" according to the present invention preferably relates to specific binding.

[0110] According to the present invention, an antibody or antibody-drug conjugate is capable of binding to a predetermined target if it has significant affinity for said target and binds to said target in a standard assay. "Affinity" or "binding affinity" is defined as the equilibrium dissociation constant (K D ) Preferably, the term "significant affinity" refers to a ratio of 10 -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 specific target.

[0111] An antibody or antibody-drug conjugate is (substantially) unable to bind to a target if it has no significant affinity for the target and does not bind significantly, in particular does not bind detectably, to said target in a standard assay. Preferably, the antibody or antibody-drug conjugate does not detectably bind to said target when present at a concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 μg / ml or more. Preferably, the antibody or antibody-drug conjugate has a K for binding to a given target to which the antibody or antibody-drug conjugate can bind. 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, when an antibody or antibody-drug conjugate binds to a target to which the antibody or antibody-drug conjugate can bind, the K D 10 -7 M, the K for binding to a target for which the antibody or antibody-drug conjugate has no significant affinity D is at least 10-6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 It's M.

[0112] An antibody or antibody-drug conjugate is specific for a given target if it can bind to the given target while it cannot bind to other targets, i.e. it has no significant affinity for other targets and does not bind significantly to other targets in standard assays. According to the present invention, an antibody or antibody-drug conjugate is specific for a tumor antigen if it can bind to the tumor antigen but cannot (substantially) bind to other targets. Preferably, an antibody or antibody-drug conjugate is specific for a tumor antigen if its affinity and binding to such other targets does not significantly exceed its affinity or binding to tumor antigen-unrelated proteins, such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or non-tumor antigenic transmembrane proteins such as MHC molecules or transferrin receptors, or any other designated polypeptides ... K for binding to a non-specific target is less than its K for binding to a non-specific target. D At least 1 / 10, 1 / 100, 1 / 10 3 1 / 10 4 1 / 10 5 1 / 10 6 K lower by a factor of 1 D For example, an antibody or antibody-drug conjugate is specific for a given target if it binds to said given target at D 10 -7 M, this is the K for binding to a non-specific target D is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 It should be M.

[0113] The binding of an antibody to a target can be experimentally determined using any suitable method, see, for example, Berzofsky et al., "Antibody-Antigen Interactions," 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 routine techniques, such as 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 of skill in the art. Affinity data can be analyzed, for example, by the method of 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, e.g., salt concentration, pH. Thus, affinity as well as other antigen binding parameters, e.g., K D ,I C 50 The measurements are preferably carried out using standardized solutions of antibody and antigen, and standardized buffers.

[0114] The term "naturally occurring" as used herein, when applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by man in a laboratory is naturally occurring.

[0115] The term "rearranged," as used herein, 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 essentially encodes a complete VH or VL domain, respectively. Rearranged immunoglobulin (antibody) loci can be identified by comparison to germline DNA, and rearranged loci will have at least one recombined heptamer / nonamer homology element.

[0116] The term "unrearranged" or "germline configuration," as used herein with reference to a V segment, refers to a configuration in which the V segment has not been recombined so that it is immediately adjacent to a D segment or a J segment.

[0117] According to the present invention, an antibody capable of binding to CLDN18.2 is an antibody capable of binding to an epitope present in CLDN18.2, preferably an epitope located in the extracellular domain of CLDN18.2, particularly the first extracellular domain, preferably within amino acids 29 to 78 of CLDN18.2. In certain embodiments, an antibody capable of binding to CLDN18.2 is an antibody capable of binding to (i) an epitope on CLDN18.2 that is not present on CLDN18.1, preferably SEQ ID NO: 3, 4, and 5; (ii) an epitope located on CLDN18.2-loop1, preferably SEQ ID NO: 8; (iii) an epitope located on CLDN18.2-loop2, preferably SEQ ID NO: 10; (iv) an epitope located on CLDN18.2-loop D3, preferably SEQ ID NO: 11; (v) an epitope encompassing CLDN18.2-loop1 and CLDN18.2-loop D3; or (vi) a non-glycosylated epitope located on CLDN18.2-loop D3, preferably SEQ ID NO: 9.

[0118] According to the present invention, an antibody capable of binding to CLDN18.2 is preferably an antibody capable of binding to CLDN18.2 but not to CLDN18.1. Preferably, an antibody capable of binding to CLDN18.2 is specific to CLDN18.2. Preferably, an antibody capable of binding to CLDN18.2 is an antibody capable of binding to CLDN18.2, preferably expressed on the cell surface. In a particular preferred embodiment, an antibody capable of binding to CLDN18.2 binds to a natural epitope of CLDN18.2 present on the surface of a living cell. Preferably, an antibody capable of binding to CLDN18.2 binds to one or more peptides selected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46, and 48-50. Preferably, an antibody capable of binding to CLDN18.2 is specific to the above-mentioned proteins, peptides, or immunogenic fragments or derivatives thereof. An antibody capable of binding to CLDN18.2 can be obtained by a method comprising the step of immunizing an animal with a protein or peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3 to 11, 44, 46, and 48 to 50, or a nucleic acid or host cell expressing said protein or peptide. Preferably, the antibody binds to cancer cells, in particular cells of the above-mentioned cancer types, and preferably does not bind to non-cancerous cells.

[0119] In a particularly preferred embodiment, the antibody having the ability of binding to CLDN18.2 is produced by the hybridoma deposited at the DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany; New Address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) and having the following name and accession number: a.182-D1106-055, accession number DSM ACC2737, deposited on October 19, 2005 b.182-D1106-056, accession number DSM ACC2738, deposited on October 19, 2005 c.182-D1106-057, accession number DSM ACC2739, deposited on October 19, 2005 d.182-D1106-058, accession number DSM ACC2740, deposited on October 19, 2005 e.182-D1106-059, accession number DSM ACC2741, deposited on October 19, 2005 f.182-D1106-062, accession number DSM ACC2742, deposited on October 19, 2005; g.182-D1106-067, accession number DSM ACC2743, deposited on October 19, 2005 h.182-D758-035, accession number DSM ACC2745, deposited on November 17, 2005 i.182-D758-036, accession number DSM ACC2746, deposited on November 17, 2005 j.182-D758-040, accession number DSM ACC2747, deposited on November 17, 2005 k.182-D1106-061, accession number DSM ACC2748, deposited on November 17, 2005 l.182-D1106-279, accession number DSM ACC2808, deposited on October 26, 2006 m.182-D1106-294, accession number DSM ACC2809, deposited on October 26, 2006; n.182-D1106-362, accession number DSM ACC2810, deposited on October 26, 2006.

[0120] Preferred antibodies according to the invention are those derived from the above mentioned hybridomas, namely 37G11 for 182-D1106-055, 37H8 for 182-D1106-056, 38G5 for 182-D1106-057, 38H3 for 182-D1106-058, 39F11 for 182-D1106-059, 43A11 for 182-D1106-062, 61C2 for 182-D1106-067, 182-D758-035, 182-D758-036, 182-D758-037, 182-D758-038, 182-D758-039, 182-D758-040, 182-D758-041, 182-D758-042, 182-D758-043, 182-D758-044, 182-D758-045, 182-D758-046, 182-D758-047, 182-D758-048, 182-D758-049, 182-D758-050, 182-D758-061, 182-D758-062, 182-D758-049, 182-D758-051, 182-D758-063, 182-D758-064, 182-D758-065, 182-D758-070, 182-D in the case of 182-D1106-362; 26B5 in the case of 182-D758-036; 26D12 in the case of 182-D758-040; 42E12 in the case of 182-D1106-061; 125E1 in the case of 182-D1106-279; 163E12 in the case of 182-D1106-294; and 175D10 in the case of 182-D1106-362; and chimeric and humanized forms thereof.

[0121] In one embodiment the antibody having the ability of binding to CLDN18.2 is an antibody selected from the group consisting of antibodies produced by and / or obtained from the clone deposited under accession numbers DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809 or DSM ACC2810, (ii) antibodies which are chimerized or humanized forms of antibodies belonging to (i), (iii) antibodies which have the specificity of antibodies belonging to (i), and (iv) antibodies which comprise an antigen-binding portion or antigen-binding site, in particular the variable region, of an antibody belonging to (i) and preferably having the specificity of an antibody belonging to (i).

[0122] Suitable antibodies, particularly chimeric antibodies, and their sequences are shown in the table below.

[0123] [Table 1]

[0124] In a preferred embodiment, the antibody, in particular a chimerized form of the antibody according to the invention, comprises an antibody comprising a heavy chain constant region (CH) comprising an amino acid sequence derived from a human heavy chain constant region, such as the amino acid sequence represented by SEQ ID NO: 13 or a fragment thereof. In a further preferred embodiment, the antibody, in particular a chimerized form of the antibody according to the invention, comprises an antibody comprising a light chain constant region (CL) comprising an amino acid sequence derived from a human light chain constant region, such as the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof. In a particularly preferred embodiment, the antibody, in particular a chimerized form of the antibody according to the invention, comprises an antibody comprising a CH comprising an amino acid sequence derived from a human CH, such as the amino acid sequence represented by SEQ ID NO: 13 or a fragment thereof, and comprising a CL comprising an amino acid sequence derived from a human CL, such as the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof.

[0125] In one embodiment, the antibody capable of binding to CLDN18.2 is a chimeric mouse / human IgG1 monoclonal antibody comprising a kappa mouse variable light chain, a human kappa light chain constant region allotype Km(3), a mouse heavy chain variable region, and a human IgG1 constant region allotype G1m(3).

[0126] In certain preferred embodiments, chimeric forms of antibodies include antibodies comprising a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 51, and fragments thereof, and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, and fragments thereof.

[0127] In certain preferred embodiments, chimerized forms of antibodies include antibodies that comprise a heavy and light chain combination selected from the following possibilities (i)-(ix): (i) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 14 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 21 or a fragment thereof; (ii) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 15 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof; (iii) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 16 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 22 or a fragment thereof; (iv) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 18 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 25 or a fragment thereof; (v) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 17 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 24 or a fragment thereof; (vi) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 23 or a fragment thereof; (vii) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 26 or a fragment thereof; (viii) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 27 or a fragment thereof; (ix) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 28 or a fragment thereof; and (x) a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 51 or a fragment thereof, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 24 or a fragment thereof.

[0128] The antibodies according to (ii), (v) or (x) are preferred embodiments of the “antibody having the ability to bind to CLDN18.2”. The antibodies according to (v) or (x) are particularly preferred.

[0129] "Fragment" or "fragment of an amino acid sequence" as used above refers to a part of an antibody sequence, i.e. an antibody sequence truncated at the N-terminus and / or C-terminus, which, when it replaces said antibody sequence in an antibody, relates to the binding of said antibody to CLDN18.2. Preferably, a fragment of an amino acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98% or 99% of the amino acid residues derived from said amino acid sequence. A fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and 51 relates to said sequences, preferably with 17, 18, 19, 20, 21, 22 or 23 amino acids at the N-terminus removed.

[0130] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, and fragments thereof.

[0131] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, and fragments thereof.

[0132] In certain preferred embodiments, an antibody capable of binding to CLDN18.2 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following possibilities (i) to (ix): (i) a VH comprising an amino acid sequence represented by SEQ ID NO: 29 or a fragment thereof, and a VL comprising an amino acid sequence represented by SEQ ID NO: 36 or a fragment thereof; (ii) a VH comprising an amino acid sequence represented by SEQ ID NO: 30 or a fragment thereof, and a VL comprising an amino acid sequence represented by SEQ ID NO: 35 or a fragment thereof; (iii) a VH comprising an amino acid sequence represented by SEQ ID NO: 31 or a fragment thereof, and a VL comprising an amino acid sequence represented by SEQ ID NO: 37 or a fragment thereof; (iv) a VH comprising an amino acid sequence represented by SEQ ID NO: 33 or a fragment thereof, and a VL comprising an amino acid sequence represented by SEQ ID NO: 40 or a fragment thereof; (v) a VH comprising an amino acid sequence represented by SEQ ID NO: 32 or a fragment thereof, and a VL comprising an amino acid sequence represented by SEQ ID NO: 39 or a fragment thereof; (vi) a VH comprising an amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and a VL comprising an amino acid sequence represented by SEQ ID NO: 38 or a fragment thereof; (vii) a VH comprising an amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and a VL comprising an amino acid sequence represented by SEQ ID NO: 41 or a fragment thereof; (viii) a VH comprising an amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and a VL comprising an amino acid sequence represented by SEQ ID NO: 42 or a fragment thereof; (ix) a VH comprising an amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and a VL comprising an amino acid sequence represented by SEQ ID NO: 43 or a fragment thereof.

[0133] The antibodies according to (ii) or (v) are preferred embodiments of the “antibody having the ability to bind to CLDN18.2”. The antibodies according to (v) are particularly preferred.

[0134] According to the invention, the term "fragment" refers in particular to one or more of the complementarity determining regions (CDRs) of the heavy chain variable region (VH) and / or the light chain variable region (VL), preferably at least the CDR3 variable region. In one embodiment, said one or more of the 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 the light chain variable region (VL).

[0135] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a VH comprising a set of complementarity determining regions CDR1, CDR2, and CDR3 selected from the following embodiments (i) to (vi): (i) CDR1: positions 45 to 52 of SEQ ID NO: 14, CDR2: positions 70 to 77 of SEQ ID NO: 14, CDR3: positions 116 to 125 of SEQ ID NO: 14, (ii) CDR1: positions 45 to 52 of SEQ ID NO: 15, CDR2: positions 70 to 77 of SEQ ID NO: 15, CDR3: positions 116 to 126 of SEQ ID NO: 15, (iii) CDR1: positions 45 to 52 of SEQ ID NO: 16, CDR2: positions 70 to 77 of SEQ ID NO: 16, CDR3: positions 116 to 124 of SEQ ID NO: 16, (iv) CDR1: positions 45 to 52 of SEQ ID NO: 17, CDR2: positions 70 to 77 of SEQ ID NO: 17, CDR3: positions 116 to 126 of SEQ ID NO: 17, (v) CDR1: positions 44 to 51 of SEQ ID NO: 18, CDR2: positions 69 to 76 of SEQ ID NO: 18, CDR3: positions 115 to 125 of SEQ ID NO: 18, and (vi) CDR1: positions 45 to 53 of SEQ ID NO:19, CDR2: positions 71 to 78 of SEQ ID NO:19, CDR3: positions 117 to 128 of SEQ ID NO:19.

[0136] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a VL comprising a set of complementarity determining regions CDR1, CDR2, and CDR3 selected from the following embodiments (i) to (ix): (i) CDR1: positions 47 to 58 of SEQ ID NO: 20, CDR2: positions 76 to 78 of SEQ ID NO: 20, CDR3: positions 115 to 123 of SEQ ID NO: 20, (ii) CDR1: positions 49 to 53 of SEQ ID NO: 21, CDR2: positions 71 to 73 of SEQ ID NO: 21, CDR3: positions 110 to 118 of SEQ ID NO: 21, (iii) CDR1: positions 47 to 52 of SEQ ID NO: 22, CDR2: positions 70 to 72 of SEQ ID NO: 22, CDR3: positions 109 to 117 of SEQ ID NO: 22, (iv) CDR1: positions 47 to 58 of SEQ ID NO: 23, CDR2: positions 76 to 78 of SEQ ID NO: 23, CDR3: positions 115 to 123 of SEQ ID NO: 23, (v) CDR1: positions 47 to 58 of SEQ ID NO: 24, CDR2: positions 76 to 78 of SEQ ID NO: 24, CDR3: positions 115 to 123 of SEQ ID NO: 24, (vi) CDR1: positions 47 to 58 of SEQ ID NO: 25, CDR2: positions 76 to 78 of SEQ ID NO: 25, CDR3: positions 115 to 122 of SEQ ID NO: 25, (vii) CDR1: positions 47 to 58 of SEQ ID NO: 26, CDR2: positions 76 to 78 of SEQ ID NO: 26, CDR3: positions 115 to 123 of SEQ ID NO: 26, (viii) CDR1: positions 47 to 58 of SEQ ID NO: 27, CDR2: positions 76 to 78 of SEQ ID NO: 27, CDR3: positions 115 to 123 of SEQ ID NO: 27, and (ix) CDR1: positions 47 to 52 of SEQ ID NO:28, CDR2: positions 70 to 72 of SEQ ID NO:28, CDR3: positions 109 to 117 of SEQ ID NO:28.

[0137] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a VH and VL combination, each of which comprises a set of complementarity determining regions CDR1, CDR2, and CDR3 selected from the following embodiments (i) to (ix): (i) VH: CDR1: positions 45 to 52 of SEQ ID NO: 14, CDR2: positions 70 to 77 of SEQ ID NO: 14, CDR3: positions 116 to 125 of SEQ ID NO: 14, VL: CDR1: positions 49 to 53 of SEQ ID NO: 21, CDR2: positions 71 to 73 of SEQ ID NO: 21, CDR3: positions 110 to 118 of SEQ ID NO: 21, (ii) VH: CDR1: positions 45 to 52 of SEQ ID NO: 15, CDR2: positions 70 to 77 of SEQ ID NO: 15, CDR3: positions 116 to 126 of SEQ ID NO: 15, VL: CDR1: positions 47 to 58 of SEQ ID NO: 20, CDR2: positions 76 to 78 of SEQ ID NO: 20, CDR3: positions 115 to 123 of SEQ ID NO: 20, (iii) VH: CDR1: positions 45 to 52 of SEQ ID NO: 16, CDR2: positions 70 to 77 of SEQ ID NO: 16, CDR3: positions 116 to 124 of SEQ ID NO: 16, VL: CDR1: positions 47 to 52 of SEQ ID NO: 22, CDR2: positions 70 to 72 of SEQ ID NO: 22, CDR3: positions 109 to 117 of SEQ ID NO: 22, (iv) VH: CDR1: positions 44 to 51 of SEQ ID NO: 18, CDR2: positions 69 to 76 of SEQ ID NO: 18, CDR3: positions 115 to 125 of SEQ ID NO: 18, VL: CDR1: positions 47 to 58 of SEQ ID NO: 25, CDR2: positions 76 to 78 of SEQ ID NO: 25, CDR3: positions 115 to 122 of SEQ ID NO: 25, (v) VH: CDR1: positions 45 to 52 of SEQ ID NO: 17, CDR2: positions 70 to 77 of SEQ ID NO: 17, CDR3: positions 116 to 126 of SEQ ID NO: 17, VL: CDR1: positions 47 to 58 of SEQ ID NO: 24, CDR2: positions 76 to 78 of SEQ ID NO: 24, CDR3: positions 115 to 123 of SEQ ID NO: 24, (vi) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 58 of SEQ ID NO: 23, CDR2: positions 76 to 78 of SEQ ID NO: 23, CDR3: positions 115 to 123 of SEQ ID NO: 23, (vii) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 58 of SEQ ID NO: 26, CDR2: positions 76 to 78 of SEQ ID NO: 26, CDR3: positions 115 to 123 of SEQ ID NO: 26, (viii) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 58 of SEQ ID NO: 27, CDR2: positions 76 to 78 of SEQ ID NO: 27, CDR3: positions 115 to 123 of SEQ ID NO: 27, and (ix) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19; VL: CDR1: positions 47 to 52 of SEQ ID NO: 28, CDR2: positions 70 to 72 of SEQ ID NO: 28, CDR3: positions 109 to 117 of SEQ ID NO: 28.

[0138] The antibodies according to (ii) or (v) are preferred embodiments of the “antibody having the ability to bind to CLDN18.2”. The antibodies according to (v) are particularly preferred.

[0139] In a further preferred embodiment, the antibody capable of binding to CLDN18.2 preferably comprises one or more, preferably at least the CDR3 variable region, of the heavy chain variable region (VH) and / or the light chain variable region (VL) of a monoclonal antibody against CLDN18.2, preferably of a monoclonal antibody against CLDN18.2 as described herein, and preferably comprises one or more, preferably at least the CDR3 variable region, of the heavy chain variable region (VH) and / or the light chain variable region (VL) as described herein. In one embodiment, said one or more of the complementarity determining regions (CDRs) are selected from the set of complementarity determining regions CDR1, CDR2 and CDR3 as described herein. In a particularly preferred embodiment, the antibody capable of binding to CLDN18.2 preferably comprises the complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL) of a monoclonal antibody against CLDN18.2, preferably a monoclonal antibody against CLDN18.2 described herein, and preferably comprises the complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL) of a monoclonal antibody against CLDN18.2 described herein.

[0140] In one embodiment, an antibody 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, this portion also comprises at least about 50% of either 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. When constructing antibodies made by recombinant DNA techniques, residues N-terminal or C-terminal to the variable region encoded by a linker can be introduced to facilitate cloning or other engineering steps, including the introduction of linkers to join the variable region of the invention to additional protein sequences, including immunoglobulin heavy chains, other variable domains (e.g., in the generation of diabodies), or protein tags.

[0141] In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of a set of CDRs described herein comprises said CDRs within a human antibody framework.

[0142] Reference herein to an antibody comprising a particular chain, or a particular region or sequence, in relation to its heavy chain, preferably relates to the situation in which all heavy chains of said antibody comprise said particular chain, region or sequence, and the same is correspondingly true for the light chains of the antibody.

[0143] In one embodiment, an antibody capable of binding to CLDN18.2, according to the present invention, refers to an antibody that recognizes, i.e. binds to, the same or essentially the same epitope as the CLDN18.2-binding antibody described herein and / or competes with said CLDN18.2-binding antibody for binding to CLDN18.2.

[0144] According to the present invention, an antibody capable of binding to CLDN18.2, particularly when present in an antibody-drug conjugate, preferably has an affinity and / or specificity for CLDN18.2 suitable to enable endocytosis of the antibody and / or antibody-drug conjugate.

[0145] The term "endocytosis" refers to the process by which eukaryotic cells internalize components from segments of the plasma membrane, cell-surface receptors, and extracellular fluids. Endocytic mechanisms include receptor-mediated endocytosis. The term "receptor-mediated endocytosis" refers to a biological mechanism in which a ligand, upon binding to its target, triggers an invagination or narrowing of the membrane, resulting in internalization and delivery into the cytosol or transfer to the appropriate intracellular compartment.

[0146] The present invention also contemplates an embodiment in which "antibody having the ability to bind to CLDN18.2" has the meaning encompassing any "binding agent to CLDN18.2". According to the present invention, "binding agent to CLDN18.2" includes any compound having the ability to bind to CLDN18.2. Preferably, such binding agent comprises at least one binding domain to CLDN18.2. The term includes all artificial binding molecules (scaffolds) having the ability to bind to CLDN18.2, including but not limited to nanobodies, affibodies, anticalins, DARPins, monobodies, avimers, and microbodies. In one embodiment, the binding agent binds to the extracellular domain of CLDN18.2. In one embodiment, the binding agent binds to a natural epitope of CLDN18.2 present on the surface of living cells. In one embodiment, the binding agent binds to the first extracellular loop of CLDN18.2. In one embodiment, the binding to CLDN18.2 is specific binding.

[0147] The term "binding domain" characterizes in the context of the present invention a structure, e.g. of an antibody, which binds to / interacts with a given target structure / antigen / epitope. A binding domain according to the present invention therefore designates an "antigen interaction site".

[0148] Any agent that exerts a therapeutic effect on cancer cells can be used as a drug for conjugation to anti-CLDN18.2 antibodies or derivatives thereof. Preferably, the conjugation of the drug does not change or does not significantly change the binding properties, particularly the specificity, of the antibody as discussed herein. Thus, the antibody-drug conjugate according to the present invention preferably has the same or essentially the same binding properties, particularly the specificity, as the antibody used for conjugation. Thus, if certain binding properties are described herein for the antibody used for conjugation, it is preferred that the antibody-drug conjugate also has such binding properties. For example, if an antibody capable of binding to CLDN18.2 is described as binding to the extracellular domain of CLDN18.2 and / or binding to the first extracellular loop of CLDN18.2, it is preferred that the antibody-drug conjugate also binds to the extracellular domain of CLDN18.2 and / or binding to the first extracellular loop of CLDN18.2.

[0149] Typically, the drug is a cytotoxic or cytostatic agent. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells, especially kills cells.

[0150] Useful classes of cytotoxic agents include, for example, antitubulin agents, DNA minor groove binders (e.g., enediynes and lexitropsins), DNA replication inhibitors, alkylating agents (e.g., platinum complexes, e.g., cisplatin, mono-, di-, and trinuclear platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophores, nitrosoureas, platinol, preforming compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes (e.g., paclitaxel and docetaxel), topoisomerase inhibitors, vinca alkaloids, and the like.

[0151] Individual cytotoxic agents include, for example, androgens, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, docetaxel, doxorubicin, estrogen, 5-fluorodeoxyglucose ... These include 5-fluordeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, plicamycin, procarbidine, streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP-16, and VM-26.

[0152] Examples of antitubulin agents include, but are not limited to, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB), maytansinoids, taxanes (e.g., paclitaxel, docetaxel), T67 (Tularik), vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colchimide, estramustine, cryptophysins, cemadotin, combretastatins, discodermolide, and eleutherobin.

[0153] In a specific embodiment, the cytotoxic or cytostatic agent is auristatin E (also known in the art as dolastatin-10) or a derivative thereof. Typically, an auristatin E derivative is an ester formed, for example, between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatin derivatives include AFP, MMAF, and MMAE.

[0154] In certain embodiments, the cytotoxic or cytostatic agent is a maytansinoid, another group of antitubulin agents, e.g., in specific embodiments, the maytansinoid is maytansine, DM-1, or DM-4.

[0155] Maytansinoids are potent microtubule-targeting compounds that inhibit the proliferation of cells in mitosis. Maytansinoids are derivatives of maytansine, a 19-membered ansamacrolide structure attached to a chlorinated benzene ring. Maytansine has the formula:

[0156] [ka]

[0157] Certain microorganisms have also been found to produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and maytansinol analogs are described, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,270; and 4,308,280, which are incorporated herein by reference. Nos. 69; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,362,663; and 4,371,533, and in Kawai et al. (1984) Chem. Pharm. Bull., 3441-3451.

[0158] Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Particularly preferred maytansinoids according to the invention are thiol-containing derivatives of maytansine, such as DM1 and DM4. Such thiol-containing derivatives of maytansine include compounds in which the methyl group attached to the carbonyl group is replaced by a group containing a free sulfhydryl group, such as a -R-SH group, where R represents an alkylene group or other carbon-containing group of atoms.

[0159] DM1, also known as mertansine, is a maytansinoid having the following formula:

[0160] [ka]

[0161] In particular, the term "mertansine" or "DM1" refers to the compound N 2' -Deacetyl-N 2'-(3-mercapto-1-oxopropyl)-maytansine.

[0162] "DM4" is compound N 2' -Deacetyl-N 2' -(4-methyl-4-mercapto-1-oxopentyl)-maytansine.

[0163] Anti-CLDN18.2 antibody-maytansinoid conjugates can be prepared by chemically linking an anti-CLDN18.2 antibody to a maytansinoid molecule without significantly reducing the biological activity of either the antibody or the maytansinoid molecule. On average, 3-4 maytansinoid molecules can be conjugated per antibody molecule, although even a single molecule of toxin / antibody is expected to enhance cytotoxicity over the use of naked antibody.

[0164] In this regard, the term "antibody covalently attached to at least one toxin drug moiety" includes the situation where one or more molecules of the same drug are covalently attached to the antibody molecule, as well as the situation where different drugs are covalently attached to the antibody molecule. In the latter situation, one or more molecules of each of the different drugs may be attached to the antibody molecule, or there may be a combination thereof (e.g., one molecule of one drug is attached while several molecules of another drug are attached).

[0165] In some embodiments of the invention, the antibody is conjugated to a dolastatin or dolostatin peptide analogs and derivatives, auristatins (U.S. Patent Nos. 5,635,483; 5,780,588, which are incorporated herein by reference). Auristatins are synthetic analogs of dolastatin 10, a natural product derived from the marine mollusc, Dolabela auricularia. Like maytansinoids, auristatins are microtubule disruptors. The dolastatin or auristatin drug moiety can be attached to the antibody through the N-terminus or C-terminus of the peptidic drug moiety.

[0166] Exemplary auristatin embodiments include monomethylauristatin drug moieties, such as MMAE and MMAF, which are preferably N-terminally linked.

[0167] MMAE, also known as monomethylauristatin E, has the formula:

[0168] [ka]

[0169] In particular, the term "MMAE" refers to the compound (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide. MMAE is actually desmethyl-auristatin E, i.e., the N-terminal amino group has only one methyl substituent instead of two as in auristatin E itself.

[0170] Antibody-vc auristatin conjugates, such as antibody-vcMMAE conjugates, are particularly preferred according to the invention. According to the invention, the term "antibody-vc auristatin" or "vcMMAE" refers to an antibody-drug conjugate (ADC) that comprises an auristatin, such as MMAE, linked to an antibody via a linker that comprises the lysosomally cleavable dipeptide, valine-citrulline (vc).

[0171] MMAF, also known as monomethylauristatin F, refers to the compound (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid.

[0172] The production of antibody-drug conjugates can be achieved by any technique known to those skilled in the art. Antibody-drug conjugates can be prepared by binding a drug to an antibody according to conventional techniques. The antibody and drug can be directly bound to each other via their own linker groups, or indirectly bound via a linker or other substance.

[0173] Several different reactions are available for the covalent attachment of drugs to antibodies. This is often accomplished by reaction of amino acid residues of the antibody molecule, including the amine groups of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine, and various moieties of aromatic amino acids. One of the most commonly used non-specific methods of covalent attachment is the carbodiimide reaction to link the carboxy (or amino) group of a compound to the amino (or carboxy) group of an antibody. Additionally, bifunctional agents such as dialdehydes or imidoesters have been used to link the amino group of a compound to the amino group of an antibody molecule. The Schiff base reaction is also available for attaching drugs to antibodies. This method involves periodate oxidation of a drug containing a glycol or hydroxy group, thus forming an aldehyde, which is then reacted with the antibody molecule. Attachment occurs via the formation of a Schiff base with the amino group of the antibody molecule. Isothiocyanates can also be used as coupling agents to covalently attach drugs to antibodies. Other techniques are known to those skilled in the art and are within the scope of the present invention.

[0174] There are many linking groups known in the art for making antibody-drug conjugates. The linker preferably contains one or more functional groups that react with either or both the antibody and the drug. Examples of functional groups include amino, carboxyl, mercapto, maleimide, and pyridinyl groups.

[0175] In one embodiment of the present invention, the antibody is linked to the drug via a bifunctional crosslinking reagent. As used herein, "bifunctional crosslinking reagent" refers to a reagent that has two reactive groups, one of which can react with the antibody, while the other can react with the drug to link the antibody to the drug, thereby forming a conjugate. Any suitable bifunctional crosslinking reagent can be used in the context of the present invention, so long as the linker reagent provides what the drug possesses, e.g., the cytotoxicity and targeting properties of the antibody. Preferably, the linker molecule joins the drug to the antibody by chemical bond, such that the drug and the antibody are chemically coupled (e.g., covalently bonded) to each other.

[0176] In one embodiment, the bifunctional cross-linking reagent comprises a non-cleavable linker. The non-cleavable linker is any chemical moiety that can link a drug, such as a maytansinoid, to an antibody in a stable covalent manner. Preferably, the non-cleavable linker is not cleavable under physiological conditions, particularly inside a cell. Thus, the non-cleavable linker is substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under conditions in which the drug or antibody remains active. Suitable cross-linking reagents that form a non-cleavable linker between a drug and an antibody are well known in the art. In one embodiment, the drug is linked to the antibody by a thioether bond.

[0177] In a particularly preferred embodiment, the linking reagent is a cleavable linker. Preferably, the cleavable linker is cleavable under physiological conditions, in particular inside a cell. Examples of suitable cleavable linkers include disulfide linkers, acid labile linkers, photolabile linkers, peptidase labile linkers, and esterase labile linkers.

[0178] Examples of linkers include, but are not limited to, N-succinimidyl-3-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS), 3-maleimidocaproic acid N-hydroxysuccinimide ester (GMBS ... ter (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB), N-(p-maleimidophenyl)isocyanate (PMPI), 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), and N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB). Peptide linkers such as valine-citrulline (Val-Cit) or alanine-phenylalanine (ala-phe) can also be used, and any of the above linkers can be used in appropriate combinations.

[0179] Disulfide-containing linkers are linkers that can be cleaved by disulfide exchange that can occur under physiological conditions. In yet other embodiments, the linker can be cleaved under reducing conditions (e.g., disulfide linkers). A variety of disulfide linkers are known in the art, including those that can be formed using, for example, SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene).

[0180] An acid-labile linker is a linker that can be cleaved at an acidic pH. For example, certain intracellular compartments, such as endosomes and lysosomes, have an acidic pH (pH 4-5), providing suitable conditions for cleaving an acid-labile linker. An acid-labile linker is relatively stable under neutral pH conditions, such as those in blood, but is unstable at pH 5.5 or below 5.0. For example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, and the like can be used.

[0181] Photolabile linkers are useful on body surfaces and in many body cavities that are accessible to light. Additionally, infrared light can penetrate tissue.

[0182] Peptidase-labile linkers can be used to cleave a particular peptide inside or outside the cell, hi one embodiment, the cleavable linker is cleaved under mild conditions, i.e., conditions within the cell where the activity of the cytotoxic agent is not affected.

[0183] The linker may be or include a peptidyl linker that is cleaved by intracellular peptidases or protease enzymes, including, but not limited to, lysosomal or endosomal proteases. Typically, the peptidyl linker is at least 2 amino acids long or at least 3 amino acids long. Cleaving agents may include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives and release active drugs inside target cells. For example, a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissues, may be used (e.g., Phe-Leu or Gly-Phe-Leu-Gly linker). In a specific embodiment, the peptidyl linker that is cleavable by intracellular proteases is a valine-citrulline (Val-Cit; vc) linker or a phenylalanine-lysine (Phe-Lys) linker. One advantage of using intracellular proteolytic release of therapeutic agents is that the agents are typically attenuated when conjugated and the serum stability of the conjugates is typically high.

[0184] In one particularly preferred embodiment, the linker according to the invention comprises or consists of the dipeptide valine (Val)-citrulline (Cit) (vc), which is cleaved by cathepsins inside tumor cells.

[0185] In one embodiment, the drug is a maytansinoid, such as DM4, coupled to an antibody capable of binding to CLDN18.2 via an amino- and sulfhydryl-reactive heterobifunctional protein crosslinker that reacts with primary amines of the antibody (found in lysine side chains or the N-terminus of proteins) and sulfhydryl groups of the maytansinoid to form a reversible disulfide bond. In one embodiment, the amino- and sulfhydryl-reactive heterobifunctional protein crosslinker is SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), which reacts with primary amines of the antibody (found in lysine side chains or the N-terminus of proteins) via its N-hydroxysuccinimide (NHS) ester and with sulfhydryl groups of DM4 via its pyridinyl disulfide group to form a reversible disulfide bond (Figure 1).

[0186] In one embodiment, the drug is an auristatin such as MMAE coupled to an antibody capable of binding to CLDN18.2 via a cathepsin-cleavable peptide linker, in particular a peptide linker such as Val-Cit(vc). In one embodiment, the antibody capable of binding to CLDN18.2 is thiolated, for example with the heterobifunctional linker 2-IT (2-iminothiolane), which reacts with the free amines of lysine residues.

[0187] In one particularly preferred embodiment, the antibody-drug conjugate according to the invention comprises an antibody comprising a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 32, or a fragment thereof, or a variant of said amino acid sequence or fragment, coupled (preferably by its amino group) to DM4 (preferably by its sulfhydryl group), and a light chain comprising the amino acid sequence represented by SEQ ID NO: 39, or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the antibody is coupled to DM4 by a SPDB linker.

[0188] In one embodiment, an antibody-drug conjugate according to the invention comprises an antibody comprising a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 17 or 51, or a fragment thereof, or a variant of said amino acid sequence or fragment, coupled (preferably by its amino group) to DM4 (preferably by its sulfhydryl group), and a light chain comprising the amino acid sequence represented by SEQ ID NO: 24, or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the antibody is coupled to DM4 by a SPDB linker.

[0189] In one particularly preferred embodiment, the antibody-drug conjugate according to the invention comprises an antibody comprising a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 32, or a fragment thereof, or a variant of said amino acid sequence or fragment, coupled (preferably by its amino group) to MMAE (preferably by its N-terminal amino group), and a light chain comprising the amino acid sequence represented by SEQ ID NO: 39, or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the antibody is coupled to MMAE by a linker comprising the dipeptide vc.

[0190] In one particularly preferred embodiment, the antibody-drug conjugate according to the invention comprises an antibody comprising a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 17 or 51, or a fragment thereof, or a variant of said amino acid sequence or fragment, coupled (preferably by its amino group) to MMAE (preferably by its N-terminal amino group), and a light chain comprising the amino acid sequence represented by SEQ ID NO: 24, or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the antibody is coupled to MMAE by a linker comprising the dipeptide vc.

[0191] The term "nucleic acid" as used herein is intended to include DNA and RNA. Nucleic acids may be single-stranded or double-stranded, but preferably are double-stranded DNA.

[0192] According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA or of RNA and proteins / peptides. It also includes partial expression of nucleic acids. Furthermore, expression can be performed transiently or stably.

[0193] The teachings given herein with respect to specific amino acid sequences, such as those shown in the sequence listing, and in particular those referred to herein by showing SEQ ID NOs, should be interpreted as also referring to variants of said specific sequences. Such variant sequences may be functionally equivalent to said specific sequences, for example amino acid sequences that exhibit the same or similar properties as those of the specific amino acid sequences. One important property is to retain the binding ability of the antibody to its target. Preferably, a sequence that is variant with respect to a specific sequence retains the binding ability of said antibody to CLDN18.2 when it replaces the specific sequence in the antibody.

[0194] In particular, it will be understood by those skilled in the art that the sequences of the CDRs, hypervariable regions and variable regions may be modified without losing the ability to bind CLDN18.2. For example, the CDR regions will be identical or highly homologous to regions of the antibodies specified herein. By "highly homologous" it is contemplated that 1-5, preferably 1-4, such as 1-3 or 1 or 2 substitutions may be made in the CDRs. Furthermore, the hypervariable and variable regions may be modified such that they exhibit substantial homology to regions of the antibodies specifically disclosed herein.

[0195] The term "variant" according to the present invention refers in particular to mutants, splice variants, conformations, isoforms, allelic variants, species variants, and species homologs, especially those that occur naturally. Allelic variants refer to changes in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies multiple allelic variants for a given gene. Species homologs are nucleic acid or amino acid sequences for a species of origin that differ from that of a given nucleic acid or amino acid sequence. The term "variant" is intended to encompass any post-translationally modified variants and conformational variants.

[0196] 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 a protein, are also referred to as N-terminal and / or C-terminal truncation variants.

[0197] Amino acid insertion variants include the insertion of a single or two or more amino acids into a particular amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a particular site in the amino acid sequence, but can also be inserted randomly with appropriate screening of the resulting products.

[0198] Amino acid addition variants include amino and / or carboxy terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

[0199] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletions can be in any position in the protein.

[0200] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another in its place. Modifications in positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or replacement of amino acids with other amino acids with similar properties are preferred. Preferably, the amino acid changes in the protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one of a family of amino acids that are related to their side chains. Naturally occurring amino acids are generally classified into four families of amino acids: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), non-polar (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 sometimes classified jointly as aromatic amino acids.

[0201] Preferably, the degree of similarity, preferably identity, between a given amino acid, such as the amino acid sequences referred to herein by setting forth a SEQ ID NO, and an amino acid sequence which is a variant of said given amino acid sequence, will be 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 which 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 for the entire length of the reference amino acid sequence. Alignment for determining 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.

[0202] "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.

[0203] The term "percentage identity" is intended to express the percentage of identical amino acid residues between the two sequences compared, obtained after the best alignment, and this percentage is purely statistical, the differences between the two sequences being distributed randomly and over their entire length. Sequence comparison between two amino acid sequences is conventionally performed by comparing these sequences after optimal alignment, said comparison being performed by segments or "windows of comparison" to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be generated 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 using 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.).

[0204] The percentage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared, and multiplying the result by 100, resulting in the percentage identity between the two sequences.

[0205] The term "transgenic animal" refers to an animal having a genome that includes one or more transgenes, preferably heavy and / or light chain transgenes, or transchromosomes (integrated or not integrated into the animal's native genomic DNA), and preferably capable of expressing the transgenes. For example, a transgenic mouse can have a human light chain transgene and a human heavy chain transgene or a human heavy chain transchromosome, such that the mouse produces human anti-CLDN18.2 antibodies when immunized with CLDN18.2 antigen and / or cells expressing CLDN18.2. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as in the case of a transgenic mouse, e.g., a HuMAb mouse, e.g., a HCo7 mouse or a HCol2 mouse, or the human heavy chain transgene can be maintained extrachromosomally, as in the case of a transchromosomal (e.g., KM) mouse described in WO02 / 43478. Such transgenic and transchromosomal mice can produce multiple isotypes of human monoclonal antibodies against CLDN18.2 (eg, IgG, IgA, and / or IgE) by undergoing VDJ recombination and isotype switching.

[0206] "Reduce", "reduce" or "inhibit" as used herein means an overall decrease or ability to cause an overall decrease in levels, e.g., levels of expression or levels of cell proliferation, preferably by 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more.

[0207] Terms such as "increase" or "enhance" preferably relate to an increase or enhancement of about at least 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 even more.

[0208] The antibodies described herein can be produced by a variety of techniques, including conventional monoclonal antibody methods, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature, 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle other techniques for producing monoclonal antibodies can also be used, e.g., viral transformation or oncogenesis of B-lymphocytes, or phage display techniques using libraries of antibody genes.

[0209] 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 and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0210] Other suitable animal systems for preparing hybridomas secreting monoclonal antibodies are the rat and rabbit systems (described, for example, in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA, 92:9348 (1995); see also Rossi et al., Am. J. Clin. Pathol., 124: 295 (2005)).

[0211] In yet another preferred embodiment, human monoclonal antibodies can be produced 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." The production of human antibodies in such transgenic mice can be performed as described in detail for CD20 in WO2004 035607.

[0212] Yet another strategy for generating monoclonal antibodies is to directly isolate the antibody-encoding genes from lymphocytes producing 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 of 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.

[0213] To generate antibodies, mice can be immunized with carrier-conjugated peptides derived from the antigen sequence, i.e., the sequence against which the antibody is directed, concentrated preparations of recombinantly expressed antigen or fragments thereof, and / or cells expressing the antigen, as described above. Alternatively, mice can be immunized with DNA encoding the antigen or fragments thereof. If immunization with a purified or concentrated preparation of the antigen does not result in antibodies, mice can also be immunized with cells expressing the antigen, e.g., cell lines, to promote an immune response.

[0214] The immune response can be monitored over the course of the immunization protocol with plasma and serum samples being obtained by tail vein or retroorbital bleeds. Mice with sufficient titers of immunoglobulin can be used for fusions. To increase the proportion of specific antibody-secreting hybridomas, mice can be boosted intraperitoneally or intravenously with antigen-expressing cells 3 days before sacrifice and removal of the spleen.

[0215] To generate hybridomas that produce monoclonal antibodies, splenocytes and lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for production of antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody-secreting hybridomas. Antibodies with specificity for the antigen can be identified by immunofluorescence and FACS analysis using antigen-expressing cells. Antibody-secreting hybridomas can be replated and screened again, and if still positive for monoclonal antibodies, can be subcloned by limiting dilution. Stable subclones can then be cultured in vitro to generate antibodies in tissue culture medium and characterized.

[0216] Antibodies can also be produced in host cell transfectomas using a combination of recombinant DNA technology and gene transfection techniques, as is well known in the art (Morrison, S. (1985) Science, 229: 1202).

[0217] For example, in one embodiment, the gene(s) of interest, e.g., antibody genes, can be ligated into an expression vector, such as a eukaryotic expression plasmid, such as those used by the GS gene expression system disclosed in WO87 / 04462, WO89 / 01036, and EP338841, or other expression systems known in the art. The purified plasmid containing the cloned antibody genes can be introduced into eukaryotic host cells, such as CHO cells, NS / 0 cells, HEK293T cells, or HEK293 cells, or alternatively other eukaryotic-like plant-derived cells, fungal cells, or yeast cells. The method used to introduce these genes can be a method described in the art, e.g., electroporation, lipofectine, lipofectamine, etc. After introducing these antibody genes into the host cells, cells expressing the antibody can be identified and selected. These cells represent transfectomas, which can then be amplified for their expression levels and upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0218] 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 transgenic non-human animals, e.g., in milk from sheep and rabbits, in eggs from hens, or in 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.

[0219] Chimerization 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 originate from different animal species, such as those with a variable region originating from a mouse antibody and a human immunoglobulin constant region. Antibody chimerization is achieved by joining the variable regions of mouse antibody heavy and light chains with human heavy and light chain constant regions (e.g., as described by Kraus et al., Methods in Molecular Biology series, Recombinant antibodies for cancer therapy, ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are produced by joining a human kappa-light chain constant region to a mouse light chain variable region. Also in a preferred embodiment, chimeric antibodies can be produced by joining a human lambda-light chain constant region to a mouse light chain variable region. Preferred heavy chain constant regions for producing chimeric antibodies are IgG1, IgG3, and IgG4. Other suitable heavy chain constant regions for generating chimeric antibodies are IgG2, IgA, IgD, and IgM.

[0220] Humanization Antibodies interact with target antigens primarily through amino acid residues located within the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Because the CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing expression vectors that contain CDR sequences from a particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, for example, 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 that contain germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they do not contain the fully assembled variable genes formed by V(D)J joining during B cell maturation. Germline gene sequences will also differ from the sequences of high affinity secondary repertoire antibodies at individual locations evenly across the variable regions.

[0221] The ability of the antibody to bind the antigen can be determined using standard binding assays (eg, ELISA, Western blot, immunofluorescence, and flow cytometric analysis).

[0222] To purify the antibodies, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. Alternatively, the antibodies can be produced in dialysis-based bioreactors. The supernatant can be filtered and concentrated if necessary before affinity chromatography with protein G-sepharose or protein A-sepharose. The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer can be exchanged into PBS and the concentration can be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibodies can be aliquoted and stored at -80°C.

[0223] To determine whether a selected monoclonal antibody binds to a unique epitope, site-directed or multi-site directed mutagenesis can be used.

[0224] To determine the antibody isotype, an isotype ELISA can be performed using various commercially available kits (e.g., Zymed, Roche Diagnostics). The wells of a microtiter plate can be coated with anti-mouse Ig. After blocking, the plate is reacted with monoclonal antibodies or purified isotype controls for 2 hours at ambient temperature. The wells can then be reacted with mouse IgG1, IgG2a, IgG2b, or IgG3, IgA, or mouse IgM specific peroxidase-conjugated probes. After washing, the plate can be developed with ABTS substrate (1 mg / ml) and analyzed at an OD of 405-650. Alternatively, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, Cat. No. 1493027) can be used as described by the manufacturer.

[0225] Flow cytometry can be used to demonstrate the presence of antibodies in the serum of immunized mice, or the binding of monoclonal antibodies to live cells expressing the antigen. Cell lines expressing the antigen naturally or after transfection, and negative controls lacking antigen expression (grown under standard growth conditions), can be mixed with various concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1% FBS and incubated for 30 minutes at 4°C. After washing, APC or Alexa 647-labeled anti-IgG antibodies can be bound to the antigen-bound monoclonal antibodies under the same conditions as the primary antibody staining. Samples can be analyzed by flow cytometry with a FACS instrument, using optical and side scatter properties to gate single live cells. To distinguish antigen-specific monoclonal antibodies from non-specific binders in a single measurement, a method of co-transfection can be used. Cells transiently transfected with plasmids encoding the antigen, and a fluorescent marker can be stained as described above. Transfected cells can be detected in a different fluorescent channel than antibody-stained cells. Since 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 to non-transfected cells in equal proportions. In addition to or instead of the flow cytometry assay, an alternative assay using fluorescent microscopy may be used. Cells can be stained exactly as described above and examined by fluorescent microscopy.

[0226] Immunofluorescence microscopy analysis can be used to demonstrate the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to live cells expressing the antigen. For example, cell lines expressing the antigen spontaneously or after transfection, and 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. The cells can then be fixed with methanol or paraformaldehyde or left untreated. The cells can then be reacted with monoclonal antibodies against the antigen for 30 minutes at 25° C. After washing, the cells can be reacted with Alexa 555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. The cells can then be examined by fluorescence microscopy.

[0227] Cell extracts can be prepared from cells expressing the antigen and appropriate negative controls 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.

[0228] The antibodies can be further tested for reactivity with the antigen by immunohistochemistry in a manner 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 spontaneously or after transfection with a cell line expressing the antigen. For immunostaining, antibodies reactive against the antigen can be incubated according to the supplier's instructions, followed by horseradish peroxidase conjugated goat anti-mouse or goat anti-rabbit antibodies (DAKO).

[0229] Antibody conjugates that bind CLDN18.2 can also be tested in in vivo models (e.g., in immune-deficient mice bearing xenograft tumors inoculated with cell lines expressing CLDN18.2, such as DAN-G, SNU-16, or KATO-III, or cell lines expressing CLDN18.2 after transfection, such as HEK293) to determine their efficacy in controlling the growth of CLDN18.2-expressing tumor cells.

[0230] The antibody conjugates can be administered to tumor-free mice, followed by injection of tumor cells, to measure the efficacy of the antibody conjugates in preventing tumor formation or tumor-related symptoms. The antibody conjugates can be administered to tumor-bearing mice to determine the therapeutic efficacy of each antibody conjugate in reducing tumor growth, metastasis, or tumor-related symptoms. The antibody conjugate application can be combined with the application of other substances, such as cystostatic drugs, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors, or other antibody conjugates, to determine the synergistic efficacy and potential toxicity of the combination. To analyze toxic side effects mediated by the antibody conjugates, animals can be inoculated with the antibody conjugates or control reagents to thoroughly investigate symptoms possibly related to CLDN18.2-antibody conjugate therapy. Potential side effects of in vivo application of CLDN18.2 antibodies include toxicity, particularly in CLDN18.2-expressing tissues, including the stomach.

[0231] Mapping of epitopes recognized by antibodies can be performed as described in detail in "Epitope Mapping Protocols" (Methods in Molecular Biology), Glenn E. Morris, ISBN-089603-375-9, and in "Epitope Mapping: A Practical Approach" Practical Approach Series, 248, Olwyn MR Westwood, Frank C. Hay.

[0232] The compounds and agents described herein may be administered in the form of any suitable pharmaceutical composition.

[0233] The pharmaceutical compositions are preferably sterile and contain an effective amount of the antibodies described herein and optionally further agents discussed herein to produce the desired reaction or desired effect.

[0234] The pharmaceutical compositions are usually provided in uniform 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 a suspension.

[0235] Pharmaceutical compositions can include salts, buffer substances, preservatives, carriers, diluents, and / or excipients, all of which are preferably pharma- ceutically acceptable. The term "pharmaceutical acceptable" refers to the non-toxicity of materials that do not interact with the action of the active ingredients of the pharmaceutical composition.

[0236] Pharmaceutically unacceptable salts may be used to prepare pharma-ceutically acceptable salts and are included in the present invention. Pharmaceutically acceptable salts of this type include, in a non-limiting manner, 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 can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0237] Suitable buffering substances for use in the pharmaceutical compositions include acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt.

[0238] Suitable preservatives for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0239] Injectable formulations may contain pharma- ceutically acceptable excipients such as Ringer's lactate.

[0240] The term "carrier" refers to an organic or inorganic component of natural or synthetic nature with which the active ingredient is combined to facilitate, enhance or enable application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a patient.

[0241] Possible carrier materials for parenteral administration are, for example, sterile water, Ringer's, Ringer's lactate, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes, and especially biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0242] The term "excipient" as used herein is intended to indicate any substance that may be present in a pharmaceutical composition and which is not an active ingredient, such as, for example, carriers, binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, flavoring agents, or coloring agents, etc.

[0243] The agents and compositions described herein may be administered via any conventional route, such as parenteral administration, including by injection or infusion. Administration is preferably parenteral, e.g., intravenous, intraarterial, subcutaneous, intradermal, or intramuscular.

[0244] Compositions suitable for parenteral administration usually include sterile aqueous or non-aqueous preparations of the active compound, preferably isotonic with the blood of the recipient. Examples of compatible carriers and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are usually used as a solution or suspension medium.

[0245] The agents and compositions described herein are administered in an effective amount. "Effective amount" refers to an amount that achieves the desired reaction or the desired effect, either alone or together with further doses. In the case of the treatment of a particular disease or a particular condition, the desired reaction preferably relates to the inhibition of the disease process. This includes the slowing down of the progression of the disease, and in particular the halting or reversal of the progression of the disease. The desired reaction in the treatment of a disease or condition can also be the delay of the onset of said disease or said condition or the prevention of the onset of said disease or said condition.

[0246] The effective amount of the agent or composition described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, duration of treatment, type of concomitant therapy (if any), specific route of administration, and similar factors. Thus, the dose of the agent described herein to be administered may depend on various such parameters. If the patient's response is inadequate at the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) can be used.

[0247] The agents and compositions provided herein can be used alone or in combination with conventional treatment regimens, such as surgery, radiation, chemotherapy, and / or bone marrow transplantation (autologous, syngeneic, allogeneic, or unrelated).

[0248] Cancer treatment represents a field in which combination strategies are particularly desirable, since the combined action of two, three, four, or even more cancer drugs / therapies often produces synergistic effects that are considerably stronger than the effects of monotherapy approaches. Thus, in another embodiment of the present invention, cancer treatment can be effectively combined with a variety of other drugs. Among these are, for example, combinations with conventional tumor therapy, multi-epitope strategies, additional immunotherapy, and treatment approaches that target angiogenesis or apoptosis (for a review, see, for example, Andersen et al., 2008: Cancer treatment: the combination of vaccination with other therapies. Cancer Immunology Immunotherapy, 57(11):1735-1743). Sequential administration of different agents can inhibit cancer cell proliferation at different checkpoints, while other agents can inhibit, for example, neovascularization, malignant cell survival, or metastasis, potentially transforming cancer into a chronic disease.

[0249] The agents and compositions described herein can be administered to a patient, for example, in vivo, to treat or prevent a variety of disorders, such as those described herein. Suitable patients include human patients having disorders that can be corrected or ameliorated by administering the agents and compositions described herein. This includes disorders involving cells characterized by an altered expression pattern of CLDN18.2.

[0250] For example, in one embodiment, the agents and compositions described herein can be used to treat a patient having a cancer disease, e.g., a cancer disease such as those described herein that is characterized by the presence of cancer cells that express CLDN18.2.

[0251] The pharmaceutical compositions and methods of treatment described by the present invention may also be used for immunization or vaccination to prevent the diseases described herein.

[0252] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. EXAMPLES

[0253] Example 1 Materials and Methods 1. Endocytosis Endocytosis of CLDN18.2-bound IMAB362 was determined using a cytotoxicity-based endocytosis assay utilizing co-internalization of target-bound antibodies and saporin-conjugated anti-human or anti-mouse IgG Fab fragments (Fab-ZAP human, Advanced Targeting Systems, IT-51, lot no. 93-23; Fab-ZAP mouse, Advanced Targeting Systems, IT-48, lot no. 93-21). Saporin is a ribosome-inactivating protein that, when internalized, inhibits protein biosynthesis, thus resulting in cell death. To ensure optimal cell lysis, Fab-ZAP antibodies were applied at least 6-fold molar concentration compared to the target antibodies.

[0254] Stably transfected HEK293-CLDN18.2 cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054) and diluted to 2.5 × 10 3 Cells / well were seeded in 50 μl of growth medium in 96-well cell culture plates. After 24 hours, 25 μl of Fab-ZAP and 25 μl of anti-CLDN18.2 mAB or isotype control antibody diluted in cell culture medium were added to the cells (Table 1). Cells were cultured for an additional 72 hours.

[0255] [Table 2]

[0256] Cell viability was analyzed as described in 6. Cells incubated in the presence of Fab-ZAP without antibody were used as control.

[0257] 2. Epitope Mapping Antigenic epitopes involved in CLDN18.2 specificity were analyzed by flow cytometry (see 5) on HEK293T cells transiently overexpressing CLDN18.2 mutants. A total of eight CLDN18.2 mutants with single amino acid substitutions in the first extracellular domain were generated by PCR. Thus, amino acids of CLDN18.2 were replaced at the corresponding positions with those of the homologous protein CLDN18.1. HEK293T cells were co-transfected with plasmids encoding specific CLDN18.2 mutants and EGFP as a reporter gene. Purified antibodies were tested at a concentration of 5 μg / ml, whereas antibodies derived from hybridoma supernatants were tested after dilution up to 1:4. To determine whether antibody binding was affected by specific amino acid substitutions, the mean fluorescence intensity (MFI) measured in the transfected (EGFP-positive) cell population was compared between the mutants exhibiting the highest MFI values ​​and the mutants of interest. Amino acid residues were characterized as essential for antibody binding and CLDN18.2 specificity if the MFI of the mutant of interest was less than 50%.

[0258] 3. Antibody-drug conjugates Conjugation of DM4 and vcMMAE to monoclonal antibody IMAB362 (batch no. p412118) and analytical characterization were carried out at Piramal Healthcare (Grangemouth, UK). Methods are briefly described in the following sections: DM4 was coupled to IMAB362 via SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate). The SPDB reagent is an amino- and sulfhydryl-reactive heterobifunctional protein crosslinker that reacts with primary amines of antibodies (found on lysine side chains or N-termini of proteins) via N-hydroxysuccinimide (NHS) esters and with sulfhydryl groups of DM4 via pyridinyl disulfide groups to generate reversible disulfide bonds (Figure 1). Briefly, for DM4 conjugation, IMAB362 was diafiltered into PBS buffer (pH 7.2) using an ultracentrifugal filter and coupled to SPDB at a molar ratio of 1:6 (IMAB362:SPDB) for 1 h at RT. The modified antibody was dialyzed against 35 mM citrate buffer (pH 5.5) to determine the linker to antibody ratio. DM4 was conjugated to IMAB362-SPDB at a molar ratio of 1:6 (IMAB362-SPDB:DM4) for 19 h at 2–8° C. The conjugated antibody was adapted to storage buffer (20 mM His, 85 mg / ml sucrose, pH 5.8) and stored at −80° C. Drug-antibody ratios were analyzed by UV spectroscopy, monomer content was analyzed by SEC-HPLC, and free drug content was analyzed by RP-HPLC.

[0259] vcMMAE was coupled to thiolated IMAB362. Thus, IMAB362 was first thiolated with the heterobifunctional linker 2-IT (2-iminothiolane), which reacts with the free amines of lysine residues. Then, vcMMAE containing the cathepsin-cleavable peptide linker Val-Cit (vc) was conjugated to the sulfhydryl groups of the thiolated antibody via valine (Figure 1). Briefly, IMAB362 was diafiltered into PBS buffer (pH 7.2) using an ultracentrifugal filter and incubated with 2-IT at a molar ratio of 1:20 (IMAB362:2-IT) for 2 hours at RT. The modified antibody was dialyzed into 35 mM citrate buffer (pH 5.5) and the linker to antibody ratio was determined. Then, vcMMAE was conjugated to thiolated IMAB362 at a molar ratio of 1:6 (IMAB362-SH:vcMMAE) by incubation for 20 h at 2-8 °C. The conjugated antibody was dialyzed into storage buffer (20 mM His, 85 mg / mL sucrose, pH 5.8) and stored at -80 °C. Drug-antibody ratios were analyzed by UV spectroscopy, monomer content was analyzed by SEC-HPLC, and free drug content was analyzed by RP-HPLC.

[0260] 4.Cell culture Cell lines were cultured at 37°C in a humidified incubator containing 5% or 7.5% CO2 according to the supplier's instructions and Ganymed's cell line data sheets (Table 2). Cell culture media and supplements were obtained from Invitrogen, Gibco, and Sigma.

[0261] [Table 3]

[0262] 5. Flow Cytometry The relative binding affinity and specificity of anti-CLDN18.2 naked antibodies and antibody-drug conjugates was determined by flow cytometry using CLDN18.2 positive and negative cell lines.

[0263] Cells from exponentially growing cultures were harvested with 0.05% trypsin / EDTA (Gibco, 25300-054) and counted using a Neubauer counting chamber. Cells were centrifuged at 1,500 rpm (468×g) for 5 min, the supernatant was discarded, and cells were resuspended in FACS buffer (PBS containing 2% FCS (Gibco, 10270-106) for analysis with toxin-conjugated antibodies, PBS containing 2% FCS and 2 mM EDTA for screening CLDN18.2-reactive naked antibodies) at 2×10 6 Resuspend cells at 100 μl (2 × 10 5 Cells were centrifuged at 1500 rpm for 1 min and the supernatant was discarded and the cells were resuspended in FACS buffer containing toxin-conjugated or naked antibodies at the appropriate concentration (up to 20 μg / ml for relative affinity measurements or 50 μg / ml for expression control) and incubated at 4°C for 30-45 min (Table 3). Cells were centrifuged at 1500 rpm for 1 min and the supernatant was discarded. After washing the cells three times with FACS buffer, they were resuspended in FACS buffer containing APC-conjugated anti-human IgG (Jackson Immuno Research, 109-136-170), or APC-conjugated goat-anti-mouse IgG (Jackson Immuno Research, 115-136-146), or protein L-FITC (1 μg / ml, analysis of chim mAB294) and incubated for 30 minutes at 4° C. (Table 3). After incubation, 100 μl of FACS buffer was added to each sample, the cells were centrifuged at 1500 rpm for 1 minute, and the supernatant was discarded. The washing step with FACS buffer was repeated twice. Finally, the cells were resuspended in 100 μl of FACS buffer and binding was determined using a BD FACS Array Bioanalyzer.

[0264] It should be noted that the toxin-conjugated and naked antibodies were applied at equal concentrations, and the difference between the molecular weights of the antibodies was ignored.

[0265] [Table 4]

[0266] 6. Viability Assay The effect of IMAB362-DM4 and IMAB362-vcMMAE on cell viability was determined using a colorimetric assay that detects cell metabolic activity. The assay is based on the ability of metabolically active cells to reduce the yellow XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) to an orange formazan compound that can be detected by spectrophotometry. The intensity of the dye is proportional to the number of live cells.

[0267] Cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054), resuspended in cell culture medium (Table 2), and 50 μl of cell suspension containing the corresponding amount of cells was seeded per well of a 96-well cell culture plate (Table 4). After 24 hours, toxin-conjugated IMAB362 or control antibody diluted in 50 μl medium at the appropriate concentration was added, and cells were cultured for another 72 hours.

[0268] [Table 5]

[0269] Cell viability was analyzed using the AppliChem Cell proliferation Kit II (AppliChem, A8088, 1000) according to the manufacturer's instructions. After 3-5 hours of incubation with XTT reagent, the absorbance at 480 nm was measured using a spectrophotometer (Tecan) (reference 630 nm). The reduction in viability was calculated using the following formula:

[0270]

number

[0271] Blank: medium control Control: cells without antibody Sample: Cells containing antibodies

[0272] EC50 values ​​were determined with GraphPad Prism 6 using nonlinear regression.

[0273] 7. Bystander Assay The bystander activity of the toxin-conjugated IMAB362 antibody against target-negative cells was analyzed in vitro by co-culturing the CLDN18.2-negative luciferase-expressing cell line PA-1(Luc) in the presence or absence of the CLDN18.2-positive cell line NUGC-4 10cE8. Thus, 1.5×10 per well were cultured. 3 PA-1(Luc) cells were cultured at 1.5 × 10 per well for monoculture or 1.5 × 10 per well for coculture in RPMI medium supplemented with 10% FCS and 1% penicillin / streptomycin. 3 NUGC-4 10cE8 cells were seeded together. After 24 hours, IMAB362-DM4, IMAB362-vcMMAE, or unconjugated IMAB362 as a negative control was added and cells were cultured for another 72 hours. Cell viability was analyzed as described in 6. Lysis of target-negative cells was determined by measuring bioluminescence of luciferase-expressing PA-1 (Luc) cells. Therefore, 50 μl of luciferin mix (1.92 mg / ml D-luciferin (Sigma, 50227) and 160 mM HEPES in ddH2O) was added per well. Plates were incubated in the dark at RT for 90 minutes and bioluminescence was measured using a luminometer (Infinite M200, TECAN). Results are expressed as integrated digital relative light units (RLU). Viability reduction was calculated as described in 6.

[0274] 8. Animal Experiments All xenograft studies were performed in accordance with national regulations and ethical guidelines for experimental animal studies. All animals were maintained under specific pathogen-free conditions in individual ventilated cages and under a 12-h artificial light / dark cycle. Food and water were provided ad libitum. Mice were allowed to acclimate for a minimum of 6 days before the start of the studies.

[0275] 8.1. Maximum Tolerated Dose (MTD) Study Xenograft tumors were grown in the flanks of female Hsd:Athymic Nude-Foxn1 mice at 8.5 × 10 6 BxPC-3-CLDN18.2 human pancreatic tumor cells were inoculated by subcutaneous injection. To determine the MTD and efficacy of the anti-CLDN18.2 antibody drug conjugates, tumor-bearing mice received different doses of IMAB362-DM4 or IMAB362-vcMMAE. The maximum applicable dose was limited by the antibody concentration and the injection volume (approximately 200 μl) recommended by GV-SOLAS for intravenous injection in mice. Both antibodies were applied at the maximum concentration (i.e., 15 and 16 mg / kg, respectively) as single and repeated doses, as well as half of this concentration (i.e., 7.5 and 8 mg / kg, respectively), or vehicle control (group size: n=5). Antibodies were injected intravenously on day 14 after grafting and again on day 21 after grafting for repeated doses. Body weight, animal health, behavior, and tumor size were monitored twice weekly with calipers, and tumor volumes were calculated according to the following formula: [length x width x (width / 2)]. All animals were 1400 mm 3Mice were dissected when the tumor reached a maximum of 100 μg / kg / day or when the tumor became ulcerated (49 days after grafting for IMAB362-DM4 and 37 days after grafting for IMAB362-vcMMAE). Blood samples for clinical chemistry were collected under general anesthesia, starting with 250 μl ip of a mixture consisting of 1.25 ml ketamine, 1 ml xylazine (2%), and 7.75 ml H2O. Subsequently, mice were perfused under general anesthesia with PBS and then with 4% formalin. Selected organs and tissues (stomach, esophagus, brain, heart, kidney, liver, lung, pancreas, spleen, duodenum, ileum, colon, uterus, and ovaries) were dissected, fixed in 4% formalin, stored at 4° C., and finally embedded in paraffin. Three micrometer tissue sections were cut from each FFPE (formalin-fixed paraffin-embedded) specimen and mounted on adhesive slides (SuperFrost Ultra Plus, Thermo Fisher Scientific). After baking at 58°C for 60 min, the FFPE tissue sections were deparaffinized using xylene and rehydrated through a graded ethanol series (2x100%, 2x96%, 2x70% ethanol for 3 min each). Nuclei were stained with Mayer's hematoxyline for 5 min at RT and then blued in tap H2O. Subsequently, cytoplasm was counterstained with 0.5% eosin in water for 2 min at RT. After dehydration through a graded ethanol series and xylene, sections were mounted using the non-aqueous mounting medium X-TRA kit.

[0276] 8.2.Clinical biochemistry To test for possible organ toxicity, relevant markers of pancreatic, nephron, and hepatotoxicity were analyzed in serum samples.

[0277] Levels of alanine aminotransferase / glutamic-pyruvic transaminase (GPT), aspartic aminotransferase / glutamic-oxaloacetic transaminase (GOT), gamma-glutamyltransferase (gamma-GT), alkaline phosphatase (AP), glutamic dehydrogenase (GLDH), creatinine, creatinine kinase (CK), urea, cholinesterase, bilirubin, lipase, alpha-amylase, lactate dehydrogenase (LDH), albumin, and total protein were determined at the Universitatsmedizin der Johannes Gutenberg Universitat (Mainz, Germany). Serum samples were prepared from blood obtained after the final bleeding. Blood was collected by retrobulbar venipuncture after mice were anesthetized with ketamine / xylazine.

[0278] 8.3. Efficacy Testing To establish human xenograft tumors, an appropriate number of cells were suspended in a volume of 200 μl of PBS and injected subcutaneously into the flank of female Hsd:Athymic Nude-Foxn1nu mice. Tumor-bearing mice were treated with a single intravenous injection of IMAB362-DM4 or IMAB362-vcMMAE at a dose not exceeding the MTD. Naked antibody controls were administered twice weekly by alternating IV / ip injections of approximately 8 mg / kg IMAB362. In early treatment studies, treatment was initiated 3 days after grafting. In advanced treatment studies, tumors were grown to a size of 50 to 200 mm 3 Tumors were allowed to grow to a volume between 100 and 1400 mm2 and mice were redistributed into control and antibody groups with homogenous tumor mean volumes before treatment. Body weight, animal health, behavior, and tumor size were monitored twice weekly with calipers. Tumor volume was calculated by the following formula: [length x width x (width / 2)]. Discontinuation criteria were length or width >16 mm or tumors >1400 mm2. 3The tumor size was greater than the calculated volume of 100 μg / kg. Further discontinuation criteria were ulcerated tumors or when animals lost more than 10% body weight. In case of complete tumor growth inhibition, mice were observed for 120 days after treatment. Persistent tumors were prepared for subsequent IHC studies and fixed in 4% formalin.

[0279] 9. Antibody-dependent cellular cytotoxicity (ADCC) Antibody-dependent cellular cytotoxicity (ADCC) was determined by measuring the intracellular ATP content in non-lysed cells after addition of human PBMC to target cells in the presence of IMAB362 toxin-conjugate. Luciferase-generated bioluminescence was used to quantify ATP.

[0280] NUGC-4 10cF7_5 sort3a p3151#10 target cells were cultured at a defined cell number (8 × 10 6 Cells) were seeded to obtain reproducible confluence.

[0281] Target cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054) and cultured at 1.6 × 10 in growth medium containing 20 mM HEPES (Gibco, 15630-056). 5 The concentration of cells / ml was adjusted to 8 × 10 per well. 3 Cells were seeded into white 96-well PP-plates and incubated at 37° C. and 5% CO 2 for approximately 5 hours.

[0282] PBMCs were prepared from fresh buffy coats obtained from healthy donors. Approximately 20–25 ml of blood was diluted (1:2) with PBS in three Falcon tubes and carefully layered onto 15 ml of Ficol-Paque Plus (GE Healthcare, 17144003) in four 50 ml Falcon tubes. The gradient was centrifuged (25 min, 700 × g, w / o brake). After centrifugation, PBMCs were collected from the interphase, washed in 50 ml of PBS / 2 mM EDTA, centrifuged (5 min, 468 × g), resuspended again in 50 ml of PBS / 2 mM EDTA, and centrifuged again (10 min, 208 × g) to remove platelets. The pellet was resuspended in 50 ml of PBS / 2 mM EDTA, and cells were counted. PBMCs were subsequently centrifuged (5 min, 468 × g), resuspended in X-Vivo-15 culture medium (Lonza, BE04-418Q) containing 5% human serum, and cultured for 1.5 h at 37 °C and 5% CO2. PBMCs were harvested, centrifuged (5 min, 468 × g), and the cell concentration was adjusted to 1.28 × 10 (for an E:T ratio of 40:1). 7 Cells / ml were resuspended in X-Vivo-15 culture medium (Lonza, BE04-418Q). IMAB362-DM4, IMAB362-vcMMAE, and IMAB362 were serially diluted 11 times (4.5-fold dilution steps) resulting in a concentration range between 160 μg / ml and 0.05 ng / ml (final concentrations of 40 μg / ml to 0.01 ng / ml). 25 μl of each dilution was added to the target cells, with quadruplicates used for each condition. PBS without antibody was added to the media and total lysis control wells. Subsequently, prepared PBMCs (3.2 × 10 525 μl of 100 mM Triton X-100 / PBS was added to each well to achieve an E:T ratio of 40:1, and the plates were incubated at 37°C, 5% CO2 for 15 hours ± 1 hour. After overnight incubation, 10 μl of 8% Triton X-100 / PBS solution was added to the maximum lysis control wells and 10 μl of PBS was added to the other wells. Finally, 50 μl of freshly prepared luciferin stock solution was added to each well (160 mM HEPES, 1× PBS, 3.84 mg / ml D-luciferin (Sigma Aldrich, 50227)) and the plates were incubated in the dark at RT for 90 minutes. Bioluminescence was measured using a luminometer (Infinite M200, TECAN). Results are expressed as integrated digital relative light units (RLU).

[0283] Specific lysis:

[0284]

number

[0285] Calculate as: (Maximum viable cells: 10 μl PBS without antibody; Total lysis: 10 μl 8% (v / v) Triton X-100 in PBS without antibody).

[0286] All ADCC data were processed with GraphPad Prism 6 using the function "log(agonist) vs response - find EC anything". Maximal lysis was defined as the span of the dose-response curve (difference from top to bottom), up to 100%.

[0287] 10. Complement-dependent cytotoxicity (CDC) Complement-dependent cytotoxicity (CDC) was determined by measuring the intracellular ATP content in non-lysed cells after addition of human complement to target cells in the presence of IMAB362 toxin-conjugate. As readout, ATP-dependent bioluminescence generated by luciferase was measured.

[0288] NUGC-4 10cF7_5 sort3a p3151#10 or KATO-III FGF-BP#12 adM p3151#25 target cells were cultured at a defined cell number (8 × 10 6 and 9 x 10 6 Cells) were seeded to obtain reproducible confluence.

[0289] Target cells were harvested using 0.05% trypsin / EDTA and cultured at 1.6 × 10 in their respective culture media containing 10% (v / v) FCS. 5 The concentration of cells / ml was adjusted to 8 × 10 3 Cells were seeded in white 96-well plates and incubated at 37°C and 5% CO2. After 24 hours, 50 μl of serially diluted antibodies in assay medium (60% RPMI containing 20 mM HEPES; 40% human serum pooled from several healthy donors) were added (final concentrations 80 μg / ml to 78.13 ng / ml) and cells were incubated for 80 min at 37°C and 5% CO2. Subsequently, 10 μl of 8% (v / v) Triton X-100 in PBS was added to all lysis controls, while 10 μl of PBS was added to all other wells (maximum viable cell control and actual samples). Luciferase reactions were initiated by adding 50 μl of luciferin mix (3.84 mg / ml D-luciferin, 160 mM HEPES in ddH2O) per well. Plates were kept in the dark at RT for 90 min and bioluminescence was measured using a luminometer (Infinite M200, TECAN). Results are expressed as integrated digital relative light units (RLU).

[0290] Specific lysis,

[0291]

number

[0292] Calculate as: (Maximum viable cells: 10 μl PBS without antibody; Total lysis: 10 μl 8% (v / v) Triton X-100 in PBS without antibody).

[0293] All CDC data were processed with GraphPad Prism 6 using the function "log(agonist) vs response - find EC anything". Maximal lysis was defined as the span of the dose-response curve (difference from top to bottom), up to 100%.

[0294] Example 2 Endocytosis screening of anti-CLDN18.2 specific antibodies In tumor therapy using naked antibodies, internalization of target-bound antibodies may reduce the number of membrane-bound antibodies accessible to major mechanisms of action, such as ADCC and CDC, but endocytosis is an essential feature in the development of antibody-drug conjugates (ADCs).One important property of ADCs is the endocytosis of target-ADC complexes.Therefore, the endocytosis rate of naked antibodies is one of the essential key factors in the development of toxin-conjugated antibodies.

[0295] The binding properties, i.e., relative affinity to CLDN18.2, cross-reactivity to CLDN18.1, and antigen epitopes mediating CLDN18.2 specificity, were determined for different murine and chimeric anti-CLDN18.2 antibodies by flow cytometry analysis (Tables 5 and 6). Antibodies showing high binding to CLDN18.2 were selected for further endocytosis screening.

[0296] The endocytosis efficiency of different CLDN18.2-specific and CLDN18.2 / CLDN18.1-reactive antibodies was tested in vitro by co-incubating the antibodies with saporin-conjugated Fab fragments (Fab-ZAP) along with CLDN18.2-expressing HEK293-CLDN18.2 cells. Upon co-internalization with the target-bound antibody, saporin inhibits cellular protein biosynthesis, leading to cell death, which can be monitored by cell viability assays. This method is an indirect way to assess endocytosis of target-antibody complexes. Antibodies were tested as chimeric antibodies, if available, otherwise endocytosis screening was performed with mouse antibodies.

[0297] Chim mAb362 (IMAB362) and chim mAB294 can efficiently internalize upon binding to CLDN18.2, reducing HEK293-CLDN18.2 cell viability even at very low antibody concentrations (IMAB362: EC50 = 11 ng / ml; chim mAB294: EC50 = 10 ng / ml). In contrast, chim mAB308 and chim mAB359 did not reduce cell viability (Figure 2). Since chim mAB294 and chim mAB359, which exhibit similar relative binding affinities, show considerable discrepancy in internalization (chim mAB294: EC50 = 10 ng / ml; chim mAB359: no endocytosis), the efficiency of endocytosis appears to not only correlate with antibody binding affinity but also depend on the binding epitope.

[0298] Even internalization of mu mAB362 was superior to all other tested murine CLDN18.2-reactive antibodies, which did not show substantial endocytosis in the Fab-Zap assay (FIG. 3).

[0299] In summary, the CLDN18.2-specific antibodies IMAB362 and chim mAB294 were efficiently internalized upon binding to CLDN18.2 and are therefore suitable for further evaluation as antibody drug conjugates.

[0300] [Table 6A]

[0301] [Table 6B]

[0302] [Table 7]

[0303] Example 3 Toxin conjugation of IMAB362 Piramal Healthcare performed the toxin conjugation, including final buffer exchange. Stability studies were performed to show that the ADCs remained within specifications for a certain period of time when stored under the defined storage conditions. IMAB362 was conjugated to MMAE via a cleavable valine-citrulline linker (vc linker) or to DM4 via a cleavable N-succinimidyl-4-(2-pyridyldithio)butyrate linker (SPDB linker). IMAB362 toxin conjugates were stored in storage buffer (20 mM histidine and 85 mg / ml sucrose, pH 5.8) at 2-8°C.

[0304] [Table 8]

[0305] Both IMAB362-DM4 and IMAB362-vcMMAE show high monomer content of over 95% and only small amounts of free drug (<1%). 28-day stability studies of toxin-conjugated IMAB362 antibody at storage temperatures of 2-8° C. show only a small decrease in monomer content and a low increase in free drug for both ADCs. Both antibodies are efficiently conjugated, exhibiting drug to antibody ratios of 3.2 for IMAB362-DM4 and 4.5 for IMAB362-vcMMAE (Table 7).

[0306] Example 4 IMAB362-ADC Combination The binding properties of IMAB362 have been previously examined in detail: · IMAB362 binds to the first extracellular loop of claudin 18 splice variant 2 (CLDN18.2). · Affinity for CLDN18.2 is in the low nanomolar range. No cross-reactivity was observed with any CLDN18.2-negative cells or tissue types. No cross-reactivity with the closest family member, claudin 18 splice variant-1 (CLDN18.1).

[0307] The relative binding affinities of DM4 and MMAE conjugated IMAB362 antibodies were compared to unconjugated IMAB362 by flow cytometry using cell lines endogenously and ectopically expressing CLDN18.2. Binding properties were tested at different antibody concentrations ranging from 0.1 to 20 μg / ml (Figure 4, Table 8).

[0308] [Table 9]

[0309] Compared to unconjugated IMAB362, DM4 and MMAE-conjugated IMAB362 showed slightly reduced relative binding affinity for cells endogenously and ectopically expressing CLDN18.2 (Figure 4, Table 8). Both toxin-conjugated antibodies had very similar EC50 values ​​but slightly different maximum binding values, with IMAB362-DM4 exhibiting higher maximum binding (Table 8).

[0310] CLDN18.2-mediated binding of IMAB362-toxin conjugated antibodies was tested against cells ectopically overexpressing CLDN18.2 and the corresponding CLDN18.2-negative parental cell lines (Figure 5, Table 8).

[0311] Binding of IMAB362-DM4 and IMAB362-vcMMAE is strictly dependent on the presence of the target molecule CLDN18.2 (Figure 5). Binding specificity was analyzed by flow cytometry using HEK293 transfectants engineered to overexpress human CLDN18.2 or the highly homologous protein human CLDN18.1. HEK293-mock cells were used as negative controls (Figure 6, Table 8).

[0312] IMAB362 and the toxin-conjugated antibodies IMAB362-DM4 and IMAB362-vcMMAE bound with similar relative affinity to HEK293-CLDN18.2 cells ectopically expressing human CLDN18.2 (Table 8). Furthermore, IMAB362, DM4, and MMAE-conjugated IMAB362 showed no cross-reactivity to human CLDN18.1 or mock-transfected cells (Figures 6B and C).

[0313] Example 5 In vitro potency and specificity of IMAB362-ADC 1. Effects on cell viability The effect of IMAB362-DM4 and IMAB362-vcMMAE on cell viability was tested with several human gastric and pancreatic cancer cell lines endogenously and ectopically expressing CLDN18.2 using a colorimetric XTT-based assay for spectrophotometric quantification of metabolically active cells. Antitumor activity was tested at different antibody concentrations ranging from 3 to 16875 ng / ml (Figure 7, Table 9).

[0314] IMAB362-DM4 and IMAB362-vcMMAE efficiently inhibited the viability of gastric cancer cell lines NUGC-4, NCI-N87≈CLDN18.2, and pancreatic cell line BxPC-3≈CLDN18.2 in vitro (Figure 7). Both IMAB362-toxin conjugates inhibited cell viability at similar concentrations in NUGC-4 cells that endogenously express CLDN18.2 (EC50 values: 155-631 ng / ml, maximum reduction in viability: ≧85%), and in BxPC-3-CLDN18.2 cells that ectopically express CLDN18.2 (EC50 values: 43-54 ng / ml, maximum reduction in viability: ≧83%) and NCI-N87-CLDN18.2 cells (EC50 values: 75-180 ng / ml, maximum reduction in viability: 45-61%).

[0315] [Table 10]

[0316] Furthermore, the target-mediated antitumor activity of IMAB362-toxin conjugates was tested in vitro using NCI-N87 CLDN18.2 negative and stably transfected NCI-N87~CLDN18.2 cell lines (Figure 8). IMAB362-vcMMAE inhibited cell viability only in CLDN18.2 positive cells, but not in CLDN18.2 negative cells. Thus, the activity of IMAB362-vcMMAE is strictly dependent on CLDN18.2 expression (Figure 8).

[0317] The specificity of the toxin-conjugated IMAB362 antibody was analyzed using HEK293 transfectants overexpressing human CLDN18.2 or the highly homologous protein human CLDN18.1. HEK293 cells stably transfected with an empty vector were used as a negative control (Figure 9). IMAB362-vcMMAE reduces cell viability only for CLDN18.2-positive cells, but not for CLDN18.2-negative cells. The effect is strictly CLDN18.2-specific, since no inhibition of cell proliferation could be observed for cells expressing the homologous protein 18.1 (Figure 9).

[0318] In summary, IMAB362-vcMMAE and IMAB362-DM4 showed similar potency in vitro, and both ADCs were highly efficient in inhibiting cell viability in several human gastric and pancreatic cancer cell lines, with efficacy strictly dependent on target expression.

[0319] 2. Bystander effect Bystander activity of IMAB362-DM4 and IMAB362-vcMMAE in vitro was determined using mixed tumor cell cultures consisting of CLDN18.2 positive and negative cell lines. Cytolysis was measured using target-negative PA-1 (Luc) cells stably expressing firefly luciferase as reporter cells.

[0320] Luciferase activity in co-cultures of luciferase-expressing PA-1(Luc) and luciferase-negative NUGC-4 cells showed that treatment with IMAB362-DM4 or IMAB362-vcMMAE very effectively eliminated target-negative PA-1(Luc) cells in the presence of target-positive NUGC-4 cells. Moreover, PA-1(Luc) cells were unaffected in the absence of CLDN18.2-expressing cells (Figure 10).

[0321] In summary, IMAB362-DM4 and IMAB362-vcMMAE ADCs were able to induce bystander effects against adjacent CLDN18.2-negative tumor cells. Both toxins were efficiently released from IMAB362 in CLDN18.2-positive cancer cells and, due to their membrane permeability, could exert cytotoxic activity against bystander cells.

[0322] Example 6 Antitumor efficacy of IMAB362-ADC in vivo 1. Maximum tolerated dose test In the first in vivo study, the maximum tolerated dose (MTD) of IMAB362-DM4 and IMAB362-vcMMAE was determined in nude mice bearing advanced human BxPC-3 to CLDN18.2 pancreatic xenograft tumors. The MTD refers to the highest dose of treatment that produces the desired effect without unacceptable toxicity.

[0323] 1.1.MTD of IMAB362-DM4 BxPC-3-CLDN8.2 cells ectopically expressing human CLDN18.2 were injected subcutaneously into the flank of female Hsd:Athymic Nude-Foxn1 mice. Tumors grew to 75±13 mm on day 13. 3 After reaching an average size (mean ± SD), mice were grouped into control and antibody groups. Mice received a single dose of 7.5 or 15 mg / kg IMAB362-DM4 by IV bolus injection on day 14, or repeated doses of 15 mg / kg IMAB362-DM4 by IV bolus injection on days 14 and 21, respectively. Mice in the control group received vehicle on day 14. On day 49 after implantation, animals were sacrificed. To test for toxicity, blood samples were collected and organs were prepared and stored for further histopathological examination.

[0324] Tumor Growth: IMAB362-DM4 inhibited tumor growth in mice bearing advanced human BxPC-3 to CLDN8.2 xenograft tumors. Single or repeated treatment with IMAB362-DM4 resulted in near complete tumor regression in all treated mice over the observation period of the study (49 days), independent of dose. Thus, a single dose of 7.5 mg / kg of IMAB362-DM4 may be sufficient for complete tumor regression (Figure 11).

[0325] Health status: Body weight, animal behavior and general health were monitored twice weekly. All animals showed normal body weight throughout the experiment (Figure 12). No behavioral abnormalities were observed. However, one animal died after the second dose of 15 mg / kg IMAB362-DM4 was applied intravenously for unknown reasons.

[0326] Clinical chemistry: We determined serum levels of alanine transaminase (GPT), aspartate transaminase (GOT), glutamate dehydrogenase (GLDH), alkaline phosphatase (AP), α-amylase, cholinesterase, creatinine kinase (CK), lactate dehydrogenase (LDH), lipase, urea, glucose, total protein, and albumin. No differences were detected between vehicle and IMAB362-DM4 groups (Figure 13). Creatinine and gamma-glutamyltransferase were below the limit of detection in all groups (data not shown). All animals in all groups showed normal serum levels of tested surrogate markers for hepatotoxicity, nephrontoxicity, or pancreatic toxicity, even after repeated doses of 15 mg / kg IMAB362-DM4.

[0327] In summary, IMAB362-DM4 at 15 mg / kg as a single dose (45 mg / m in humans) 2(equivalent to ) was well tolerated in mice and showed high antitumor efficacy in treating CLDN18.2-positive xenografts. Due to concentration and injection volume limitations, intravenous injection of higher doses was not feasible and the maximum tolerated single dose could not be determined.

[0328] Tissue analysis: For tissue analysis, paraffin sections from brain, heart, kidney, liver, lung, pancreas, spleen, and stomach were stained with hematoxylin-eosin and examined microscopically for IMAB362-vcMMAE-mediated morphological changes. No morphological changes could be observed in tissue sections from IMAB362-DM4-treated animals compared to vehicle-treated mice. Notably, the stomach, the only tissue expressing mouse Cldn18.2, did not show antibody therapy-mediated tissue damage (Figure 14).

[0329] 1.2.MTD of IMAB362-vcMMAE The MTD of IMAB362-vcMMAE was tested using the same mouse model as IMAB362-DM4 (1.1). Mice were cultured until tumors reached 111±27 mm on day 13. 3 After reaching the mean size (mean ± SD), the animals were divided into groups and administered 8 or 16 mg / kg of IMAB362-vcMMAE (24 and 48 mg / m 2 Mice were treated with a single IV bolus injection of 10 mg / kg (equivalent to 100 mg / kg of IMAB362-vcMMAE) or multiple doses of 16 mg / kg IV bolus injections of IMAB362-vcMMAE on days 14 and 21. A control group of mice received vehicle control on day 14. Animals were sacrificed on day 37. Clinical biochemistry was determined and organs were collected and stored for further histopathological examination.

[0330] Tumor Growth: IMAB362-vcMMAE treatment induced tumor regression and further inhibited tumor growth in mice bearing advanced human BxPC-3 to CLDN8.2 xenograft tumors. At the end of the study (day 37), single or repeated IMAB362-vcMMAE treatment led to near complete tumor regression in all treated mice, independent of dose. Thus, a single dose of 8 mg / kg IMAB362-vcMMAE may be sufficient for complete tumor regression (Figure 15).

[0331] Health status: Body weight, animal behavior and general health were monitored twice weekly. All animals showed normal body weight throughout the experiment (Figure 16). Two animals (one mouse from the SD group and one from the RD group) were apathetic for a short time immediately after the first injection of 16 mg / kg IMAB362-vcMMAE. However, this abnormal behavior was not observed in any other animals or after the second application of IMAB362-vcMMAE.

[0332] Clinical chemistry: We determined serum levels of surrogate markers of hepatotoxicity, nephrotoxicity, or pancreatic toxicity (alanine transaminase (GPT), aspartate transaminase (GOT), glutamate dehydrogenase (GLDH), alkaline phosphatase (AP), alpha-amylase, cholinesterase, creatinine kinase (CK), lactate dehydrogenase (LDH), lipase, urea, glucose, total protein, and albumin). No major deviations of serum surrogate markers were observed in animals treated with IMAB362-vcMMAE compared to the vehicle control group (Figure 17). Creatinine and gamma-glutamyltransferase were below the detection limit in all groups. Thus, no signs of hepatotoxicity, pancreatic toxicity, or nephrotoxicity were observed in clinical biochemistry within the dose range evaluated.

[0333] Tissue analysis: For histological analysis, paraffin sections from brain, heart, kidney, liver, lung, pancreas, spleen, and stomach were stained with hematoxylin-eosin and examined microscopically for IMAB362-vcMMAE-mediated morphological changes.

[0334] No IMAB362-vcMMAE-associated morphological changes could be observed in tissue sections from IMAB362-vcMMAE-treated animals compared to vehicle-treated mice, indicating that IMAB362-vcMMAE did not induce tissue damage or inflammation. Notably, the stomach, the only tissue expressing mouse Cldn18.2, did not show antibody therapy-mediated tissue damage (Figure 22).

[0335] 2. Efficacy Testing The antitumor effects of IMAB362-DM4 and IMAB362-vcMMAE were evaluated in athymic Nude-Foxn1 mice subcutaneously implanted with human cancer cells endogenously or ectopically expressing CLDN18.2. nu Further analysis was performed in vivo in mice. The optimal therapeutic doses of IMAB362-DM4 and IMAB362-vcMMAE in animal tumor models were determined in dose range-finding studies (Figures 18 and 20, respectively). Further efficacy studies against human xenograft tumors were performed with the optimal doses of IMAB362-DM4 and IMAB362-vcMMAE (Figures 19 and 21, respectively).

[0336] IMAB362-DM4 and IMAB362-vcMMAE were administered in different early stage xenograft models (therapy started 3 days after tumor implantation) and in the treatment of advanced solid tumors (approximately 100 mm 3 In tumor-bearing mice (treatment was initiated when the tumor size was 100 μg / mL), it highly significantly inhibited tumor growth and improved survival.

[0337] Treatment of advanced human NCI-N87-CLDN18.2 gastric xenograft tumors: In a dose range finding study, the antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE was analyzed in mice bearing advanced CLDN18.2 positive NCI-N87 to CLDN18.2 xenograft tumors. Thirteen days after implantation, animals were treated with 15.2, 7.6, or 3.8 mg / kg of IMAB362-DM4, or 16, 8, or 4 mg / kg of IMAB362-vcMMAE, or vehicle control administered as a single IV bolus injection. Animals from the control group received 8 mg / kg of unconjugated IMAB362 (twice weekly, IV / ip).

[0338] IMAB362-DM4 and IMAB362-vcMMAE highly significantly inhibited tumor growth, mediated tumor regression, and extended survival in tumor-bearing mice in a dose-dependent manner, whereas IMAB362 naked antibody did not exhibit a statistically significant anti-tumor effect in this advanced treatment model (Figure 18). Both IMAB362 toxin-conjugated antibodies extended survival of tumor-bearing mice (median survival: 73 days in the vehicle group compared to 143 days in the IMAB362-vcMMAE 16 mg / kg and 136 days in the IMAB362-DM4 15.2 mg / kg groups) (Figure 18).

[0339] Treatment of early stage human NUGC-4 10cF7-5 sort3a gastric xenograft tumors: The antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE was analyzed in mice implanted subcutaneously with NUGC-4 10cF7-5 sort3a gastric cancer cells that endogenously express CLDN18.2. Animals were treated 3 days after implantation with a single dose IV injection of 15.2 mg / kg IMAB362-DM4, 16 mg / kg IMAB362-vcMMAE, or vehicle control.

[0340] IMAB362-DM4 and IMAB362-vcMMAE prevented tumor growth in treated animals, while all mice in the control group developed tumors (p<0.0001) (Figure 19). After the predetermined observation period of 120 days, 9 of 10 mice receiving IMAB362-DM4 or IMAB362-vcMMAE were alive and tumor-free, while all animals from the vehicle control group had to be euthanized due to the latest discontinuation criterion of 41 days post-implantation (median survival 34 days, p<0.0003) (Figure 19).

[0341] Treatment of advanced human BxPC-3 to CLDN18.2 pancreatic xenograft tumors: The dose-dependent antitumor activity of IMAB362-DM4 and IMAB362-vcMMAE in vivo was analyzed in a dose-range-finding study in mice bearing advanced human BxPC-3 to CLDN18.2 pancreatic xenograft tumors. Animals were treated on day 14 with 15.2, 7.6, or 3.8 mg / kg IMAB362-DM4, 16, 8, or 4 mg / kg IMAB362-vcMMAE administered as a single bolus IV injection, vehicle, or with a repeat dose of 8 mg / kg IMAB362 (twice weekly, IV / ip).

[0342] IMAB362-DM4 and IMAB362-vcMMAE highly significantly inhibited tumor growth, mediated tumor regression, and extended survival in tumor-bearing mice in a dose-dependent manner. In contrast, unconjugated IMAB362 did not exhibit statistically significant antitumor activity in this advanced tumor model (Figure 20). Both IMAB362 toxin-conjugated antibodies highly significantly extended survival of tumor-bearing mice (median survival: 48 days in the vehicle group compared to 98.5 days in the IMAB362-vcMMAE 16 mg / kg group and 81 days in the IMAB362-DM4 15.2 mg / kg group) (Figure 20).

[0343] Treatment of early stage human DAN-G 1C5F2 pancreatic xenograft tumors: The antitumor activity of IMAB362-DM4 and IMAB362-vcMMAE in vivo was tested in mice subcutaneously implanted with DAN-G 1C5F2 pancreatic cancer cells that endogenously express CLDN18.2. DAN-G 1C5F2 cells have very low amounts of CLDN18.2 on the cell surface, but substantial amounts of protein or RNA can be detected by immunoblot or qRT-PCR. IHC analysis of DAN-G 1C5F2 xenograft tumors demonstrated that only a subpopulation of tumor cells exhibited moderate to strong membrane-associated CLDN18.2 staining. Thus, DAN-G 1C5F2 xenograft tumors may be amenable to treatment with antibody-drug conjugates that exhibit bystander killing. Animals were treated on day 3 post-implantation with a single dose IV injection of 15.2 mg / kg IMAB362-DM4, 16 mg / kg IMAB362-vcMMAE, or vehicle control.

[0344] IMAB362-DM4 and IMAB362-vcMMAE highly significantly inhibited tumor growth and extended survival in tumor-bearing mice compared to vehicle controls (Figure 21). In the majority of mice (>50%), tumor growth was completely prevented. After a 120-day observation period, 2 of 7 animals in the IMAB362-DM4 (median survival 87 days, p=0.0002) and 4 of 7 animals in the IMAB362-vcMMAE treatment group were still alive (survival undetermined, p=0.0006), whereas all animals in the vehicle group had to be euthanized within 31 days due to discontinuation criteria such as cancer cachexia (median survival 24 days) (Figure 21). Both IMAB362-DM4 and IMAB362-vcMMAE significantly inhibited tumor growth and prolonged survival of mice bearing xenograft tumors and exhibiting heterologous CLDN18.2 expression.

[0345] In summary, tumors with low and / or heterologous expression of CLDN18.2 (e.g., NUGC-4 and DAN-G xenograft tumors) could be efficiently treated with IMAB362-DM4 or IMAB362-vcMMAE, and the majority of tumor-bearing animals were cured. The antitumor activity of both ADCs can be explained on the basis of the bystander effect: the release of cell membrane-permeable forms of DM4 and MMAE after cell processing promotes the killing of adjacent tumor cells, even if these are target-negative. Thus, both ADCs are highly effective in eradicating tumors that contain only a portion of CLDN18.2-positive cells.

[0346] Example 7 Induction of apoptosis The cytotoxicity of the toxin conjugate IMAB362 was evaluated by apoptosis assays measuring caspase 3 / 7 activity and externalization of phosphatidylserine. Caspase activation represents one of the earliest measurable markers of apoptosis, which is important for the initiation of programmed cell death (Henkart, 1996). Caspase 3 / 7 activity was determined in a luciferase-based assay by cleavage of a caspase 3 / 7-specific pro-luminogenic substrate. Another early event in apoptosis was monitored by flow cytometry using fluorescently conjugated annexin V (Vermes et al., 1995). Annexin V specifically binds to phosphatidylserine, which is translocated from the inner to the outer leaflet of the plasma membrane immediately after induction of apoptosis. To distinguish between live and dead cells, cells are co-stained with the DNA dye propidium iodide (PI).

[0347] To analyze the induction of apoptosis, CLDN18.2-positive NUGC4 cells were treated with a single dose of IMAB362-toxin conjugate for several days. Untreated cells and cells treated with unconjugated IMAB362 served as controls (Figure 23). After 3 days, cells treated with IMAB362-DM4 or IMAB362-vcMMAE showed increased caspase 3 / 7 activity, whereas incubation with naked antibody did not affect caspase activity (Figure 23A). Co-staining with Annexin V and PI was used as an independent parameter to verify the induction of apoptosis by the toxin-conjugated IMAB362 antibody. After 4 days of treatment, approximately 50% of cells treated with IMAB362-DM4 or IMAB362-vcMMAE were found to be Annexin V or Annexin V / PI positive, indicating that cell death occurs via the induction of apoptosis. In contrast, naked IMAB362 without cross-linking does not induce apoptosis within the applied concentration range (FIG. 23B).

[0348] In summary, treatment of CLDN18.2-positive tumor cells with IMAB362 conjugated to DM4 or vcMMAE induces apoptosis.

[0349] Example 8 Treatment of advanced human NUGC-4 10cF7-5 sort3a gastric xenograft tumors The antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE was analyzed in mice subcutaneously implanted with NUGC-4 10cF7-5 sort3a gastric cancer cells that endogenously express CLDN18.2. 3 Animals bearing 10-30% IMAB362-DM4, 16 mg / kg IMAB362-vcMMAE, or vehicle control were treated 10 days after implantation with an IV injection of 15.2 mg / kg IMAB362-DM4, 16 mg / kg IMAB362-vcMMAE, or vehicle control, and after tumor recurrence in the IMAB362 conjugate treatment group, with a second injection of the respective drug (day 38).

[0350] IMAB362-DM4 and IMAB362-vcMMAE significantly inhibited tumor growth and mediated tumor regression in all treated animals (day 52), whereas all mice in the control group developed tumors (IMAB362-DM4: p<0.05; IMAB362-vcMMAE: p<0.001) (FIG. 24). After 28 days of therapy (day 38 post-implantation), there was recurrent tumor growth (tumor size ≧100 mm 3 ) was observed in 50% of the animals in the IMAB362-DM4 treatment group. Again, a second injection of each of IMAB362-DM4 and IMAB362-vcMMAE resulted in partial or complete regression of the tumors. Recurrent tumor growth was eventually observed in 7 of 8 animals in the IMAB362-DM4 group and 4 of 8 animals in the IMAB362-vcMMAE group. After the designated time point for the end of treatment (108 days post-graft), 4 of 8 animals in the IMAB362-vcMMAE group and 1 animal in the IMAB362-DM4 and IMAB362 groups were alive, while all animals in the vehicle group died at the latest 52 days post-graft (median survival 32.5 days for vehicle, 90 days for IMAB362-DM4 (p<0.0003 vs. vehicle), and undetermined for IMAB362-vcMMAE (p<0.0003 vs. vehicle)) (Figure 24).

[0351] Example 9 Induction of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) Antibody-dependent cellular cytotoxicity (ADCC): The ADCC activity of DM4 and MMAE conjugated IMAB362 antibodies was compared to unconjugated IMAB262 using NUGC-4 10cF7_5 sort3a p3151#10 human gastric cancer cells, which endogenously express CLDN18.2 (Figure 25, Table 10).

[0352] [Table 11]

[0353] Both toxin-conjugated antibodies, IMAB362-DM4 and IMAB362-vcMMAE, exhibited similar EC50 and maximum lysis values ​​compared to the unconjugated antibody IMAB362, indicating that ADCC activity is retained following drug conjugation.

[0354] Complement-dependent cytotoxicity (CDC): The CDC activity of IMAB362-DM4 and IMAB362-vcMMAE was analyzed on NUGC-4 10cF7_5 sort3A p3151#10 and KATO-III FGF-BP#12 adM p3151#25 human gastric cancer cells that endogenously express CLDN18.2 (Figure 26, Table 11).

[0355] [Table 12]

[0356] CDC activity was not affected by conjugation of the toxin to the antibody IMAB362. Both toxin-conjugated antibodies, IMAB362-DM4 and IMAB362-vcMMAE, exhibited at least similar EC50 and maximum lysis values ​​compared to the unconjugated antibody IMAB362.

[0357] Thus, IMAB362-DM4 and IMAB362-vcMMAE combine toxin-mediated cytotoxicity with the primary mechanisms of action of unconjugated IMAB362, antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, thereby improving overall therapeutic activity.

[0358] [Table 13]

[0359] New International Patent Applications Ganymed Pharmaceuticals AG et al. "Combination therapy including antibodies against claudin 18.2" Our reference: 342-84 PCT Additional sheets for biological materials Further deposit identification: 1) Name and address of the depositary institution for the deposited materials (DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748): DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Mascheroder Weg 1b 38124 Braunschweig DE 2) Name and address of the depository institution for the deposited materials (DSM ACC2808, DSM ACC2809, DSM ACC2810): DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Inhoffenstr. 7 B 38124 Braunschweig DE

[0360] [Table 14]

[0361] Additional notice for all above mentioned deposits: - Mouse (Mus musculus) myeloma P3X63Ag8U.1 fused to mouse (Mus musculus) splenocytes - Hybridoma secreting antibody against human claudin-18A2 3) Depositor: All of the above mentioned deposits were made by: Ganymed Pharmaceuticals AG Freiligrathstrasse 12 55131 Mainz DE

[0362] [Deposit Certificate] TIFF2024036444000022.tif239170TIFF2024036444000023.tif236170TIFF2024036444000024.tif236170TIFF2024036444000025.tif235170TIFF2024036444000026.tif235170TIFF2024036444000027.tif235170TIFF2024036444000028.tif235170TIFF2024036444000029.tif235170TIFF2024036444000030.tif235170TIFF2024036444000031.tif234170TIFF2024036444000032.tif233170TIFF2024036444000033.tif228170TIFF2024036444000034.tif228170TIFF2024036444000035.tif228170TIFF2024036444000036.tif133152TIFF2024036444000037.tif132146TIFF2024036444000038.tif133146TIFF2024036444000039.tif133147TIFF2024036444000040.tif133147TIFF2024036444000041.tif133147TIFF2024036444000042.tif133147TIFF2024036444000043.tif133147TIFF2024036444000044.tif133146TIFF2024036444000045.tif133147TIFF2024036444000046.tif133147TIFF2024036444000047.tif133147TIFF2024036444000048.tif133146TIFF2024036444000049.tif132146

Claims

1. The use of an antibody conjugate comprising an antibody capable of binding to CLDN18.2 covalently attached to a therapeutic portion for the manufacture of a pharmaceutical product for treating or preventing CLDN18.2-expressing cancer in cancer patients, The antibody comprises a heavy chain variable region (VH) including VH CDR1 at positions 45-52 of SEQ ID NO: 17, VH CDR2 at positions 70-77 of SEQ ID NO: 17, and VH CDR3 at positions 116-126 of SEQ ID NO: 17, and a light chain variable region (VL) including VL CDR1 at positions 47-58 of SEQ ID NO: 24, VL CDR2 at positions 76-78 of SEQ ID NO: 24, and VL CDR3 at positions 115-123 of SEQ ID NO:

24. The antibody conjugate is used in which the antibody conjugate is internally transported into cells that express CLDN18.2 on its surface.

2. The use according to claim 1, wherein the antibody having the ability to bind to CLDN18.2 specifically binds to CLDN18.

2.

3. The use according to claim 1 or 2, wherein the antibody capable of binding to CLDN18.2 is a monoclonal antibody or its antigen-binding fragment, a bispecific antibody, a humanized or chimeric antibody.

4. The use according to claim 3, wherein the heavy chain of the antibody comprises the antibody heavy chain sequence of SEQ ID NO: 17, and the light chain of the antibody comprises the antibody light chain sequence of SEQ ID NO:

24.

5. The use according to claim 3, wherein the heavy chain of the antibody comprises the antibody heavy chain sequence of SEQ ID NO: 17, and the light chain of the antibody comprises the antibody light chain sequence of SEQ ID NO: 24, with the N-terminal 17, 18, 19, 20, 21, 22, or 23 amino acids removed.

6. The use according to any one of claims 1 to 5, wherein the antibody comprises a human heavy chain constant region selected from the group consisting of IgG1 and IgG3.

7. The use according to any one of claims 1 to 5, wherein the antibody comprises a human kappa light chain constant region and a human IgG1 heavy chain constant region.

8. The use according to claim 7, wherein the human kappa light chain constant region is allotype Km(3) and / or the human IgG1 heavy chain constant region is allotype G1m(3).

9. The use according to any one of claims 1 to 8, wherein the treatment area is permeable to cell membranes.

10. The use according to any one of claims 1 to 9, wherein the therapeutic portion is a cytotoxic agent or a cell proliferation inhibitor.

11. The use according to any one of claims 1 to 10, wherein the therapeutic portion is meitansinoid or auristatin.

12. The use according to claim 11, wherein the maytansinoid is selected from the group consisting of DM1 and DM4.

13. The use according to claim 11, wherein auristatin is selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

14. The use according to any one of claims 1 to 13, wherein an antibody capable of binding to CLDN18.2 is covalently attached to the therapeutic portion by a linker.

15. The use according to claim 14, wherein the linker is a severable linker.

16. The use according to claim 14 or 15, wherein the linker is cleavable under intracellular conditions.

17. The use according to any one of claims 14 to 16, wherein the linker is hydrolyzable at a pH of less than 5.

5.

18. The use according to any one of claims 14 to 17, wherein the linker is cleavable by an intracellular protease.

19. The use according to any one of claims 14 to 18, wherein the linker is a cathepsin-cleavable linker.

20. The use according to any one of claims 14 to 19, wherein the linker comprises a dipeptide.

21. The use according to claim 20, wherein the dipeptide is val-cit or phe-lys.

22. The use according to any one of claims 1 to 21, wherein the antibody conjugate is administered in an amount effective for the treatment or prevention of CLDN18.2-expressing cancer.

23. The use according to any one of claims 1 to 22, wherein the antibody conjugate is administered in a dose between 3 and 30 mg / kg of body weight.

24. The antibody conjugate is administered at a dose of 9-90 mg per 1 m² of the human patient's body surface. 2 The use according to any one of claims 1 to 22, administered in a dose between [a certain value].

25. The use according to any one of claims 1 to 24, wherein a single dose of antibody conjugate or two or more doses of antibody conjugate are administered.

26. The use according to any one of claims 1 to 25, wherein the antibody conjugate is administered by intravenous injection.

27. The use according to any one of claims 1 to 26, wherein the pharmaceutical is formulated for further administration of surgery, chemotherapy, and / or radiotherapy.

28. The use according to any one of claims 1 to 27, wherein the cancer is adenocarcinoma, in particular advanced adenocarcinoma.

29. The use according to any one of claims 1 to 28, wherein 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, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof, Krukenberg tumor, peritoneal metastases, and / or lymph node metastases.

30. The use according to any one of claims 1 to 29, wherein the cancer is selected from the group consisting of gastric cancer, esophageal cancer, particularly lower esophageal cancer, esophagogastric junction cancer, and gastroesophageal cancer.

31. The use according to any one of claims 1 to 30, wherein the patient is a HER2 / neu-negative patient or a patient who is HER2 / neu-positive but is not eligible for trastuzumab therapy.

32. The use according to any one of claims 1 to 31, wherein CLDN18.2 has the amino acid sequence according to SEQ ID NO:

1.

33. An antibody conjugate comprising an antibody having the ability to bind to CLDN18.2 covalently attached to the treatment site, The antibody comprises a heavy chain variable region (VH) including VH CDR1 at positions 45-52 of SEQ ID NO: 17, VH CDR2 at positions 70-77 of SEQ ID NO: 17, and VH CDR3 at positions 116-126 of SEQ ID NO: 17, and a light chain variable region (VL) including VL CDR1 at positions 47-58 of SEQ ID NO: 24, VL CDR2 at positions 76-78 of SEQ ID NO: 24, and VL CDR3 at positions 115-123 of SEQ ID NO:

24. An antibody conjugate in which the antibody conjugate is internally transported into cells expressing CLDN18.2 on its surface.

34. The antibody conjugate according to claim 33, wherein the antibody having the ability to bind to CLDN18.2 specifically binds to CLDN18.

2.

35. The antibody conjugate according to claim 33 or 34, wherein the antibody capable of binding to CLDN18.2 is a monoclonal antibody or its antigen-binding fragment, a bispecific antibody, a humanized or chimeric antibody.

36. The antibody conjugate according to claim 35, wherein the heavy chain of the antibody comprises the antibody heavy chain sequence of SEQ ID NO: 17, and the light chain of the antibody comprises the antibody light chain sequence of SEQ ID NO:

24.

37. The antibody conjugate according to claim 36, wherein the heavy chain of the antibody comprises the antibody heavy chain sequence of SEQ ID NO: 17, and the light chain of the antibody comprises the antibody light chain sequence of SEQ ID NO: 24, with the N-terminal amino acids 17, 18, 19, 20, 21, 22, or 23 removed.

38. The antibody conjugate according to any one of claims 33 to 37, wherein the antibody comprises a human heavy chain constant region selected from the group consisting of IgG1 and IgG3.

39. The antibody conjugate according to any one of claims 33 to 37, wherein the antibody comprises a human kappa light chain constant region and a human IgG1 heavy chain constant region.

40. The antibody conjugate according to claim 39, wherein the human kappa light chain constant region is allotype Km(3) and / or the human IgG1 heavy chain constant region is allotype G1m(3).

41. The antibody conjugate according to any one of claims 33 to 40, wherein the therapeutic portion is permeable to the cell membrane.

42. The antibody conjugate according to any one of claims 33 to 41, wherein the therapeutic portion is a cytotoxic agent or a cell proliferation inhibitor.

43. The antibody conjugate according to any one of claims 33 to 42, wherein the therapeutic portion is a meitansinoid or auristatin.

44. The antibody conjugate according to claim 43, wherein the maytansinoid is selected from the group consisting of DM1 and DM4.

45. The antibody conjugate according to claim 44, wherein the auristatin is selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

46. The antibody conjugate according to any one of claims 33 to 45, wherein an antibody having the ability to bind to CLDN18.2 is covalently attached to the therapeutic portion by a linker.

47. The antibody conjugate according to claim 46, wherein the linker is a cleavable linker.

48. The antibody conjugate according to claim 46 or 47, wherein the linker is cleavable under intracellular conditions.

49. The antibody conjugate according to any one of claims 46 to 48, wherein the linker is hydrolyzable at a pH of less than 5.

5.

50. The antibody conjugate according to any one of claims 46 to 49, wherein the linker is cleavable by an intracellular protease.

51. The antibody conjugate according to any one of claims 46 to 50, wherein the linker is a cathepsin-cleavable linker.

52. The antibody conjugate according to any one of claims 46 to 51, wherein the linker comprises a dipeptide.

53. The antibody conjugate according to claim 52, wherein the dipeptide is val-cit or phe-lys.

54. A pharmaceutical formulation comprising an antibody conjugate according to any one of claims 33 to 53, and a pharmaceutically acceptable diluent, carrier, or excipient.

55. A pharmaceutical preparation comprising an antibody conjugate according to any one of claims 33 to 53.

56. The pharmaceutical preparation according to claim 55, which exists in the form of a kit comprising a container containing an antibody conjugate.

57. Use of an antibody conjugate according to any one of claims 33 to 53, a pharmaceutical formulation according to claim 54, or a pharmaceutical preparation according to claim 55 or 56 for the manufacture of a pharmaceutical for use in the treatment or prevention of CLDN18.2-expressing cancer.

58. The use according to claim 57, wherein the pharmaceutical preparation further comprises printed instructions for the use of the formulation in the treatment or prevention of CLDN18.2-expressing cancer.

59. The use according to claim 57 or 58, wherein the pharmaceutical preparation is formulated to treat or prevent cancer.

Images