RU2841168C2 - Drug conjugates containing anti-claudin 18.2 antibodies - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: present invention relates to biotechnology and can be used in medicine. Invention discloses conjugates of an anti-CLDN18.2 antibody with a drug, specifically a cytotoxic or cytostatic agent.

EFFECT: invention can be used for treating or preventing oncological diseases associated with cells expressing CLDN18.2, including stomach cancer, oesophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gall bladder cancer and their metastases.

31 cl, 9 ex, 11 tbl, 26 dwg

Description

Monoclonal antibodies (mAB) have revolutionized cancer treatment over the past two decades (Sliwkowski, M. X. et al. (2013) Science 341 (6151), 1192-1198). A critical feature of mAB is their high specificity and ability to target tumor cells, marking them for immune effector-mediated cell killing (complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC)) and/or leading to decreased proliferation and apoptosis (Kubota, T. et al. (2009) Cancer Sci. 100 (9), 1566-1572). Conjugation with cytotoxic drugs may expand the utility of mABs and enhance their activity and efficacy (Goldmacher, V.S. et al. (2011) Ther. Deliv. 2 (3), 397-416; Sievers, E. L. (2013) Annu. Rev. Med. 64, 15-29).

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

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 for an ADC include the antibody, the target tumor antigen, the cytotoxic drug, and the linker (Panowksi, S. et al. (2014) MAbs 6 (1), 34-45).

As a basic prerequisite for the development of ADCs, the tumor target antigen should be localized on the cell surface and accessible to the circulating antibody. In addition, tumor selectivity and the expression level of the target antigen are critical parameters for the development of safe and effective ADCs. Currently, various tumor-associated cell surface antigens are being evaluated as ADC targets for the treatment of cancer (Trail, P. A. (2013) Antibodies 2 (1), 113-129; Teicher, B. A. (2009) Curr. Cancer Drug Targets 9 (8), 982-1004).

The efficacy of ADCs also depends on the cytotoxic drug. Since the amount of antibody that localizes to the tumor is very small compared to the administered dose, toxic compounds with subnanomolar potency are required. Auristatins and maytansinoids are two classes of highly potent cytotoxins currently used in ADC development (Trail, P. A. (2013) Antibodies 2 (1), 113-129). Both are antimitotic agents that block tubulin polymerization, causing cell death by cell cycle arrest in the G2/M phase (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 an important element in ADC design. The linker must be stable to exploit the pharmacokinetic half-life of the mAB and should not release the cytotoxic drug prior to antigen-mediated internalization. Linkers can be classified by 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 by degradation of the mAB in lysosomes after internalization (Dosio, F. et al. (2011) Toxins (Basel) 3 (7), pp. 848-883).

Depending on the linker arrangement, membrane-permeable (lipophilic) toxins that are released within target-positive cells can cross the cell membrane and kill other nearby cells, including neighboring malignant tumor cells that do not express the antigen (the "bystander effect", non-specific cytolysis) (Kovtun, Y.V. et al. (2006) Cancer Res. 66 (6), 3214-3221). The ability of these cytotoxic drugs to mediate local non-specific cytolysis is an important selection criterion for those ADCs that are directed against antigens heterogeneously expressed in tumors.

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 containing four transmembrane domains with two small extracellular loops (loop 1 spanned by hydrophobic region 1 and hydrophobic region 2, loop 2 spanned by hydrophobic regions 3 and 4). CLDN18.2 is a highly selective gastric-derived antigen exclusively expressed on short-lived differentiated gastric epithelial cells and is not detected in any other normal human tissue. The antigen is ectopically expressed at significant levels in a variety of human malignancies, including gastroesophageal cancer and pancreatic cancer (Sahin, U., et al., Clin Cancer Res, 2008. 14 (23): p.7624-34). CLDN18.2 protein is also frequently detected in lymph node metastases from gastric cancer and in distant metastases. CLDN18.2 appears to be involved in the proliferation of CLDN18.2-positive tumor cells, since downregulation of the target by siRNA technology results in inhibition of gastric cancer cell proliferation.

IMAB362 is a chimeric IgG1 monoclonal antibody 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 member of the claudin family, including the closely related claudin 18 splice variant 1 (CLDN18.1). In mice bearing human xenografts expressing CLDN18.2, IMAB362 treatment resulted in increased survival and tumor regression. No toxicity was observed in gastric tissue when administered intravenously to relevant species because the target epitope is not accessible. However, the tumor target becomes accessible to IMAB362 during malignant transformation. IMAB362 combines four independent potent mechanisms of action: (i) antibody-dependent cellular cytotoxicity (ADCC), (ii) complement-dependent cytotoxicity (CDC), (iii) induction of apoptosis mediated by tumor surface target cross-linking, and (iv) direct inhibition of proliferation. A previous phase I study evaluated IMAB362 as a single-dose monotherapy in patients with advanced gastroesophageal cancer. This study shows that a single dose of this antibody is safe and well tolerated at a dose of up to 1000 mg/ ^m2 , as no significant differences in the AE profile or other safety parameters were observed between the dose groups (AE=adverse event). The best results in terms of antitumor activity were obtained for the 300 mg/ ^m2 and 600 mg/ ^m2 groups. This phase IIa clinical study was conducted to determine the safety, tolerability, and antitumor activity of repeated doses of IMAB362 in patients with metastatic, refractory, or recurrent advanced adenocarcinoma of the stomach or lower esophagus proven by histology.

As described above, CLDN18.2 has a restricted expression pattern in normal cells and thus appears to be an ideal target for antibody-mediated therapy of CLDN18.2-expressing malignancies. Accordingly, there is a need for a therapy directed against CLDN18.2-expressing malignant tumor cells that is capable of exerting a clinically useful cytotoxic or cytostatic effect on CLDN18.2-expressing cells, in particular without having an undesirable effect on CLDN18.2-non-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 enhance 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 chemotherapy is associated with immunosuppression. In addition, isotopically labeled agents are difficult to obtain, and patients often relapse after initial treatment with isotopically labeled agents.

The present invention demonstrates the existence of monoclonal antibodies against CLDN18.2 that can be highly efficiently internalized upon binding to CLDN18.2 on CLND18.2 expressing cells and are therefore useful for the development of ADCs. Furthermore, successful conjugation of such antibodies to the drugs DM4 and MMAE using cleavable linkers SPDB or Val-Cit (vc), respectively, is 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 or affect the viability of CLDN18.2 negative cells. Both DM4 and vcMMAE conjugates exert non-specific cytolytic effects on CLDN18.2-negative cancer cells co-cultured in vitro with CLDN18.2-positive cancer cells. Furthermore, in vivo, intravenous administration of the antibody conjugates to nude mice bearing CLDN18.2-positive gastric or pancreatic tumor xenografts results in dose-dependent inhibition of tumor growth, survival, and even complete regression of early and advanced tumors. Significant therapeutic effects are observed with a single intravenous administration of ~4-8 mg/kg; optimal therapeutic effects are achieved at 15-16 mg/kg. The maximum tolerated single dose of both conjugates cannot be determined, since the highest possible tested doses of 15.2 and 16 mg/kg do not result in hepatotoxicity or other toxic effects.

From the data presented herein, it can be concluded that anti-CLDN18.2 antibody-drug conjugates, such as those described herein, are potent drugs for the treatment of CLDN18.2-positive human carcinomas, such as gastric and pancreatic carcinomas.

The essence of the invention

The present invention generally provides a therapy for the effective treatment and/or prevention of cancers associated with CLDN18.2 expressing cells, 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, gallbladder cancer and metastases, in particular gastric cancer metastases such as Kruckenberg tumors, peritoneal metastasis and lymph node metastases. Particularly preferred cancers are adenocarcinomas of the stomach, esophagus, pancreatic ducts, bile ducts, lungs and ovary.

In one aspect, the present invention relates to a method of treating or preventing a malignant neoplasm expressing CLDN18.2, comprising administering an antibody-drug conjugate comprising an antibody capable of binding to CLDN18.2 covalently linked to at least one toxin drug moiety, to a patient with an oncological disease.

In one embodiment, the antibody-drug conjugate is internalized into cells upon binding to CLDN18.2, which is expressed by the cells.

In one embodiment, an antibody having the ability to bind to CLDN18.2 specifically binds to CLDN18.2. In one embodiment, the antibody-drug conjugate specifically binds to CLDN18.2.

In one embodiment, the antibody having the ability to bind to CLDN18.2 is a monoclonal, chimeric or humanized antibody or antibody fragment. In one embodiment, the antibody having the ability to bind to CLDN18.2 is a monoclonal antibody.

In one embodiment, an antibody having the ability to bind to CLDN18.2 binds to native epitopes of CLDN18.2 present on the surface of living cells. In one embodiment, an antibody having the ability to bind to CLDN18.2 binds to the extracellular domain of CLDN18.2. In one embodiment, an antibody having the ability to bind to CLDN18.2 binds to the first extracellular loop of CLDN18.2.

In one embodiment, the antibody having the ability of binding to CLDN18.2 is an antibody selected from the group consisting of (i) an antibody produced and/or obtainable from a clone deposited under accession number 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) an antibody that is a chimerized or humanized form of the antibody of (i), (iii) an antibody having the specificity of the antibody of (i), and (iv) an antibody comprising an antigen-binding portion or an antigen-binding site, in particular the variable region of the antibody (i) and preferably with the specificity of the antibody (i). In one embodiment, the antibody having the ability of binding to CLDN18.2 comprises 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, 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 having the ability of binding to CLDN18.2 comprises 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, 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, an antibody having the ability of binding to CLDN18.2 comprises a heavy chain comprising the 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 the 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, an antibody having the ability 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, an antibody having the ability of binding to CLDN18.2 recognizes the same or substantially the same epitope as a CLDN18.2-binding 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, 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, and/or competes with said CLDN18.2-binding antibody for binding to CLDN18.2. In one embodiment, an antibody having the ability of binding to CLDN18.2 recognizes the same or substantially 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, an antibody having the ability of binding to CLDN18.2 recognizes the same or substantially the same epitope as a CLDN18.2-binding antibody comprising a heavy chain comprising the 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 the 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, an antibody having the ability of binding to CLDN18.2 recognizes the same or substantially the same epitope as a CLDN18.2-binding antibody comprising 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, 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 an antagonist of said second antibody.

In one embodiment, the toxin portion of the drug is permeable to the cell membrane. In one embodiment, the toxin portion of the drug is a cytotoxic or cytostatic agent. In one embodiment, the toxin portion of the drug 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 monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

In one embodiment, the antibody having the ability to bind to CLDN18.2 is covalently attached to the drug-toxin moiety via 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 hydrolyzed at a pH of less than 5.5. In one embodiment, the linker is cleavable by an intracellular protease. 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 amino groups, in particular amino groups of lysine residues of the antibody.

In one embodiment, the antibody-drug conjugate is administered in an amount effective to treat or prevent a cancer that expresses CLDN18.2. In one embodiment, the antibody-drug conjugate is administered at a dose of 3 to 30 mg/kg body weight, such as 4 to 25, 5 to 20, 10 to 18, or 15 to 16 mg/kg body weight. In one embodiment, the antibody-drug conjugate is administered at a dose of 8 to 150, 9 to 100, or 9 to 90 mg/ ^m2 of body surface area of a human patient, such as 12 to 75, 15 to 60, 30 to 54, or 45 to 48 mg/m2 of body surface ^area of a human patient. In one embodiment, a single dose of the antibody-drug conjugate or two or more doses of the antibody-drug conjugate are administered. In one embodiment, the antibody-drug conjugate is administered by intravenous injection.

In one embodiment, the method of the invention further comprises performing surgery, chemotherapy and/or radiation therapy.

In one embodiment, CLDN18.2 is expressed on the cell surface of malignant tumor cells. In one embodiment, the cancer is adenocarcinoma, in particular advanced adenocarcinoma. In one embodiment, the malignancy 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, gallbladder cancer and metastases, in particular gastric cancer metastases such as Kruckenberg tumors, peritoneal metastasis and lymph node metastases. In one embodiment, the malignancy is selected from the group consisting of gastric cancer, esophageal cancer, in particular lower esophagus, gastroesophageal junction cancer and gastroesophageal cancer. In one embodiment, the patient is a HER2/neu negative patient or a HER2/neu positive patient who is not eligible for trastuzumab therapy.

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

In a further aspect, the present invention relates to an antibody-drug conjugate comprising an antibody having the ability to bind to CLDN18.2 covalently attached to at least one toxin drug moiety.

In one embodiment, the antibody-drug conjugate is internalized into cells upon binding to CLDN18.2 expressed by the cells.

In one embodiment, an antibody having the ability to bind to CLDN18.2 specifically binds to CLDN18.2. In one embodiment, the antibody-drug conjugate specifically binds to CLDN18.2.

In one embodiment, the antibody having the ability to bind to CLDN18.2 is a monoclonal, chimeric or humanized antibody or antibody fragment. In one embodiment, the antibody having the ability to bind to CLDN18.2 is a monoclonal antibody.

In one embodiment, an antibody having the ability to bind to CLDN18.2 binds to native epitopes of CLDN18.2 present on the surface of living cells. In one embodiment, an antibody having the ability to bind to CLDN18.2 binds to the extracellular domain of CLDN18.2. In one embodiment, an antibody having the ability to bind to CLDN18.2 binds to the first extracellular loop of CLDN18.2.

In one embodiment, the antibody having the ability of binding to CLDN18.2 is an antibody selected from the group consisting of (i) an antibody produced and/or obtainable from a clone deposited under accession number 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) an antibody that is a chimerized or humanized form of the antibody of (i), (iii) an antibody having the specificity of the antibody of (i), and (iv) an antibody comprising an antigen-binding portion or an antigen-binding site, in particular the variable region of the antibody (i) and preferably with the specificity of the antibody (i). In one embodiment, the antibody having the ability to bind to CLDN18.2 comprises 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, 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 having the ability to bind to CLDN18.2 comprises 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, 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, an antibody having the ability of binding to CLDN18.2 comprises a heavy chain comprising the 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 the 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, an antibody having the ability 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, an antibody having the ability of binding to CLDN18.2 recognizes the same or substantially the same epitope as a CLDN18.2-binding 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, 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, and/or competes with said CLDN18.2-binding antibody for binding to CLDN18.2. In one embodiment, an antibody having the ability of binding to CLDN18.2 recognizes the same or substantially 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, an antibody having the ability of binding to CLDN18.2 recognizes the same or substantially the same epitope as a CLDN18.2-binding antibody comprising a heavy chain comprising the 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 the 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, an antibody having the ability of binding to CLDN18.2 recognizes the same or substantially the same epitope as a CLDN18.2-binding antibody comprising 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, 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 an antagonist of said second antibody.

In one embodiment, the toxin portion of the drug is cell membrane penetrating. In one embodiment, the toxin portion of the drug is a cytotoxic or cytostatic agent. In one embodiment, the toxin portion of the drug 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 monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

In one embodiment, the antibody having the ability of binding to CLDN18.2 is covalently attached to the drug-toxin moiety via 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 hydrolyzed at a pH of less than 5.5. In one embodiment, the linker is cleavable by an intracellular protease. 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 amino groups, in particular amino groups of lysine residues of the antibody.

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

In a further aspect, the present invention relates to a pharmaceutical composition comprising an antibody-drug conjugate of the invention and a pharmaceutically acceptable diluent, carrier or excipient.

In a further aspect, the present invention relates to a medicinal product comprising an antibody-drug conjugate of the invention. In one embodiment, the medicinal product is present in the form of a kit comprising a container comprising an antibody-drug conjugate. In one embodiment, the medicinal product further comprises printed instructions for using the product in a method for treating or preventing oncological diseases, in particular a malignant neoplasm expressing CLDN18.2.

In a further aspect, the present invention provides an antibody-drug conjugate of the invention, a pharmaceutical composition of the invention or a medicinal product of the invention for use in therapy, in particular for use in a method for treating or preventing an oncological disease, in particular a CLDN18.2-expressing malignant neoplasm. In one embodiment, the method for treating or preventing an oncological disease is a method for treating or preventing a CLDN18.2-expressing malignant neoplasm according to the invention.

Other features and advantages of the present invention will be apparent from the following detailed description and claims.

Brief description of the drawings

Figure 1. Conjugation of drug and antibody.

Figure 2. Decreased viability after co-incubation of HEK293~CLDN18.2 cells with chimeric anti-CLDN18.2 mAbs and Fab-ZAP (indirect assessment of internalization)

HEK293~CLDN18.2 cells were incubated for 72 h with anti-CLDN18.2 specific antibodies and saporin-conjugated anti-human IgG Fab (human Fab-ZAP). Endocytosis of IMAB362, chim mAB294, chim mAB308, and chim mAB359 was determined indirectly by measuring cell viability. Data points (n=3 replicates) are reported as mean±SD.

Figure 3. Decreased viability after co-incubation of HEK293~CLDN18.2 cells with mouse anti-CLDN18.2 antibodies and Fab-ZAP (indirect assessment of internalization)

HEK293~CLDN18.2 cells were incubated with reactive mouse anti-CLDN18.2 antibodies and saporin-conjugated anti-mouse IgG Fab (mouse Fab-ZAP) for 72 h. Endocytosis of different anti-CLDN18.2 reactive mouse antibodies was indirectly determined by measuring cell viability.

Figure 4. Relative binding affinities of IMAB362-DM4 and IMAB362-vcMMAE to CLDN18.2-positive cells

Relative binding affinities of IMAB362-toxin conjugates compared to unconjugated IMAB362 were determined in (A) NUGC-4 10cF7-5 sort3a and (B) DAN-G 1C5F2 cells endogenously expressing CLDN18.2, (C) NCI-N87~CLDN18.2 and (D) BxPC-3~CLDN18.2 cells ectopically overexpressing CLDN18.2 by flow cytometry at antibody concentrations up to 20 μg/mL. Data points (n=2 replicates) are reported as mean±SD.

Figure 5. CLDN18.2-mediated binding of IMAB362-DM4 and IMAB362-vcMMAE

CLDN18.2-mediated binding of IMAB362-toxin conjugates was analyzed in (A) NCI-N87~CLDN18.2 cells ectopically overexpressing CLDN18.2 and (B) the corresponding CLDN18.2-negative human tumor cell line by flow cytometry with antibody concentrations up to 20 μg/mL. Data points (n=2 replicates) are reported as mean±SD.

Figure 6. 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 with antibody concentrations up to 20 μg/mL. Data points (n=2 replicates) are reported as mean±SD.

Figure 7. Effect of IMAB362-DM4 and IMAB362-vcMMAE on the viability of CLDN18.2-expressing human carcinoma cell lines

Dose-response curves of IMAB362-DM4- and IMAB362-vcMMAE-mediated reduction in viability of (A) NUGC-4 10cF7-5 sort 3a, (B) NCI-N87~CLDN18.2, and (C) BxPC-3~CLDN18.2 cells. IMAB362 was used as a negative control (no effect in viability assays under these conditions). Cells were incubated for 72 h in the presence of antibodies at concentrations up to 16,875 ng/mL. Reduction in cell viability was measured using an XTT-based viability assay. Data points (n=3 replicates) are reported as mean±SD.

Figure 8. Dependence of CLDN18.2 on IMAB362-vcMMAE-mediated reduction in tumor cell viability

The target dependence of IMAB362-vcMMAE-mediated reduction in cell viability was determined in NCI-N87 (CLDN18.2 negative) and NCI-N87-CLDN18.2 cells ectopically expressing the target. Cells were incubated for 72 h with IMAB362-vcMMAE or unconjugated IMAB362 at concentrations up to 16,875 ng/mL. IMAB362 is known to be inactive under the experimental conditions used here. Reduction in cell viability was measured using an XTT-based viability assay. Data points (n=3 replicates) are reported as mean±SD.

Figure 9. Specificity of IMAB362-vcMMAE-mediated reduction in cell viability

The target specificity of IMAB362-vcMMAE-mediated reduction in cell viability was tested in stably transfected HEK293~CLDN18.2, HEK293~CLDN18.1, and HEK293~mock cells. Cells were incubated for 72 h in the presence of IMAB362-vcMMAEat concentrations up to 16,875 ng/mL. Reduction in cell viability was measured using an XTT-based viability assay. Data points (n=3 replicates) are reported as mean±SD.

Figure 10. Bystander effect activities in IMAB362-DM4 and IMAB362-vcMMAE

IMBA362-DM4- and IMAB362-vcMMAE-mediated induction of bystander effects was determined in co-culture experiments using PA-1(Luc) (CLX18.2-positive/luciferase) and NUGC-4 10cE8 (CLDN18.2-positive/luciferase-negative) cells. As a 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 200 ng/ml IMAB362-DM4, 800 ng/ml IMAB362-vcMMAE, or 800 ng/ml IMAB362. Luciferase activity was measured.

Figure 11. Inhibition of growth of progressive BxPC-3~CLDN18.2 xenograft tumors by IMAB362-DM4

CLDN18.2-positive BxPC-3~CLDN18.2 cells were inoculated subcutaneously into the flank of female nude mice. On day 14, mice were divided into 4 groups and administered intravenous injections of a single dose of vehicle, 7.5 mg/kg, 15 mg/kg IMAB362-DM4, or a repeated dose of 15 mg/kg IMAB362-DM4 (days 14 and 21). Subcutaneous tumor size was measured twice a week (mean+SEM). Group size n=5. SD: single dose, RD: repeated dose.

Figure 12. Average body weight of mice treated with IMAB362-DM4

The body weight of BxPC-3~CLDN18.2 tumor-bearing nude mice treated with a single dose of vehicle control, 7.5 mg/kg or 15 mg/kg or repeated doses of 15 mg/kg IMAB362-DM4, respectively, was monitored twice a week. The body weight of the 4 groups is presented as the mean. Group size n=5.

Figure 13. Clinical chemistry parameters after single and repeated doses of IMAB362-DM4 in xenograft-bearing nude mice

Clinical chemistry of female nude mice bearing BxPC-3~CLDN18.2 tumors treated 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 was analyzed on day 49 post-engraftment. 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. Histological analysis of gastric sections from IMAB362-DM4 and vehicle-treated mice.

Mice bearing BxPC-3~CLDN18.2 xenograft tumors were treated with IMAB362-DM4. On day 49 post-tumor transplantation, mice were sacrificed and individual organs were dissected and fixed in formalin. Sections of these FFPE tissues were stained with hematoxylin and eosin and examined microscopically for morphological changes. (A, C) Gastric tissues of a representative mouse from the treatment group with the highest IMAB362-DM4 exposure (15 mg/kg IMAB362-DM4 on day 14 and day 21 post-transplantation). (B, D) Gastric tissue from a control mouse treated with vehicle only. Magnification: see scale.

Figure 15. Inhibition of growth of progressive BxPC-3~CLDN18.2 xenograft tumors by IMAB362-vcMMAE

CLDN18.2-positive BxPC-3~CLDN18.2 cells were transplanted subcutaneously into the flank of female nude mice. On day 14, mice were divided into 4 groups and injected intravenously with a single dose of vehicle, 8 mg/kg, 16 mg/kg IMAB362-vcMMAE, or a repeated 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. Average body weight of mice treated with IMAB362-vcMMAE

Body weights of tumor-bearing female nude mice treated with a single control dose of 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 4 groups are presented as means. Group size n=5.

Figure 17. Clinical chemistry parameters following single and repeated dosing of IMAB362-vcMMAE in xenograft-bearing mice

Clinical chemistry of female nude mice bearing BxPC-3~CLDN18.2 tumors intravenously treated with a single dose of vehicle, 8 mg/kg, 16 mg/kg IMAB362-vcMMAE, or a repeat dose of 16 mg/kg IMAB362-vcMMAE were analyzed on day 37 post-transplantation. 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. Dose-dependent antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE in the human NCI-N87~CLDN18.2 advanced tumor xenograft model

NCI-N87~CLDN18.2 cells ectopically expressing human CLDN18.2 were injected subcutaneously into the flank of female nude mice. On day 10 post-transplantation, mice were arranged 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 13. Another control group received repeated doses of ~8 mg/kg IMAB362 twice weekly, alternating i.v. and i.p. Tumor volumes were measured twice weekly. Animals were sacrificed when tumor volume exceeded 1400 ^mm3 or when tumors ulcerated. Statistical analysis of tumor growth was performed using the Kruskal-Wallis and post-hoc Dunn tests. Survival was analyzed using the Mantel Cox test comparing the vehicle control group with IMAB362-DM4 and IMAB362-vcMMAE, respectively. (AH) Tumor growth curves, (I, K) mean tumor growth (±SEM), and (J, L) survival graphs of mice treated with vehicle control, IMAB362 or IMAB362-DM4 or IMAB362-vcMMAE. Group size: n=11; *: p<0.05; *** p<0.001. Arrow indicates the start of treatment.

Figure 19. Antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE in an early human gastric tumor xenograft model NUGC-4 10cF7-5 sort3a

NUGC-4 10cF7-5 sort3a cells endogenously expressing CLDN18.2 were subcutaneously transplanted 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 i.v. injection. Tumor volumes were measured twice weekly. Animals were sacrificed when tumor volume exceeded 1400 ^mm3 or when tumors ulcerated, or after a predetermined observation period of 120 days. Statistical analysis of tumor growth was performed using the Kruskal-Wallis and post-hoc Dunn tests. Survival was analyzed using the Mantel-Cox test. (AC) Tumor growth curves, (D) mean tumor growth (±SEM), and (E, F) survival graphs of mice treated with vehicle control, IMAB362-DM4, or IMAB362-vcMMAE. Group size: n=10; ***: p<0.001; ****: p<0.0001. Arrow indicates the start of treatment.

Figure 20. Dose-dependent antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE in an advanced human pancreatic xenograft tumor model BxPC-3~CLDN18.2

BxPC-3~CLDN18.2 cells ectopically expressing human CLDN18.2 were transplanted subcutaneously into the flank of female nude mice. On day 13, mice were group-organized 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. Antibody control mice received ~8 mg/kg unconjugated IMAB362 twice weekly by alternating i.v. and i.p. Tumor size was measured twice weekly. Animals were sacrificed when tumor volume exceeded 1400 ^mm3 or when tumors were ulcerated. Statistical analysis of tumor growth was performed using the Kruskal-Wallis and post-hoc Dunn tests. Survival was analyzed using the Mantel Cox test comparing the vehicle control group with IMAB362-DM4 and IMAB362-vcMMAE, respectively. (AH) Tumor growth curves, (I, K) mean tumor growth (±SEM) and (J, L) survival graphs 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. Arrow indicates the start of treatment.

Figure 21. Antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE in the early human pancreatic xenograft tumor model DAN-G 1C5F2.

DAN-G 1C5F2 cells endogenously expressing CLDN18.2 were transplanted subcutaneously into the flank of female nude mice. On day 3 post-transplantation, mice were treated with a single intravenous 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% of body weight due to cancer cachexia, when tumors ulcerated, or after a predetermined observation period of 120 days. Statistical analysis of tumor growth was performed using the Kruskal-Wallis and post-hoc Dunn tests. Survival was analyzed using the Mantel-Cox test. (A-C) Tumor growth curves (D) represent plots of tumor growth (±SEM) and (E, F) survival of mice treated with vehicle control, IMAB362-DM4 or IMAB362-vcMMAE. Group size: n=10; **: p<0.01; ***: p<0.001. Arrow indicates the start of treatment.

Figure 22. Histological analysis of gastric sections from mice treated with IMAB362-vcMMAE and vehicle.

Mice bearing BxPC-3~CLDN18.2 xenograft tumors were treated with IMAB362-vcMMAE. On day 37 post-transplantation, mice were sacrificed and individual organs were dissected and formalin-fixed. FFPE sections of these tissues were stained with hematoxylin and eosin and examined microscopically for morphological changes. (A, C) Gastric tissue of a representative mouse from the treatment group with the highest IMAB362-vcMMAE exposure (16 mg/kg IMAB362-vcMMAE on day 14 and day 21 post-transplantation). (B, D) Gastric tissue of control mice treated with vehicle only. Magnification: see scale.

Figure 23. Induction of apoptosis by IMAB362-DM4 and IMAB362-vcMMAE

IMAB362-DM4- and IMAB362-vcMMAE-mediated apoptosis induction was determined by measuring caspase 3/7 activity and annexin V staining using target-positive NUGC-4 10cE8 cells. A) Caspase 3/7 activity was analyzed after cells were incubated for 3 days in the presence of 2.5 μg/ml IMAB362 antibody (n=3 replicates, mean±SD). B) Flow cytometric analysis of annexin V and propidium iodide (PI)-stained cells was performed 4 days after treatment with 2.5 μg/ml IMAB362 antibody (n=3 replicates). Untreated cells served as a control.

Figure 24. Antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE in a xenograft model of advanced human gastric tumor NUGC-4 10cF7-5

NUGC-4 10cF7-5 sort3a cells endogenously expressing CLDN18.2 were subcutaneously transplanted into the flank of female nude mice. On day 10, mice were treated with vehicle, 15.2 mg/kg IMAB362-DM4, or 16 mg/kg IMAB362-vcMMAE by a single i.v. injection. Tumor volumes were measured twice weekly. Animals were sacrificed when tumor volume exceeded 1400 mm ³ , when tumors ulcerated, or after a predetermined observation period of 120 days. Statistical analysis of tumor growth was performed using the Kruskal-Wallis and post-hoc Dunn tests. Survival was analyzed using the Mantel-Cox test. (AC) Tumor growth curves, (D) mean tumor growth score (±SEM) and (E, F) survival of mice treated with vehicle control, IMAB362-DM4 or IMAB362-vcMMAE. Group size: n=10; *: p<0.05; ***: p<0.001. Arrow indicates the start time of treatment.

Figure 25. IMAB362-DM4 and IMAB362-vcMMAE-mediated ADCC 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 ADCC on endogenously CLDN18.2-expressing NUGC-4 10cF7_5 sort3a p3151#10 gastric carcinoma cells. Experiments were performed using an effector to target ratio of ~40:1. Data points (n=4 replicates) are reported as mean±SD. B) Flow cytometric analysis of CLDN18.2 expression on NUGC-4 10cF7_5 sort3a p3151 #10 cells. Gray filled histogram: anti-CLDN18.2 (IMAB362, 50 μg/ml). Black dotted line: isotype control.

Figure 26. 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 endogenously CLDN18.2-expressing KATO-III FGF BP#12 adM p3151#25 (left) and NUGC-4 10cF7_5 sort3a p3151#10 human gastric carcinoma cells (right). Luciferase expressing target cells were incubated for 90 min with 20% human serum (healthy donor pool) and the corresponding antibodies at the indicated concentrations. Data points (n=3 replicates) are reported as mean±SD. B) Flow cytometric analysis of CLDN18.2 expression on KATO-III FGF BP#12 adM p3151#25 (left) and NUGC-4 10cF7_5 sort3a p3151#10 (right) cells. Gray filled histogram: anti-CLDN18.2 (IMAB362, 50 μg/ml). Black dotted line: isotype control.

Detailed description of the invention

Although the present invention is described in detail below, it should be understood that the invention is not limited to the specific methodologies, protocols and reagents described herein, as they may vary. Furthermore, it should be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

The elements of the present invention will now be described. These elements are listed in specific embodiments, but it should be understood that they can be combined in any way and in any number to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the present invention to only the expressly described embodiments. This description should be understood as supporting and covering embodiments that combine the expressly described embodiments with any number of the disclosed and/or preferred elements. In addition, any permutations and combinations of all of the described elements in this appendix should be considered disclosed by the description of the present application, unless the context indicates otherwise.

Preferably, the terms used in this document are defined in accordance with the description of “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, cell biology, immunology, and recombinant DNA, as explained in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, ^2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the following claims, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" shall be construed as including the stated element, integer or step or group of members, integers or steps, but not excluding any member, integer or step or group of members, integers or steps, although in some embodiments such other element, integer or step or group of members, integers or steps may be excluded, i.e., the subject matter of the invention is the inclusion of the stated member, integer or step or group of members, integers or steps. The terms "a" and "an" and "the" and similar references used in the context of the specification of the invention (especially in the context of the claims) are to be construed as embracing both the singular and the plural unless otherwise indicated herein or clearly contradicted by the context. The recitation of ranges of values herein is merely intended to serve as a shorthand way of referring individually to each distinct value included within the range. Unless otherwise indicated, each individual value is included in the description as if it were individually described herein. All methods described herein may 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., "such as") provided herein is intended merely to better illustrate the invention and does not constitute a limitation on the scope of the invention otherwise claimed. No language in the description should be construed as indicating any unreported element as essential in the practice of the invention.

Several documents are cited throughout this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether listed above or below, is hereby incorporated by reference in its entirety. Nothing contained herein should be construed as an admission that the invention may not antedate the present specification by virtue of prior invention.

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 cross the membrane 4 times, with an N- and C-termini that are both localized 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 are particularly suitable as targets for antibody-mediated immunotherapy of malignancies due to their selective expression (lack of expression in toxicity-relevant normal tissue) and localization in the plasma membrane.

As used herein, the term "CLDN" means claudin and includes CLDN18.2. Preferably, the claudin is human claudin.

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 (claudin18.2 (CLDN18.2)).

The term "CLDN18.1" preferably refers to human CLDN18.1 and in particular to a protein comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 2 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. Said first and second extracellular loops or domains preferably form an extracellular portion or domain of CLDN18.2.

CLDN18.2 is selectively expressed in normal tissues in differentiated epithelial cells of the gastric mucosa. CLDN18.2 is expressed in malignancies of various origins, such as pancreatic carcinoma, esophageal carcinoma, gastric carcinoma, bronchial carcinoma, breast carcinoma 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, gallbladder cancer and metastases, in particular gastric cancer metastases such as Kruckenberg tumors, peritoneal metastases and lymph node metastases.

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

The term "variant" according to the invention refers in particular to mutants, splice variants, conformations, isoforms, allelic variants, species and species homologues, in particular those that occur in nature. An allelic variant refers to a change in the normal sequence of a gene, the meaning of which is often unclear. Sequencing of a complete gene often identifies multiple allelic variants for a given gene. A species homologue is a nucleic acid or amino acid sequence of a species of different origin derived from a given nucleic acid sequence or amino acid sequence. The term "variant" shall cover any post-translationally modified variants and conformational variants.

According to the invention, the term "CLDN18.2-expressing malignant neoplasm" or "CLDN18.2-positive malignant neoplasm" means a malignant neoplasm comprising malignant tumor cells expressing CLDN18.2, preferably on the surface of said malignant tumor cells.

"Cell surface" is used in accordance with its normal meaning in the art and thus includes the outer surface of a cell that is accessible to binding by proteins and other molecules.

CLDN18.2 is expressed on the surface of cells if it is located on the surface of said cells and is accessible for binding to CLDN18.2-specific antibodies added to the cells.

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 outside said cell, for example, to antigen-binding molecules such as antibodies located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or a fragment thereof.

According to the invention, CLDN18.2 is not significantly expressed in a cell if the expression level is lower compared to the expression in stomach cells or stomach tissue. Preferably, the expression level is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% of the expression in stomach cells or stomach tissue or even lower. Preferably, CLDN18.2 is not significantly expressed in a cell if the expression level exceeds the expression level in non-malignant tissue other than the stomach by no more than 2-fold, preferably 1.5-fold, and preferably does not exceed the expression level in said non-malignant tissue. Preferably, CLDN18.2 is not substantially expressed in a cell if the expression level is below the detection limit and/or if the expression level is too low to allow binding by CLDN18.2-specific antibodies added to the cells.

According to the invention, CLDN18.2 is expressed in a cell if the expression level exceeds the expression level in non-malignant tissue other than the stomach, preferably more than 2-fold, preferably 10-fold, 100-fold, 1000-fold or 10,000-fold. Preferably, CLDN18.2 is expressed in a cell if the expression level is above the detection limit and/or if the expression level is high enough to allow binding to CLDN18.2-specific antibodies added to the cells. Preferably, CLDN18.2 expressed in a cell is expressed or exposed on the surface of said cell.

The term "disease" refers to an abnormal condition that affects the human body. Disease is often construed as a medical condition associated with specific signs and symptoms. Disease may be caused by factors originating from an external source, such as an infectious disease, or may be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to individual sufferers or similar problems to those in contact with the person. In this broader sense, disease sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behavior, and atypical variations in structure and function, while in other contexts and for other purposes they may be considered distinct categories. Diseases typically affect people not only physically but also emotionally, as acquiring and living with many diseases can change an individual's outlook on life and personality. According to the invention, the term "disease" includes a malignant neoplasm, in particular those forms of malignant neoplasms that are described herein. Any reference herein to a malignant neoplasm or specific forms of malignant neoplasms also includes metastases of malignant neoplasms. In a preferred embodiment, the disease to be treated according to the present application includes cells expressing CLDN18.2.

"A disease involving cells expressing CLDN18.2" or "a disease associated with cells expressing CLDN18.2" or similar expressions mean according to the invention that CLDN18.2 is expressed in cells of a diseased tissue or organ. In one embodiment, the expression of CLDN18.2 in cells of a diseased tissue or organ is increased compared to the state in the corresponding healthy tissue or organ. The 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 even more. In one embodiment, the expression is detected only in the diseased tissue, while the expression in the corresponding healthy tissue is repressed. According to the invention, diseases associated with cells expressing CLDN18.2 include oncological diseases. Furthermore, according to the invention, oncological diseases are preferably those in which malignant tumor cells express CLDN18.2.

The terms "oncological disease" or "malignant neoplasm" refer to or describe a physiological condition of an individual that is typically characterized by unregulated cell growth. Examples of malignant neoplasms include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. In particular, examples of such malignant neoplasms include malignant neoplasms of bone, blood, lung, liver, pancreas, skin, head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, carcinoma of the genital and reproductive organs, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis carcinoma, central nervous system (CNS) neoplasias, neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma. The term "malignant neoplasm" according to the invention also includes metastases of malignant neoplasms. Preferably, the "oncological disease" is characterized by cells expressing CLDN18.2, and the malignant tumor cell expresses CLDN18.2. The cell expressing CLDN18.2 is preferably a malignant tumor cell, preferably a malignant neoplasm as described herein.

According to the invention, the term "tumor" or "tumor disease" refers to the abnormal growth of cells (called neoplastic cells, tumorigenic cells or tumor cells), preferably forming a tumor or lesion. By "tumor cell" is meant an abnormal cell that grows by rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth have ceased. Tumors exhibit a partial or complete lack of structural organization and functional coordination with normal tissue and usually form a distinct mass of tissue that can be either benign, premalignant or malignant. According to the invention, "oncological disease" is preferably a "tumor disease". However, as a rule, the terms "malignant tumor" and "tumor" are used interchangeably herein.

In one embodiment, a malignant neoplasm according to the invention comprises malignant neoplasm cells expressing CLDN18.2. In one embodiment, the malignant neoplasm is CLDN18.2 positive. In one embodiment, CLDN18.2 is expressed on the cell surface. In one embodiment, at least 50%, preferably 60%, 70%, 80% or 90% of the malignant neoplasm cells are CLDN18.2 positive and/or at least 40%, preferably at least 50% of the malignant neoplasm cells are positive for CLDN18.2 surface expression. In one embodiment, at least 95% or at least 98% of the malignant neoplasm cells are CLDN18.2 positive. In one embodiment, at least 60%, at least 70%, at least 80%, or at least 90% of the malignant tumor cells are positive for CLDN18.2 surface expression.

In one embodiment, the CLDN18.2-expressing malignant neoplasm comprising malignant tumor cells expressing CLDN18.2 or a CLDN18.2-positive malignant neoplasm is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastases, Kruckenberg tumor, peritoneal metastases and/or lymph node metastasis. In one embodiment, the malignant neoplasm is an adenocarcinoma, in particular an advanced adenocarcinoma. Particularly preferred oncological diseases are adenocarcinomas of the stomach, esophagus, pancreatic ducts, bile ducts, lungs and ovary. In one embodiment, the malignant neoplasm is selected from the group consisting of gastric cancer, esophageal cancer, in particular lower esophagus, gastroesophageal junction cancer and gastroesophageal cancer. In a particularly preferred embodiment, the malignant neoplasm is gastroesophageal cancer, such as metastatic, refractory or recurrent progressive gastroesophageal cancer.

According to the invention, "carcinoma" is a malignant tumor derived from epithelial cells. This group represents the most common types of malignant neoplasms, including common forms of breast, prostate, lung, and colon cancer.

"Adenocarcinoma" is a malignancy that occurs in glandular tissue. This tissue is also part of a larger category of tissues known as epithelial tissue. Epithelial tissue includes skin, glands, and many other tissues that line the cavities and organs of the body. Epithelium is derived embryologically from the ectoderm, endoderm, and mesoderm. To be classified as an adenocarcinoma, the cells do not have to be part of a gland as long as they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while poorly differentiated ones may not. By staining the cells from the biopsy, a pathologist will determine whether the tumor is an adenocarcinoma or another type of malignancy. Adenocarcinomas can occur in many tissues of the body due to the ubiquitous nature of glands in the body. Although each gland cannot secrete the same substance, as long as it has exocrine function in the cell, it is considered glandular, so its malignant form is called adenocarcinoma. Malignant adenocarcinomas invade other tissues and often metastasize if given enough time. Ovarian adenocarcinoma is the most common type of ovarian carcinoma. It includes serous and mucinous adenocarcinoma, renal cell carcinoma, and endometrioid adenocarcinoma.

By "metastasis" is meant the spread of malignant tumor cells from the original site to another part of the body. The formation of metastases is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration through the endothelial basement membranes into the body cavity and vessels, and then, after transport by the blood, infiltration of target organs. Finally, the growth of a new tumor at the target site depends on angiogenesis. Tumor metastases often occur even after removal of the primary tumor, because tumor cells or components can remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the invention refers to "distant metastasis", which refers to metastasis distant from the primary tumor and the regional lymph node system. In one embodiment, the term "metastasis" according to the invention refers to metastasis to the lymph nodes. One particular form of metastasis that is treatable using the therapy of the invention is metastasis originating from gastric cancer as a primary site. In preferred embodiments, such gastric cancer metastasis is Kruckenberg tumors, peritoneal metastasis, and/or lymph node metastasis.

Kruckenberg tumor is an uncommon metastatic tumor of the ovary, accounting for 1% to 2% of all ovarian tumors. The prognosis of Kruckenberg tumor remains very poor, and there is no cure for Kruckenberg tumors. Kruckenberg tumor is a metastatic adenocarcinoma of the finger cell of the ovary. The stomach is the primary site in most cases of Kruckenberg tumors (70%). Carcinomas of the colon, appendix, and breast (predominantly invasive lobular carcinoma) are the most common primary sites. Rare cases of Kruckenberg tumor have been reported arising from carcinomas of the gallbladder, biliary tract, pancreas, small bowel, ampulla of Vater, cervix, and bladder/urachus. The interval between diagnosis of primary carcinoma and subsequent discovery of ovarian involvement is usually 6 months or less, but longer periods have been reported. In many cases, the primary tumor is very small and may escape detection. A history of previous gastric or other organ carcinoma can be obtained in only 20-30% of cases.

Patients with Kruckenberg tumors have a significantly higher overall mortality rate. Most patients die within 2 years (median survival, 14 months). Several studies show that the prognosis is poor when the primary tumor is identified after metastasis to the ovary is detected, and the prognosis worsens if the primary tumor remains occult.

The optimal treatment strategy for Krukenberg tumors is not clearly defined in the literature. Whether surgical resection should be performed has not yet been decided. Chemotherapy or radiotherapy do not significantly affect the prognosis of patients with Krukenberg tumors.

The term "therapeutic treatment", particularly in connection with the treatment of a malignant neoplasm, as used herein, refers to any treatment that is intended to improve the health status and/or prolong (increase) the life expectancy of a patient. Said treatment may eliminate a malignant neoplasm, reduce the size or number of tumors in a patient, stop or slow the development of a malignant neoplasm in a patient, inhibit or slow the development of a new malignant neoplasm in a patient, reduce the frequency or severity of symptoms in a patient, and/or reduce recurrence in a patient who currently suffers from or who previously had a malignant neoplasm. The (therapeutic) intervention on a malignant neoplasm may be selected from the group consisting of surgery, chemotherapy, radiation therapy, and targeted therapy.

The term "surgery" as used in this document includes the removal of tumors during surgery. This is a common treatment for malignant tumors. The surgeon may remove tumors using local excision.

As used herein, the term "chemotherapy" refers to the use of chemotherapeutic agents or combinations of chemotherapeutic agents, preferably to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. When chemotherapy is taken by mouth or injected into a vein or muscle, the drugs enter the bloodstream and can reach cancer cells throughout the body (systemic chemotherapy). When chemotherapy is placed directly into the cerebrospinal fluid, an organ, or a body cavity such as the abdomen, the drugs primarily affect cancer cells in those areas (regional chemotherapy).

The chemotherapeutic agents of the invention include cytostatic compounds and cytotoxic compounds. Conventional chemotherapeutic agents act by killing cells that divide rapidly, which is one of the main properties of most malignant tumor 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 leads to the most common side effects of chemotherapy. According to the invention, the term "chemotherapy" preferably does not include antibodies that target proteins that are abnormally expressed in malignant tumor cells (tumor antigens, such as CLDN18.2) and act by recruiting the patient's immune system to destroy tumor cells. However, antibodies that target proteins that are abnormally expressed in malignant tumor cells (tumor antigens, such as CLDN18.2) and act via a therapeutic moiety or agent conjugated to the antibody may be considered a form of chemotherapy. However, in the strict sense, the term "chemotherapy" according to the invention does not include targeted therapy.

According to the invention, the term "targeted therapy" refers to any therapy that can be used to target preferentially diseased cells, such as cancer cells, and that does not target or targets to a lesser extent unaffected cells. Targeting diseased cells preferably results in killing and/or impairing the proliferation or viability of diseased cells. Such therapy includes: i) antibodies, antibody fragments and proteins, which are either uncoated or conjugated to a therapeutic moiety, that target specific cell surface targets on diseased cells, such as tumor antigens, such as CLDN18.2 (e.g., antibodies or antibody conjugates against CLDN18.2 as described herein) 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 higher level on patients than on normal stem cells. In a specific embodiment, the agent binds specifically to a tumor antigen. Conventional chemotherapy or radiation therapy are not considered "targeted therapy", although they are often targeted to tumors. In addition, the term "antibody therapy" according to the invention preferably does not include therapy with antibodies, fragments or derivatives thereof that are conjugated to a therapeutic moiety, but simply refers to therapy with antibodies, fragments or derivatives thereof that act by recruiting the patient's immune system to kill tumor cells.

In the context of the present invention, terms such as "protection", "prevention" or "prophylaxis" refer to preventing the occurrence and/or spread of a disease in a subject and, in particular, to minimizing the likelihood that the subject will develop a disease or to delaying the development of a disease. For example, a subject at risk of cancer would be a candidate for therapy for the prevention of cancer.

By "at risk" is meant an entity that is identified as having a higher than normal chance of developing a disease, particularly a malignancy, compared to the general population. In addition, an entity that has had or currently has a disease, particularly a malignancy, is an entity that is at increased risk of developing the disease because such an entity may continue to develop the disease. Entities that currently have or have had a malignancy also have an increased risk of developing metastases from the malignancy.

The terms "individual" and "subject" are used interchangeably herein. They refer to humans, non-human primates or other mammals (e.g., mice, rats, rabbits, dogs, cats, cows, pigs, sheep, horses, or non-human primates) that may be affected by or susceptible to a disease or disorder (e.g., a cancer), but may or may not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a specific age and therefore include adults, the elderly, children, and neonates. In preferred embodiments of the present invention, the "individual" or "subject" is a "patient". The term "patient" means, according to the invention, an object to be treated, in particular a diseased object.

The term "antigen" refers to an agent, such as a protein or peptide, containing an epitope against which an immune response is directed and/or should be directed. In a preferred embodiment, the antigen is a tumor-associated antigen, such as CLDN18.2, i.e. an element of malignant tumor cells that can be obtained from the cytoplasm, cell surface and cell nucleus, in particular those antigens that are produced, preferably in large quantities, intracellularly or as surface antigens on malignant tumor cells.

In the context of the present invention, the term "tumor-associated antigen" or "tumor antigen" preferably refers to proteins that are normally specifically expressed in a limited number of tissues and/or organs or at specific stages of development and are expressed or aberrantly expressed in one or more tumor or neoplastic tissues. In the context of the present invention, a tumor-associated antigen preferably binds to the surface of a malignant tumor cell and is preferably not expressed or rarely expressed in normal tissues.

The term "epitope" refers to an antigenic determinant in a molecule, i.e., a part of the molecule that is recognized by the immune system, such as that recognized by an antibody. For example, epitopes are discrete three-dimensional sites on an antigen that are recognized by the immune system. Epitopes are typically composed of chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes differ in that binding to the former, but not to 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 from 5 to 100, preferably from 5 to 50, more preferably from 8 to 30, most preferably from 10 to 25 amino acids in length, for example, the epitope may be preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.

The term "antibody" includes a glycoprotein comprising at least two heavy (H) chains and two light chains (L) linked together by disulfide bonds, and any molecule comprising an antigen-binding portion of such a glycoprotein. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, molecules comprising binding fragments or derivatives of antibodies, including, but not limited to, single-chain antibodies such as scFv fragments and antigen-binding antibodies such as Fab and Fab', and also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding fragments and derivatives of antibodies as described herein. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists 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 regions that are more conserved, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with antigen. The constant regions of antibodies can mediate the binding of immunoglobulin 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.

As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules with a single molecular composition. A monoclonal antibody exhibits a single binding specificity and affinity. In one embodiment, monoclonal antibodies are produced by a hybridoma that includes a B cell derived from a non-human animal, such as a mouse, fused with an immortalized cell.

As used herein, the term "recombinant antibody" includes all antibodies that have been produced, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes, or a hybridoma derived therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, such as from a transfectoma, (c) antibodies isolated from a recombinant combinatorial antibody library, and (d) antibodies produced, expressed, created, or isolated by any other means that involves splicing of immunoglobulin gene sequences with other DNA sequences.

As used herein, the term "human antibody" 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 somatic mutation in vivo).

The term "humanized antibody" refers to a molecule having an antigen-binding site that is substantially derived from an immunoglobulin from a non-human species in which the remaining immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin. The antigen-binding site may either comprise the entire variable domains fused to the constant domains or only the complementarity determining regions (CDRs) grafted into the corresponding framework regions in the variable domains. The antigen-binding sites may be wild-type or may be modified by one or more amino acid substitutions, such as modified to more closely resemble human immunoglobulins. Some forms of humanized antibodies retain all of the CDR sequences (e.g., a humanized murine antibody, which contains all six CDRs from a murine antibody). Other forms have one or more CDRs that are altered relative to the parent antibody.

The term "chimeric antibody" refers to antibodies in which one portion of each of the amino acid sequences of the heavy and light chains is homologous to corresponding sequences in antibodies obtained from one species or belonging to a particular class, while the remaining segment of the chain is homologous to corresponding sequences in another species or class. Typically, the variable region of both the light and heavy chains mimics the variable regions of antibodies obtained from one mammalian species, while the constant portions are homologous to sequences of antibodies obtained from another organism. One distinct advantage of such chimeric forms is that the variable region can be conveniently obtained from currently known sources, using readily available B cells or hybridomas from non-human host organisms in combination with constant regions obtained, for example, from human cell preparations. Although the variable region has the advantage of being easy to obtain and the specificity is not affected by the source, the constant region from a human is less likely to elicit an immune response in a human when the antibody is administered than a constant region from a non-human source. However, this definition is not limited to this specific example.

The terms "antigen-binding portion" of an antibody (or simply "binding portion") or "antigen-binding fragment" of an antibody (or simply "binding fragment") or similar terms refer 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 included within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH domains; (ii) F(ab') ₂ fragments, divalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments, consisting of the VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of one arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of the VH domain; (vi) isolated complementarity determining regions (CDRs), and (vii) combinations of two or more isolated CDRs, which may optionally be joined by a synthetic linker. Furthermore, although the two regions of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant techniques, by a synthetic linker that produces a single protein chain in which the VL and VH pairs form monovalent molecules (known as single-chain Fv (scFv), see, e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1989) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody. Another example is immunoglobulin-binding domain fusion proteins comprising (i) a binding domain polypeptide that is fused to an immunoglobulin hinge 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 may be a heavy chain variable region or a light chain variable region. Binding domain-immunoglobulin fusion proteins are further described in US 2003/0118592 and US 2003/0133939. These antibody fragments are prepared by conventional methods known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

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

The term "bispecific molecule" is intended to include an agent that has two different binding specificities. For example, the molecule may bind to (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 that has more than two different binding specificities. For example, the molecule may 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. Accordingly, the term "anti-tumor antigen antibody" includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules that are directed to a tumor antigen and to other targets, such as Fc receptors on effector cells. The term "bispecific antibodies" also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but 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 the complementary domains of another chain and creating two antigen-binding sites (see, e.g., Holliger, P., et al. (1989) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123).

Antibodies can be obtained from a variety of species, including, but not limited to, mice, rats, rabbits, guinea pigs, and humans.

As used herein, the term "isotype" refers to the class of antibodies (e.g., IgM or IgG1) that are encoded by the heavy chain constant region genes.

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

The antibodies described herein include IgA, such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments of the invention, the antibody is an IgG1 antibody, more particularly an IgG1, kappa or IgG1, lambda isotype (i.e., IgG1, κ, λ), an IgG2a antibody (e.g., IgG2a, κ, λ), an IgG2b antibody (e.g., IgG2b, κ, λ), an IgG3 antibody (e.g., IgG 3, κ, λ), or an IgG4 antibody (e.g., IgG4, κ, λ).

As used herein, the term "heterologous antibody" is defined with respect to a transgenic organism producing such an antibody. This term refers to an antibody that has an amino acid sequence or a nucleic acid coding sequence corresponding to that found in an organism that is not the same as the transgenic organism, and is typically obtained from a species different from the transgenic organism.

As used herein, the term "heterohybrid antibody" refers to an antibody having light and heavy chains of different origin. For example, an antibody having a human heavy chain linked to a mouse light chain is a heterohybrid antibody.

The antibodies described herein are preferably isolated. "Isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to a tumor antigen is substantially free of antibodies that specifically bind antigens of another tumor antigen). An isolated antibody that specifically binds to an epitope, isoform, or variant of a human tumor antigen may, however, have cross-reactivity with other related antigens, such as from other species (e.g., homologues of tumor antigens). Furthermore, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies refers to antibodies having different specificities and combined in a well-defined composition or mixture.

In the context of the present invention, an antibody is capable of acting by recruiting the patient's immune system to destroy tumor cells if the antibody, in particular when bound to its target, such as a tumor antigen on an affected cell, induces immune effector functions as described herein. Preferably, said immune effector functions are directed against cells, such as malignant tumor cells, bearing a tumor antigen, such as CLDN18.2, on their surface.

The term "immune effector functions" in the context of the present invention includes any functions mediated by components of the immune system that lead, for example, to inhibition of tumor growth and/or inhibition of tumor development, including inhibition of tumor spread and metastasis. Preferably, the immune effector functions lead to the destruction of malignant tumor 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 a tumor antigen, cytolysis of cells bearing a tumor antigen and/or inhibition of proliferation of cells bearing a tumor antigen.

Antibody-dependent cell-mediated cytotoxicity

ADCC describes the ability of effector cells, particularly lymphocytes that preferentially require a target cell to be marked with an antibody, to kill cells.

ADCC occurs preferentially when antibodies bind to antigens on tumor cells and the Fc domains of the antibody interact with Fc receptors (FcRs) on the surface of immune effector cells. Several families of Fc receptors have been identified, and specific cell populations characteristically express certain Fc receptors. ADCC can be viewed as a mechanism to directly induce variable degrees of immediate tumor destruction, leading to antigen presentation and induction of tumor-induced T cell responses. Preferably, in vivo induction of ADCC will result in tumor-induced T cell and host antibody responses.

Complement-dependent cytotoxicity

CDC is another mode of cell killing that can be antibody-driven. IgM is the most efficient complement activating isotype. IgG1 and IgG3 are also very efficient at driving CDC via the classical complement activation pathway. Preferably in this cascade, the formation of antigen-antibody complexes results in the exposure of multiple C1q binding sites in close proximity to the CH2 domains of participating antibody molecules, such as IgG molecules (C1q is one of the three complement C1 subcomponents). Preferably, these exposed C1q binding sites convert the previously low-affinity C1q-IgG interaction into a high-avidity interaction that initiates a cascade of events involving a number of other complement proteins and results in the proteolytic release of the effector cell chemotactic/activating agents C3a and C5a. Preferably, the complement cascade ends with the formation of a membrane attack complex, which creates pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell.

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

As used herein, an antibody is "derived from" a particular germline sequence if the antibody is obtained from the system by immunization of an animal or by screening a library of immunoglobulin genes and wherein 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. Typically, an antibody derived from a particular germline sequence will have 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 differences from the amino acid sequence encoded by the germline immunoglobulin gene.

As used herein, the term "heteroantibodies" refers to two or more antibodies, derivatives thereof, or linked to each other with antigen-binding regions, at least two of which have different specificities. These different specificities include binding specificity to an Fc receptor on an effector cell and binding specificity to an antigen or epitope on a target cell, such as a tumor cell.

As used herein, the term "transfectoma" includes recombinant eukaryotic host cells expressing an antibody, such as CHO cells, NS/0 cells, HEK293 cells, HEK293T cells, plant cells, or fungi, including yeast cells.

The invention includes all antibodies and antibody derivatives described herein that are encompassed by the term "antibody" for purposes of the invention. The term "antibody derivative" refers to any modified form of an antibody, such as a conjugate of an antibody and another agent or antibody, or an antibody fragment.

The term "anti-tumor antigen antibody" or similar terms refers to an antibody directed to or capable of binding to a tumor antigen. The term "binding" according to the invention preferably refers to specific binding.

According to the present invention, an antibody or antibody-drug conjugate is capable of binding to a given target if it has significant affinity for said given target and binds to said given target in standard assays. "Affinity" or "binding affinity" is often measured by the equilibrium dissociation constant ( _KD ). Preferably, the term "significant affinity" refers to binding to a given target with a dissociation constant ( _KD ) of ^10-5 M or lower, ^10-6 M or lower, ^10-7 M or lower, ^10-8 M or lower, ^10-9 M or lower, ^10-10 M or lower, ^10-11 M or lower, or ^10-12 M or lower.

An antibody or antibody-drug conjugate is (substantially) incapable of binding to a target if it does not have significant affinity for said target and does not bind significantly, in particular does not bind detectably, to said target in standard assays. Preferably, the antibody or antibody-drug conjugate does not detectably bind to said target if present at a concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 μg/ml or higher. Preferably, the antibody or antibody-drug conjugate does not have significant affinity for a target if it binds to said target with a _KD that is at least 10-fold, 100-fold, ^103- fold, ^104- fold, ^105- fold or ^106- fold higher than the _KD for binding to a given target to which the antibody or antibody-drug conjugate is capable of binding. For example, if the K _D for binding of an antibody or antibody-drug conjugate to a target to which the antibody or drug conjugate is capable of binding is 10 ^-7 M, the K _D for binding to a target for which the antibody or antibody-drug conjugate does not have significant affinity will be at least 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M, or 10 ^-1 M.

An antibody or antibody-drug conjugate is specific for a predetermined target if it is capable of binding to said predetermined target, while being unable to bind to other targets, i.e., it does not have significant affinity for other targets and does not have significant binding to other targets in standard assays. According to the invention, an antibody or antibody-drug conjugate is specific for a tumor antigen, such as CLDN18.2, if it is capable of binding to a tumor antigen, but is (substantially) unable to bind to other targets. Preferably, an antibody or antibody-drug conjugate is specific for a tumor antigen if the affinity and binding to such other targets is slightly greater than the affinity or binding to proteins unbound to the tumor antigen, such as bovine serum albumin (BSA) casein, human serum albumin (HSA) or transmembrane proteins that are not a tumor antigen, such as MHC molecules or the transferrin receptor or any other said polypeptide. Preferably, an antibody or antibody-drug conjugate is specific for a predetermined target if it binds to said target with a _KD that is at least 10-fold, 100-fold, ^103- fold, ^104- fold, ^105- fold or ^106- fold lower than the _KD for binding to a target for which it is not specific. For example, if the K _D for binding of an antibody or antibody-drug conjugate to a target for which it is specific is 10 ^-7 M, the K _D for binding to a target for which it is not specific will be at least 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M, or 10 ^-1 M.

Binding of an antibody to a target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, NY (1992), and the methods described herein. Affinities can be readily determined using conventional methods such as equilibrium dialysis; with a BIAcore 2000 instrument using the general procedures specified by the manufacturer; by radioimmunoassay using radiolabeled antigen; or by other method known to one skilled 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 may change when measured under different conditions, such as salt concentration, pH. Therefore, measurements of affinity and other antigen-binding parameters, such as _KD , _IC50 , are preferably made with standardized solutions of antibody and antigen and a standardized buffer.

The term "naturally occurring" as used herein to apply 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 viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in a laboratory is naturally occurring.

As used herein, the term "rearranged" refers to a configuration of the heavy chain or light chain immunoglobulin locus wherein the V segment is positioned immediately adjacent to the D-J or J segment in a conformation encoding substantially the entire VH or VL domain, respectively. A rearranged immunoglobulin (antibody) gene locus can be identified by comparison with germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element.

The term "non-rearranged" or "germline configuration," when used herein in relation to the V segment, refers to a configuration in which the V segment is not recombined to be directly adjacent to the D or J segment.

According to the invention, an antibody having the ability to bind to CLDN18.2 is an antibody capable of binding to an epitope present in CLDN18.2, preferably an epitope located in the extracellular domains of CLDN18.2, in particular the first extracellular domain, preferably at amino acid positions 29-78 in CLDN18.2. In particular embodiments, an antibody having the ability 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 NOs: 3, 4 and 5, (ii) an epitope located in CLDN18.2 loop 1, preferably SEQ ID NO: 8, (iii) an epitope localized in CLDN18.2 loop 2, preferably SEQ ID NO: 10, (iv) an epitope localized in CLDN18.2 loop D3, preferably SEQ ID NO: 11, (v) an epitope that spans CLDN18.2 loop 1 and CLDN18.2 loop D3, or (vi) a non-glycosylated epitope localized on CLDN18.2 loop D3, preferably SEQ ID NO: 9.

According to the invention, an antibody having the ability of binding to CLDN18.2 is preferably an antibody having the ability of binding to CLDN18.2 but not to CLDN18.1. Preferably, the antibody having the ability of binding to CLDN18.2 is specific for CLDN18.2. Preferably, the antibody having the ability of binding to CLDN18.2 is preferably an antibody having the ability of binding to CLDN18.2 expressed on the surface of a cell. In preferred embodiments, the antibody having the ability of binding to CLDN18.2 binds to native epitopes of CLDN18.2 present on the surface of living cells. Preferably, the antibody having the ability 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, the antibody having the ability to bind to CLDN18.2 is specific for the above-mentioned proteins, peptides or immunogenic fragments or derivatives thereof. The antibody having the ability to bind 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, 3-11, 44, 46 and 48-50, or a nucleic acid or host cell expressing said protein or peptide. Preferably, the antibody binds to malignant tumor cells, in particular to cells of the above-mentioned types of malignant neoplasms, and preferably does not bind substantially to cells that are not malignant tumor cells.

In a particularly preferred embodiment, the antibody having the ability to bind to CLDN18.2 is obtained using a hybridoma deposited at DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) and has the following designation and accession number:

a. 182-D1106-055, accession number DSM ACC2737, deposited October 19, 2005

b. 182-D1106-056, accession number DSM ACC2740, deposited October 19, 2005

c. 182-D1106-057, accession number DSM ACC2740, deposited October 19, 2005

d. 182-D1106-058, accession number DSM ACC2740, deposited October 19, 2005

e. 182-D1106-059, accession number DSM ACC2740, deposited October 19, 2005

f. 182-D1106-062, accession number DSM ACC2742, deposited October 19, 2005,

g. 182-D1106-067, accession number DSM ACC2740, deposited October 19, 2005

h. 182-D758-035, accession number DSM ACC2745, deposited November 17, 2005.

i. 182-D758-036, accession number DSM ACC2746, deposited November 17, 2005.

j. 182-D758-040, accession number DSM ACC2747, deposited November 17, 2005.

k. 182-D1106-061, accession number DSM ACC2748, deposited November 17, 2005

l. 182-D1106-279, accession number DSM ACC2808, deposited October 26, 2006.

m. 182-D1106-294, accession number DSM ACC2809, deposited October 26, 2006,

n. 182-D1106-362, accession number DSM ACC2810, deposited October 26, 2006.

Preferred antibodies according to the invention are those produced and obtainable from the above-described hybridomas; i.e. 37G11 in case of 182-D1106-055, 37H8 in case of 182-D1106-056, 38G5 in case of 182-D1106-057, 38H3 in case of 182-D1106-058, 39F11 in case of 182-D1106-059, 43A11 in case of 182-D1106-062, 61C2 in case of 182-D1106-067, 26B5 in case of 182-D758-035, 26D12 in case of 182-D758-036, 28D10 in case of 182-D758-040, 42E12 in case 182-D1106-061, 125E1 in case of 182-D1106-279, 163E12 in case of 182-D1106-294 and 175D10 in case of 182-D1106-362; and chimerized and humanized forms thereof.

In one embodiment, the antibody having the ability of binding to CLDN18.2 is an antibody selected from the group consisting of (i) an antibody produced and/or obtainable from a clone deposited under accession number 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) an antibody that is a chimerized or humanized form of the antibody of (i), (iii) an antibody having the specificity of the antibody of (i), and (iv) an antibody comprising an antigen-binding portion or an antigen-binding site, in particular the variable region, of the antibody (i) and preferably with the specificity of the antibody (i).

Preferred antibodies, particularly chimerized antibodies, and their sequences are shown in the following table.

clone mAb Isotype Variable region Chimerized antibody Heavy chain 43A11 182-D1106-062 IgG2a SEQ ID NO:29 SEQ ID NO:14 163E12 182-D1106-294 IgG3 SEQ ID NO:30 SEQ ID NO:15 125E1 182-D1106-279 IgG2a SEQ ID NO:31 SEQ ID NO:16 166E2 182-D1106-308 IgG3 SEQ ID NO:33 SEQ ID NO:18 175D10 182-D1106-362 IgG1 SEQ ID NO:32 SEQ ID NO:17 45C1 182-D758-187 IgG2a SEQ ID NO:34 SEQ ID NO:19 light chain 43A11 182-D1106-062 IgK SEQ ID NO:36 SEQ ID NO:21 163E12 182-D1106-294 IgK SEQ ID NO:35 SEQ ID NO:20 125E1 182-D1106-279 IgK SEQ ID NO:37 SEQ ID NO:22 166E2 182-D1106-308 IgK SEQ ID NO:40 SEQ ID NO:25 175D10 182-D1106-362 IgK SEQ ID NO:39 SEQ ID NO:24 45C1 182-D758-187 IgK SEQ ID NO:38 SEQ ID NO:23 45C1 182-D758-187 IgK SEQ ID NO:41 SEQ ID NO:26 45C1 182-D758-187 IgK SEQ ID NO:42 SEQ ID NO:27 45C1 182-D758-187 IgK SEQ ID NO:43 SEQ ID NO:28

In preferred embodiments, antibodies, in particular chimerized forms of antibodies according to the invention, include antibodies 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 presented in SEQ ID NO: 13 or a fragment thereof. In other preferred embodiments, antibodies, in particular chimerized forms of antibodies according to the invention, include antibodies 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 presented in SEQ ID NO: 12 or a fragment thereof. In a particular preferred embodiment, the antibodies, in particular the chimerized forms of the antibodies according to the invention, include antibodies that comprise a CH comprising an amino acid sequence derived from a human CH, such as the amino acid sequence presented in SEQ ID NO: 13 or a fragment thereof, and comprise a CL comprising an amino acid sequence derived from a human CL, such as the amino acid sequence presented in SEQ ID NO: 12 or a fragment thereof.

In one embodiment, an antibody having the ability to bind to CLDN18.2 is a chimeric mouse/human IgG1 monoclonal antibody comprising a mouse kappa light chain variable region, a human kappa light chain constant region of the Km(3) allotype, a mouse heavy chain variable region, a human IgG1 constant region of the G1m(3) allotype.

In some preferred embodiments, chimerized forms of antibodies include antibodies comprising a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 15, 16, 17, 18, 19, 51 and a fragment thereof and/or comprising a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, 28 and a fragment thereof.

In some preferred embodiments, chimerized forms of antibodies comprise antibodies comprising a combination of heavy chains and light chains selected from the following options (i)-(ix):

(i) the heavy chain comprises the amino acid sequence presented in SEQ ID NO: 14 or a fragment thereof, and the light chain comprises the amino acid sequence presented in SEQ ID NO: 21 or a fragment thereof,

(ii) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 15 or a fragment thereof, and the light chain comprises an amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof,

(iii) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 16 or a fragment thereof, and the light chain comprises an amino acid sequence represented by SEQ ID NO: 22 or a fragment thereof,

(iv) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 18 or a fragment thereof, and the light chain comprises an amino acid sequence represented by SEQ ID NO: 25 or a fragment thereof,

(v) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 17 or a fragment thereof, and the light chain comprises an amino acid sequence represented by SEQ ID NO: 24 or a fragment thereof,

(vi) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and the light chain comprises an amino acid sequence represented by SEQ ID NO: 23 or a fragment thereof,

(vii) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and the light chain comprises an amino acid sequence represented by SEQ ID NO: 26 or a fragment thereof,

(viii) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and the light chain comprises an amino acid sequence represented by SEQ ID NO: 27 or a fragment thereof,

(ix) the heavy chain comprises an amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and the light chain comprises an amino acid sequence represented by SEQ ID NO: 28 or a fragment thereof, and

(x) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 51 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 24 or a fragment thereof.

Antibodies according to (ii), (v) or (x) are preferred embodiments of the "antibody having the ability to bind to CLDN18.2". Particularly preferred is an antibody according to (v) or (x).

"Fragment" or "fragment of an amino acid sequence" as used above refers to a portion of an antibody sequence, i.e. a sequence that represents an antibody sequence truncated at the N- and/or C-terminus, which, when it replaces said antibody sequence in an antibody, maintains the binding of said antibody to CLDN18.2. Preferably, the fragment of the amino acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98% or 99% of the amino acid residues of said amino acid sequence. A fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 15, 16, 17, 18, 19, 51, 20, 21, 22, 23, 24, 25, 26, 27 and 28, preferably refers to said sequence, wherein 17, 18, 19, 20, 21, 22 or 23 amino acids at the N-terminus are removed.

In a preferred embodiment, an antibody having the ability to bind to CLDN18.2 comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 30, 31, 32, 33, 34 and a fragment thereof.

In a preferred embodiment, an antibody having the ability to bind to CLDN18.2 comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43 and a fragment thereof.

In some preferred embodiments, an antibody having the ability to bind to CLDN18.2 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following options (i)-(ix):

(i) VH comprises the amino acid sequence represented by SEQ ID NO: 29 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 36 or a fragment thereof,

(ii) VH comprises the amino acid sequence represented by SEQ ID NO: 30 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 35 or a fragment thereof,

(iii) VH comprises the amino acid sequence represented by SEQ ID NO: 31 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 37 or a fragment thereof,

(iv) VH comprises the amino acid sequence represented by SEQ ID NO: 33 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 40 or a fragment thereof,

(v) VH comprises the amino acid sequence represented by SEQ ID NO: 32 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 39 or a fragment thereof,

(vi) VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 38 or a fragment thereof,

(vii) VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 41 or a fragment thereof,

(viii) VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 42 or a fragment thereof,

(ix) VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 43 or a fragment thereof.

Antibodies according to (ii) or (v) are preferred embodiments of the "antibody having the ability to bind to CLDN18.2". Particularly preferred is the antibody according to (v).

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

In a preferred embodiment, the antibody having the ability to bind to CLDN18.2 comprises a VH comprising a set of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following options (i)-(vi):

(i) CDR1: positions 45-52 of SEQ ID NO: 14, CDR2: positions 70-77 of SEQ ID NO: 14, CDR3: positions 116-125 of SEQ ID NO: 14,

(ii) CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: positions 70-77 of SEQ ID NO: 15, CDR3: positions 116-126 of SEQ ID NO: 15,

(iii) CDR1: positions 45-52 of SEQ ID NO: 16, CDR2: positions 70-77 of SEQ ID NO: 16, CDR3: positions 116-124 of SEQ ID NO: 16,

(iv) CDR1: positions 45-52 of SEQ ID NO: 17, CDR2: positions 70-77 of SEQ ID NO: 17, CDR3: positions 116-126 of SEQ ID NO: 17,

(v) CDR1: positions 44-51 of SEQ ID NO: 18, CDR2: positions 69-76 of SEQ ID NO: 18, CDR3: positions 115-125 of SEQ ID NO: 18 and

(vi) CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19.

In a preferred embodiment, the antibody having the ability to bind 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 47-58 of SEQ ID NO: 20, CDR2: positions 76-78 of SEQ ID NO: 20, CDR3: positions 115-123 of SEQ ID NO: 20,

(ii) CDR1: positions 49-53 of SEQ ID NO: 21, CDR2: positions 71-73 of SEQ ID NO: 21, CDR3: positions 110-118 of SEQ ID NO: 21,

(iii) CDR1: positions 47-52 of SEQ ID NO: 22, CDR2: positions 70-72 of SEQ ID NO: 22, CDR3: positions 109-117 of SEQ ID NO: 22,

(iv) CDR1: positions 47-58 of SEQ ID NO: 23, CDR2: positions 76-78 of SEQ ID NO: 23, CDR3: positions 115-123 of SEQ ID NO: 23,

(v) CDR1: positions 47-58 of SEQ ID NO: 24, CDR2: positions 76-78 of SEQ ID NO: 24, CDR3: positions 115-123 of SEQ ID NO: 24,

(vi) CDR1: positions 47-58 of SEQ ID NO: 25, CDR2: positions 76-78 of SEQ ID NO: 25, CDR3: positions 115-122 of SEQ ID NO: 25,

(vii) CDR1: positions 47-58 of SEQ ID NO: 26, CDR2: positions 76-78 of SEQ ID NO: 26, CDR3: positions 115-123 of SEQ ID NO: 26,

(viii) CDR1: positions 47-58 of SEQ ID NO: 27, CDR2: positions 76-78 of SEQ ID NO: 27, CDR3: positions 115-123 of SEQ ID NO: 27 and

(ix) CDR1: positions 47-52 of SEQ ID NO: 28, CDR2: positions 70-72 of SEQ ID NO: 28, CDR3: positions 109-117 of SEQ ID NO: 28.

In a preferred embodiment, the antibody having the ability to bind to CLDN18.2 comprises a combination of VH and VL, 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 45-52 of SEQ ID NO: 14, CDR2: positions 70-77 of SEQ ID NO: 14, CDR3: positions 116-125 of SEQ ID NO: 14, VL: CDR1: positions 49-53 of SEQ ID NO: 21, CDR2: positions 71-73 of SEQ ID NO: 21, CDR3: positions 110-118 of SEQ ID NO: 21,

(ii) VH: CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: positions 70-77 of SEQ ID NO: 15, CDR3: positions 116-126 of SEQ ID NO: 15, VL: CDR1: positions 47-58 of SEQ ID NO: 20, CDR2: positions 76-78 of SEQ ID NO: 20, CDR3: positions 115-123 of SEQ ID NO: 20,

(iii) VH: CDR1: positions 45-52 of SEQ ID NO: 16, CDR2: positions 70-77 of SEQ ID NO: 16, CDR3: positions 116-124 of SEQ ID NO: 16, VL: CDR1: positions 47-52 of SEQ ID NO: 22, CDR2: positions 70-72 of SEQ ID NO: 22, CDR3: positions 109-117 of SEQ ID NO: 22,

(iv) VH: CDR1: positions 44-51 of SEQ ID NO: 18, CDR2: positions 69-76 of SEQ ID NO: 18, CDR3: positions 115-125 of SEQ ID NO: 18, VL: CDR1: positions 47-58 of SEQ ID NO: 25, CDR2: positions 76-78 of SEQ ID NO: 25, CDR3: positions 115-122 of SEQ ID NO: 25,

(v) VH: CDR1: positions 45-52 SEQ ID NO: 17, CDR2: positions 70-77 SEQ ID NO: 17, CDR3: positions 116-126 SEQ ID NO: 17, VL: CDR1: positions 47-58 SEQ ID NO: 24, CDR2: positions 76-78 SEQ ID NO: 24, CDR3: positions 115-123 SEQ ID NO: 24,

(vi) VH: CDR1: positions 45-53 SEQ ID NO: 19, CDR2: positions 71-78 SEQ ID NO: 19, CDR3: positions 117-128 SEQ ID NO: 19, VL: CDR1: positions 47-58 SEQ ID NO: 23, CDR2: positions 76-78 SEQ ID NO: 23, CDR3: positions 115-123 SEQ ID NO: 23,

(vii) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58 of SEQ ID NO: 26, CDR2: positions 76-78 of SEQ ID NO: 26, CDR3: positions 115-123 of SEQ ID NO: 26,

(viii) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58 of SEQ ID NO: 27, CDR2: positions 76-78 of SEQ ID NO: 27, CDR3: positions 115-123 of SEQ ID NO: 27 and

(ix) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-52 of SEQ ID NO: 28, CDR2: positions 70-72 of SEQ ID NO: 28, CDR3: positions 109-117 of SEQ ID NO: 28.

Antibodies according to (ii) or (v) are preferred embodiments of the "antibody having the ability to bind to CLDN18.2". Particularly preferred is the antibody according to (v).

In other preferred embodiments, the antibody having the ability of binding to CLDN18.2 preferably comprises one or more complementarity determining regions (CDRs), preferably at least the variable region CDR3 of the variable region of the heavy chain (VH) and/or the variable region of the light chain (VL) of an anti-CLDN18.2 monoclonal antibody, preferably the anti-CLDN18.2 monoclonal antibody described herein, and preferably comprises one or more complementarity determining regions (CDRs), preferably at least the variable region CDR3 of the variable regions of the heavy chain (VH) and/or the variable regions of the light chain (VL) described herein. In one embodiment, said one or more complementarity determining regions (CDRs) are selected from the set of complementarity determining regions CDR1, CDR2 and CDR3 described herein. In a particularly preferred embodiment, the antibody having the ability of binding to CLDN18.2 preferably comprises the CDR1, CDR2 and CDR3 regions of complementarity of the variable region of the heavy chain (VH) and/or the variable region of the light chain (VL) of an anti-CLDN18.2 monoclonal antibody, preferably the anti-CLDN18.2 monoclonal antibody described herein, and preferably comprises the CDR1, CDR2 and CDR3 regions of complementarity of the variable regions of the heavy chain (VH) and/or the variable regions of the light chain (VL) described herein.

In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs as described herein comprises said CDRs together with their intervening framework regions. Preferably, this portion will also comprise at least about 50% of one or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. The engineering of antibodies produced by recombinant DNA techniques may result in the introduction of N- or C-terminal residues into the variable regions encoded by linkers introduced to facilitate cloning or other manipulations, including the introduction of linkers for attaching the variable regions of the invention to additional protein sequences, including immunoglobulin heavy chains, other variable domains (e.g., in the production of diabodies) or protein tags.

In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs as described herein comprises said CDRs in a human antibody framework.

Reference in this document to an antibody comprising, with respect to its heavy chain, a specific chain or a specific region or sequence, preferably refers to the situation where all the heavy chains of said antibody comprise said specific chain, region or sequence. This applies correspondingly to the light chain of the antibody.

In one embodiment, an antibody having the ability to bind to CLDN18.2 according to the invention refers to an antibody that recognizes, i.e. binds to the same or substantially 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.

According to the invention, an antibody having the ability to bind to CLDN18.2, in particular when it is present in an antibody-drug conjugate, preferably has an affinity and/or specificity for CLDN18.2 suitable for endocytosis of the antibody and/or the antibody-drug conjugate.

The term endocytosis refers to the process by which eukaryotic cells internalize plasma membrane segments, cell surface receptors, and components from the extracellular fluid. The mechanisms of endocytosis include receptor-mediated endocytosis. The term receptor-mediated endocytosis refers to the biological mechanism by which a ligand, upon binding to its target, triggers membrane invagination and constriction, becoming internalized and delivered into the cytosol or transported to appropriate intracellular compartments.

The present invention also provides embodiments in which "an antibody having the ability to bind to CLDN18.2" has a meaning that encompasses any "binding agent to CLDN18.2". According to the invention, "a binding agent to CLDN18.2" includes any compound that has the ability to bind to CLDN18.2. Preferably, such a binding agent comprises at least one binding domain for CLDN18.2. The term includes all artificial binding molecules (scaffolds) having the ability to bind to CLDN18.2, including, but not limited to, nanoparticles, affibody (antibody mimetic protein created by Affibody AB), anticalins, ankyrin repeat engineered proteins, monomers, avimers and microbodies. In one embodiment, said binding agent binds to the extracellular domain of CLDN18.2. In one embodiment, said binding agent binds to native epitopes of CLDN18.2 present on the surface of living cells. In one embodiment, said binding agent binds to the first extracellular loop of CLDN18.2. In one embodiment, binding to CLDN18.2 is specific binding.

The term "binding domain" characterizes in connection with the present invention a structure, for example of an antibody, which binds/interacts with a given target structure/antigen/epitope. Thus, the binding domain according to the invention denotes the "antigen interaction site".

Any agent that has a therapeutic effect on cancer cells can be used as a drug for conjugation with an anti-CLDN18.2 antibody or derivative thereof. Preferably, the drug conjugation does not change or does not substantially change the binding characteristics, in particular the specificity of the antibody discussed herein. Thus, the antibody-drug conjugate according to the invention preferably has the same or substantially the same binding characteristics, in particular the specificity, as the antibody used for the conjugation. Accordingly, if certain binding characteristics are described herein for the antibody used for the conjugation, it is preferred that the antibody-drug conjugate has the same binding characteristics. For example, if an antibody having the ability to bind to CLDN18.2 is described to bind to the extracellular domain of CLDN18.2 and/or bind 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 binds to the first extracellular loop of CLDN18.2.

Typically, the drug is a cytotoxic or cytostatic agent. A cytotoxin or cytotoxic agent includes any agent that is harmful to cells and, in particular, kills cells.

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 such as cis-platinum, mono(platinum), bis(platinum), and trinuclear platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophores, nitrosoureas, platinones, preforming compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes (e.g., paclitaxel and docetaxel), topoisomerase inhibitors, vinca alkaloids, and the like.

Individual cytotoxic agents include, for example, androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, butoquinone 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-fluorodeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, plicamycin, procarbizine, streptozotocin, tenoside, 6-thioguanine, thiotepa, topotecan, vinblastine, vincristine, vinorelbine, VP-16 and VM-26.

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 (tularic), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, camadotin, combretastatins, discodermolide, and eleutherobin.

In particular embodiments, the cytotoxic or cytostatic agent is auristatin E (also known in the art as dolastatin-10) or a derivative thereof. Typically, a derivative of auristatin E is, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with para-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical derivatives of auristatin include AFP, MMAF, and MMAE.

In some embodiments, the cytotoxic or cytostatic agent is a maytansinoid, another group of antitubulin agents. For example, in certain embodiments, the maytansinoid is maytansine, DM-1, or DM-4.

Maytansinoids are potent microtubule-targeting compounds that inhibit cell proliferation in mitosis. Maytansinoids are derivatives of maytansine, which is a 19-membered ansa-macrolide structure attached to a chlorinated benzene ring. Maytansine has the following formula:

Some microbes 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 disclosed, for example, in U.S. Pat. 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; 4,308,269; 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; 4362663; and 4371533, and Kawai et al (1984) Chem. Pharm. Bull. 3441-3451, incorporated herein by reference.

Maytansinoids are well known in the art and can be synthesized by known methods 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 bound to the carbonyl group is replaced by a group containing a free sulfhydryl group, such as the group -R-SH, wherein R is an alkylene group or other carbon-containing group of atoms.

DM1, also known as mertansine, is a maytansinoid with the following formula:

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

"DM4" refers to the compound N ^2' -deacetyl-N ^2' -(4-methyl-4-mercapto-1-oxopentyl)-maytansine.

Anti-CLDN18.2 antibody-maytansinoid conjugates can be prepared by chemically coupling 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 one toxin molecule per antibody is expected to enhance cytotoxicity compared to using unarmed antibody.

In this regard, the term "antibody covalently linked to at least one toxin drug component" includes situations where one or more molecules of the same drug are covalently attached to the antibody molecule, as well as where different drugs are covalently attached to the antibody molecule. In the latter case, one or more molecules of each of the different drugs may be attached to the antibody molecule or a combination thereof (e.g., one molecule of one drug is attached when several molecules of another drug are attached).

In some embodiments of the invention, the antibody is conjugated to dolastatins or peptide analogs and derivatives of dolastatin, auristatins (U.S. Patent Nos. 5,635,483, 5,780,588, incorporated herein by reference). Auristatins are synthetic analogs of dolastatin 10, a natural product obtained from the marine mollusk, Dolabela auricularia. Like maytansinoids, auristatins disrupt microtubules. Dolastatin or a drug moiety of auristatin can be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptide portion of the drug.

Typical auristatin variants include monomethyl auristatin drug moieties such as MMAE and MMAF, which are preferentially linked to the N-terminus.

MMAE, also known as monomethyl auristatin E, has the following formula:

Specifically, 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)butanamido)butanamide. MMAE is actually desmethyl auristatin E, meaning the N-terminal amino group has only one methyl substituent instead of the two found in auristatin E itself.

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

MMAF, also known as monomethyl auristatin 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.

The preparation of antibody-drug conjugates can be carried out by any method known to a person skilled in the art. Antibody-drug conjugates can be prepared by linking a drug to an antibody according to a conventional technique. The antibody and drug can be directly linked to each other through their own linker groups or indirectly through a linker or other substance.

There are many different reactions for covalent attachment of drugs to antibodies. This is often achieved by reaction of amino acid residues of the antibody molecule, including amino groups of lysine, free carboxyl groups of glutamic and aspartic acids, sulfhydryl groups of cysteine, and various aromatic amino acid moieties. One of the most commonly used non-specific methods of covalent attachment is the carbodiimide reaction to couple the carboxy (or amino) group of the compound to the amino (or carboxy) groups of the antibody. In addition, bifunctional agents such as dialdehydes or imidoesters have been used to couple the amino group of the compound to the amino groups of the antibody molecule. The Schiff base reaction is also available for the attachment of drugs to antibodies. This method involves the oxidation of the periodate of the drug, which contains glycol or hydroxyl groups, thereby forming an aldehyde, which is then reacted with the antibody molecule. The attachment occurs by forming a Schiff base with the amino groups of the antibody molecule. Isothiocyanates can also be used as coupling agents for covalent attachment of drugs to antibodies. Other methods are known to those skilled in the art and within the scope of the present invention.

There are many linking groups known in the art for preparing 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.

In one embodiment of the invention, the antibody is linked to the drug via a bifunctional crosslinking reagent. As used herein, the term "bifunctional crosslinking reagent" refers to a reagent that has two reactive groups, one of which is capable of reacting with the antibody, while the other is capable of reacting with the drug to link the antibody to the drug, thereby forming a conjugate. Any suitable bifunctional crosslinking reagent may be used in connection with the invention as long as the linker reagent ensures that the properties of the drug, such as cytotoxicity and target characteristics of the antibody, are maintained. Preferably, the linker molecule connects the drug to the antibody via chemical bonds such that the drug and the antibody are chemically linked (e.g., covalently linked) to each other.

In one embodiment, the bifunctional cross-linking reagent comprises non-cleavable linkers. A non-cleavable linker is any chemical moiety that is capable of linking 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, in particular inside a cell. Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, light-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 non-cleavable linkers between a drug and an antibody are well known in the art. In one embodiment, the drug is linked to the antibody via a thioether bond.

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

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), zinc N-hydroxysuccinimide 3-maleimidocapropyl ester (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). A peptide linker such as valine-citrulline (Val-Cit) or alanine-phenylalanine (ala-phe) can also be used, and any of the above mentioned linkers can be used in adequate combination.

Disulfide-containing linkers are linkers that are cleavable by disulfide exchange, which can occur under physiological conditions. In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). Various disulfide linkers are known in the art, including, for example, those that can be prepared using 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-pyridyldithio)toluene).

Acid-labile linkers are linkers that are cleaved at acidic pH. For example, some intracellular compartments such as endosomes and lysosomes have an acidic pH (pH 4-5) and provide conditions suitable for the cleavage of acid-labile linkers. Acid-labile linkers are relatively stable at neutral pH conditions, such as in blood, but are unstable at pH below 5.5 or 5.0. For example, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitate amide, orthoester, acetal, ketal, etc. can be used.

Photolabile linkers are useful on the body surface and in many body cavities that are accessible to light. In addition, infrared light can penetrate tissue.

Peptidase-labile linkers can be used to cleave certain peptides inside or outside cells. In one embodiment, the cleavable linker is cleaved under mild conditions, i.e., under conditions inside the cell that do not affect the activity of the cytotoxic agent.

The linker may comprise or may comprise, for example, a peptidyl linker that is cleavable by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. Typically, the peptidyl linker is at least two amino acids in length or at least three amino acids in length. Cleaving agents may include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide-drug derivatives, resulting in the release of the active drug within target cells. For example, a peptidyl linker may be used that is cleavable by a thiol-dependent protease, cathepsin B, which is highly expressed in cancer tissue (e.g., Phe-Leu or Gly-Phe-Leu-Gly ligand). In particular embodiments, the intracellular protease cleavable peptidyl linker is a valine-citrulline (Val-Cit; vc) linker or a phenylalanine-lysine (Phe-Lys) linker. One advantage of using intracellular proteolytic release of a therapeutic agent is that the agent is typically attenuated upon conjugation, and the stability of the conjugates in serum is typically high.

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 cathepsin within tumor cells.

In one embodiment, the drug is a maytansinoid, such as DM4, which is linked to an antibody having the ability to bind to CLDN18.2 via an amino group and a sulfhydryl reactive heterobifunctional protein cross-linker that reacts with primary amines (as found in lysine side chains or at the N-terminus of proteins) of the antibody and with the sulfhydryl group 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 via the N-hydroxysuccinimide (NHS) ester with primary amines (which are found in lysine side chains or at the N-terminus of proteins) of the antibody and via the pyridinyl disulfide group with the sulfhydryl group of DM4 to form a reversible disulfide bond (Figure 1).

In one embodiment, the drug is an auristatin, such as MMAE, which is linked to an antibody having the ability to bind to CLDN18.2 via a peptide linker, such as a cathepsin cleavable peptide linker, in particular Val-Cit (vc). In one embodiment, the antibody having the ability to bind to CLDN18.2 is thiolated, such as with a heterobifunctional linker 2-IT (2-iminothiolane), which reacts with free amines of lysine residues.

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, 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, linked (preferably via their amino groups) to DM4 (preferably via its sulfhydryl group). In one embodiment, the antibody is linked to DM4 via an SPDB linker.

In one embodiment, the antibody-drug conjugate according to the invention comprises an 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, linked (preferably via their amino groups) to DM4 (preferably via its sulfhydryl group). In one embodiment, the antibody is linked to DM4 via an SPDB linker.

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, 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, linked (preferably via their amino groups) to MMAE (preferably via its N-terminal amino group). In one embodiment, the antibody is linked to MMAE via a linker comprising the vc dipeptide.

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, 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, linked (preferably via their amino groups) to MMAE (preferably via its N-terminal amino group). In one embodiment, the antibody is linked to MMAE via a linker comprising the vc dipeptide.

As used herein, the term "nucleic acid" is intended to include DNA and RNA. Nucleic acid may be single-stranded or double-stranded, but is preferably double-stranded DNA.

According to the invention, the term "expression" is used in its most general meaning and includes the production of RNA or RNA and protein/peptide. It also includes partial expression of nucleic acids. In addition, expression can be transient or stable.

The description given herein with respect to specific amino acid sequences, such as those shown in the sequence listing, in particular those referred to herein by reference to SEQ ID NO:, shall be construed to also apply to variants of said specific sequences. Such variant sequences may be functionally equivalent to said specific sequences, such as amino acid sequences having properties identical to or similar to those of the specific amino acid sequences. One important property is the retention of binding of the antibody to its target. Preferably, a sequence that is a variant with respect to a certain sequence, when it replaces a specific sequence in an antibody, retains the binding of said antibody to CLDN18.2.

It will be understood by those skilled in the art that, in particular, the sequences of the CDRs, hypervariable and variable regions can be altered without loss of the ability to bind CLDN18.2. For example, the CDRs will be either identical to or highly homologous to regions of the antibodies described herein. By "highly homologous" it is meant that the CDRs may have substitutions in an amount of 1 to 5, preferably 1 to 4, such as 1 to 3 or 1 or 2 substitutions. In addition, the hypervariable and variable regions can be modified so that they exhibit substantial homology to regions of the antibodies specifically disclosed herein.

The term "variant" according to the invention refers in particular to mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologues, in particular those occurring in nature. An allelic variant refers to a change in the normal sequence of a gene, the significance of which is often unclear. Sequencing of a complete gene often identifies multiple allelic variants for a given gene. A species homologue is a nucleic acid or amino acid sequence originating from a species different from the species of the nucleic acid sequence or amino acid sequence in question. The term "variant" shall cover any post-translationally modified variants and conformational variants.

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 that include a deletion at the N-terminus and/or C-terminus of a protein are also referred to as N-terminal and/or C-terminal truncation variants.

Amino acid insertion variants include insertions of one or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are introduced at a specific site in the amino acid sequence, although random insertion is also possible with appropriate screening of the resulting product.

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.

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

Amino acid variants are characterized by at least one residue in the sequence to be deleted and another residue inserted in its place. Preference is given to modifications that are at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and/or to substitutions of amino acids for others that have similar properties. Preferably, amino acid substitutions in protein variants are conservative amino acid substitutions, i.e., substitutions of equally charged or uncharged amino acids. A conservative amino acid substitution includes a substitution for one of a family of amino acids that are related by their side chains. Natural amino acids are generally divided into four families: acidic (aspartate, glutamate), 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 as aromatic amino acids.

Preferably, the degree of similarity, preferably identity, between a given amino acid sequence, such as an amino acid sequence designated herein by SEQ ID NO:, and an amino acid sequence that is a variant of said 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 that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 amino acids, preferably contiguous amino acids. In preferred embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using known tools, preferably using the best sequence alignment, such as using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, gap penalty 10.0, extension penalty 0.5.

"Sequence similarity" refers to the percentage of amino acids that are either identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences.

The term "percent identity" is intended to refer to the percentage of amino acid residues that are identical between two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two amino acid sequences are usually performed by comparing the sequences after they have been optimally aligned, said comparison being made using a segment or "comparison window" to identify and compare local regions of sequence similarity. An optimal alignment of sequences to be compared can also be obtained manually by using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Needleman 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 using computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

Percent identity is calculated by determining the number of identical positions between two compared sequences, dividing this number by the number of compared positions, and multiplying the result by 100 to obtain the percentage identity between the two sequences.

The term "transgenic animal" refers to an animal having a genome comprising one or more transgenes, preferably heavy and/or light chain transgenes or transchromosomes (either integrated or unintegrated into the animal's natural genomic DNA) and which is preferably capable of expressing the transgenes. For example, a transgenic mouse may have a human light chain transgene and either a human heavy chain transgene or a human heavy chain transchromosome, such that the mouse produces human antibodies against CLDN18.2 when immunized with CLDN18.2 antigen and/or cells expressing CLDN18.2. The human heavy chain transgene may be integrated into the chromosomal DNA of the mouse, as is the case for transgenic mice, for example HuMAb mice such as HCo7 or HCol2 mice, or the human heavy chain transgene may be maintained extrachromosomally, as in the case of transchromosomal (e.g. KM) mice as described in WO 02/43478. Such transgenic and transchromosomal mice may be capable of producing multiple isotypes of human monoclonal antibodies to CLDN18.2 (e.g., IgG, IgA, and/or IgE) through VDJ recombination and isotype switching.

“Reduce,” “reduce,” or “inhibit,” as used herein, means an overall decrease or the ability to cause an overall decrease, preferably by 5% or more, by 10% or more, by 20% or more, more preferably by 50% or more, and most preferably by 75% or more, in a level, such as an expression level or a proliferation level of cells.

Terms such as "increase" or "increase" preferably refer to an increase or enhancement by about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80% and most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more.

The antibodies described herein can be produced by a variety of methods, including conventional monoclonal antibody methodology, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, other methods for producing monoclonal antibodies could in principle be used, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using antibody gene libraries.

The preferred animal system for producing hybridomas that secrete monoclonal antibodies is the murine system. Hybridoma production in mice is a very well-established procedure. Immunization protocols and methods for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

Other preferred animal systems for producing hybridomas that secrete monoclonal antibodies are the rat and rabbit systems (e.g., described in Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995); see also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

In another preferred embodiment, human monoclonal antibodies can be produced using transgenic or transchromosomal mice that carry 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 accomplished as described in detail for CD20 in WO2004 035607.

Another strategy for producing monoclonal antibodies is to directly isolate the genes encoding antibodies from lymphocytes producing antibodies of defined specificity, for example, see Babcock et al., 1996; A new strategy for producing monoclonal antibodies from single isolated lymphocytes producing antibodies of defined specificity. For details on the development of recombinant antibodies, see also Welschof and Kraus, Recombinant antibodes for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

To generate antibodies, mice can be immunized with carrier-conjugated peptides derived from an antigen sequence, i.e., a sequence against which antibodies are to be directed, from an enriched preparation of recombinantly expressed antigen or fragments thereof and/or cells expressing the antigen, as described. Alternatively, mice can be immunized with DNA encoding the antigen or fragments thereof. In the event that immunization using a purified or enriched preparation of the antigen does not result in the production of antibodies, mice can also be immunized with cells expressing the antigen, such as a cell line, to stimulate immune responses.

The immune response can be monitored during the immunization course using plasma and serum samples obtained from the tail vein or retro-orbital bleeding. Mice with sufficient immunoglobulin titers can be used for fusion. Mice can be stimulated intraperitoneally or intravenously with antigen-expressing cells 3 days before sacrifice and splenectomy to increase the percentage of hybridomas secreting specific antibodies.

To generate monoclonal antibody-producing hybridomas, splenocytes and lymph node cells can be isolated from immunized mice and fused with an appropriate immortalized cell line, such as a murine myeloma cell line. The resulting hybridomas can then be screened for antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody-secreting hybridomas. Immunofluorescence and FACS analysis using cells expressing the antigen can identify antibodies with specificity for the antigen. Antibody-secreting hybridomas can be replicated, screened again, and if still positive for the monoclonal antibody, can be subcloned by limiting dilution. Stable subclones can then be cultured in vitro to produce antibodies in tissue culture medium for characterization.

Antibodies can also be produced in a host cell transfectome using, for example, a combination of recombinant DNA techniques and gene transfection methods that are well known in the art (Morrison, S. (1985) Science 229: 1202).

For example, in one embodiment, the gene(s) of interest, such as antibody genes, can be ligated into an expression vector, such as a eukaryotic expression plasmid, such as that used by the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expression systems well known in the art. The purified plasmid with the cloned antibody genes can be introduced into eukaryotic host cells, such as CHO cells, NS/0 cells, HEK293T cells or HEK293 cells, or otherwise into other eukaryotic cells, such as cells derived from plants, fungi or yeast. The method used to introduce these genes can be a method described in the art, such as electroporation, the use of lipofectin, lipofectamine or others. Once these antibody genes are introduced into host cells, cells expressing the antibody can be identified and selected. These cells constitute transfectomas, which can then be amplified for their expression levels and produced to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and/or cells.

Alternatively, cloned antibody genes can be expressed in other expression systems, including prokaryotic cells such as microorganisms such as E. coli. Additionally, antibodies can be produced in transgenic non-human animals such as sheep and rabbit milk or chicken eggs 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.

Chimerization

The immunogenicity of murine antibodies in humans can be reduced or completely eliminated if the corresponding antibodies are chimerized or humanized. Chimeric antibodies are antibodies, different portions of which are derived from different animal species, such as those having a variable region derived from a murine antibody and constant regions of a human immunoglobulin. Chimerization of antibodies is achieved by fusing the variable regions of the heavy and light chain of a murine antibody to the constant region of a human heavy and light chain (e.g., as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are formed by fusing the constant region of a human kappa light chain to the variable region of a mouse light chain. In a further preferred embodiment, chimeric antibodies can be prepared by fusing the constant region of a human lambda light chain to the variable region of a mouse light chain. Preferred heavy chain constant regions for producing chimeric antibodies are IgG1, IgG3, and IgG4. Other preferred heavy chain constant regions for producing chimeric antibodies are IgG2, IgA, IgD, and IgM.

Humanization

Antibodies interact with target antigens primarily through amino acid residues that are located in the six complementarity-determining regions (CDRs) of the heavy and light chains. For this reason, amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that incorporate CDR sequences from a specific naturally occurring antibody grafted onto framework sequences from another antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332: 323–327; Jones, P. et al. (1986) Nature 321: 522–525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U. S. A. 86: 10029–10033). Such framework sequences can be obtained from publicly available DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include fully assembled variable genes that are formed by V(D)J joining during B cell maturation. Germline gene sequences will also differ from those of antibodies with high affinity for the secondary repertoire when individually distributed evenly across the variable region.

The ability of antibodies to bind antigen can be determined using standard binding assays (e.g., ELISA, Western blot, immunofluorescence, and flow cytometric analysis).

For antibody purification, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. Alternatively, antibodies can be produced in dialysis-based bioreactors. Supernatants can be filtered and, if necessary, concentrated before affinity chromatography on Protein G Sepharose or Protein A Sepharose. Eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography for purity. The buffer can be replaced with PBS, and the concentration can be determined by OD280 using an absorbance coefficient of 1.43. Monoclonal antibodies can be aliquoted and stored at -80°C.

To determine whether selected monoclonal antibodies can bind to unique epitopes, site-directed or multisite-directed mutagenesis can be used.

To determine the isotype of antibodies, isotype ELISA can be performed with various commercial kits (e.g., Zymed, Roche Diagnostics). Wells of microplates can be coated with anti-mouse Ig. After blocking, the plates are reacted with monoclonal antibodies or purified isotype controls at ambient temperature for two hours. The well contents can then be reacted with IgG1, IgG2a, IgG2b or IgG3, IgA or mouse IgM-specific peroxidase-conjugated probes. After washing, the plates can be developed with ABTS substrate (1 mg/ml) and analyzed at OD 405-650. Alternatively, the IsoStrip Mouse Anti-Mouse Monoclonal Isotyping Kit (Roche, Cat.# 1493027) can be used as described by the manufacturer.

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 antigen. Cell lines expressing 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 at 4°C for 30 minutes. After washing, APC- or Alexa647-labeled anti-IgG antibody can bind to the antigen-bound monoclonal antibody under the same conditions as primary antibody staining. Samples can be analyzed by flow cytometry using a FACS instrument using the properties of light and side scatter with single-cell passbanding of live cells. To distinguish antigen-specific monoclonal antibodies from non-specific binders in a single measurement, a co-transfection method 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 fluorescence channel than antibody-stained cells. Since most transfected cells express both transgenes, antigen-specific monoclonal antibodies bind preferentially to cells expressing the fluorescent marker, whereas non-specific antibodies bind in a comparable ratio to untransfected cells. An alternative assay using fluorescence microscopy can be used in addition to or instead of flow cytometric analysis. Cells can be stained exactly as described above and examined by fluorescence microscopy.

Immunofluorescence microscopy 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 antigen. For example, cell lines expressing either spontaneously or after transfection of the antigen and negative controls lacking antigen expression are grown on 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 min at 25°C. After washing, the cells can be reacted with Alexa555-labeled anti-mouse IgG (Molecular Probes) under the same conditions. The cells can then be examined by fluorescence microscopy.

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

Antibodies may be further tested for reactivity with the antigen by immunohistochemistry methods well known to those skilled in the art, such as using paraformaldehyde- or acetone-fixed cryosections or paraformaldehyde-fixed paraffin-embedded sections from non-tumor tissue samples or tumor tissue obtained from patients during routine surgical procedures or from mice bearing xenotransformed tumors inoculated with cell lines expressing the antigen spontaneously or after transfection. For immunostaining, antibodies reactive with the antigen may then be incubated with goat anti-mouse antibodies or goat anti-rabbit antibodies conjugated with horseradish peroxidase (DAKO), according to the supplier's instructions.

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

The antibody conjugates can be administered to tumor-free mice followed by injection of tumor cells to measure the effect of the antibody conjugates on preventing tumor formation or tumor-related symptoms. The antibody conjugates can be administered to tumor-bearing mice to determine the therapeutic efficacy of the respective antibody conjugates in reducing tumor growth, metastasis, or tumor-related symptoms. The use of the antibody conjugate can be combined with the use of other substances such as cystostatic drugs, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors, or other antibodies to determine synergistic efficacy and potential toxicity of the combinations. To analyze the toxic side effects mediated by the antibody conjugates, animals can be inoculated with the antibody conjugates or control reagents and carefully examined for the presence of symptoms that may be associated with anti-CLDN18.2 antibody conjugate therapy. Potential adverse effects of in vivo use of CLDN18.2 antibody conjugates include, in particular, toxicity in CLDN18.2-expressing tissue, including the stomach.

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

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

The pharmaceutical compositions are preferably sterile and contain an effective amount of the antibody conjugates described herein, and optionally additional agents as discussed herein, to produce the desired reaction or effect.

The pharmaceutical compositions are usually provided in a single dosage form and can be prepared in a known manner. The pharmaceutical composition can, for example, be presented in the form of a solution or suspension.

The pharmaceutical composition may contain salts, buffers, preservatives, carriers, diluents and/or excipients, each of which is preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to the non-toxicity of the material, which does not interact with the action of the active component of the pharmaceutical composition.

Salts that are not pharmaceutically acceptable can be used to prepare pharmaceutically acceptable salts and are included in the invention. Pharmaceutically acceptable salts of this type include, without limitation, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic acids and the like. 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.

Suitable buffering agents for use in the pharmaceutical composition include acetic acid in salt, citric acid in salt, boric acid in salt, and phosphoric acid in salt.

Suitable preservatives for use in the pharmaceutical composition include benzalkonium chloride, chlorobutanol, paraben and thimerosal.

The injection formulation may contain a pharmaceutically acceptable excipient such as Ringer's lactate.

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

Possible carrier substances for parenteral administration are, for example, sterile water, Ringer's solution, Ringer's lactate, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide-glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers.

As used herein, the term "excipient" is intended to designate all substances that may be present in a pharmaceutical composition and that are not active ingredients, such as, for example, carriers, binders, lubricants, thickeners, surface-active agents, preservatives, emulsifiers, buffers, flavorings or colorings.

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

Compositions suitable for parenteral administration typically comprise a sterile aqueous or non-aqueous preparation of the active compound which is 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 commonly employed as a solution or suspension medium.

The agents and compositions described herein are administered in effective amounts. "Effective amount" refers to an amount that achieves the desired response or effect, alone or in combination with additional doses. In the case of treating a specific disease or a specific condition, the desired response preferably refers to inhibition of the progress of the disease. This includes slowing the progress of the disease and, in particular, stopping or reversing the progress of the disease. The desired response in the treatment of a disease or condition may also be a delay in the onset or prevention of the onset of the disease or said condition.

The effective amount of an agent or composition described herein will depend on the condition being treated, the severity of the condition, individual patient parameters including age, physiological condition, size and weight, duration of treatment, type of concomitant therapy (if any), the particular route of administration and similar factors. Accordingly, the doses administered with the agents described herein may depend on various such parameters. In the event that the patient's response is inadequate with the initial dose, higher doses (or effective higher doses achieved by another, more localized route of administration) may be used.

The agents and compositions provided herein may be used alone or in combination with traditional therapeutic regimens such as surgery, radiation, chemotherapy, and/or bone marrow transplantation (autologous, isogeneic, allogeneic, or unrelated).

The treatment of cancer is an area where combination strategies are particularly desirable, since often the combined action of two, three, four or even more anticancer drugs/therapeutic agents creates synergistic effects that are significantly stronger than the effect of a monotherapeutic approach. Thus, in another embodiment of the present invention, the treatment of cancer can be effectively combined with various other drugs. These include, for example, combinations with conventional anticancer therapies, multiple epitope strategies, adjunctive immunotherapy and treatment approaches targeting 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 may inhibit tumor cell growth at different checkpoints, while other agents may, for example, inhibit neoangiogenesis, cancer cell survival or metastasis, potentially converting malignancy into a chronic disease.

The agents and compositions described herein can be administered to patients, such as in vivo, to treat or prevent various disorders, such as those described herein. Preferred patients include patients suffering from disorders that can be corrected or improved by administering the agents and compositions described herein. This includes disorders associated with cells characterized by an altered pattern of CLDN18.2 expression.

For example, in one embodiment, the agents and compositions described herein can be used to treat a patient with an oncological disease, such as an oncological disease described herein, characterized by the presence of malignant tumor cells expressing CLDN18.2.

The pharmaceutical compositions and methods of treatment described in accordance with the invention can also be used for immunization or vaccination to prevent the disease described herein.

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

EXAMPLES

Example 1: MATERIALS AND METHODS

1. Endocytosis

Endocytosis of CLDN18.2-bound IMAB362 was determined using a cytotoxicity-based endocytosis assay that relies on the co-internalization of target-bound antibody and saporin-conjugated anti-human or anti-mouse IgG Fab fragment (Human Fab-ZAP, Advanced Targeting Systems, IT-51, Lot: #93-23; Mouse Fab-ZAP, Advanced Targeting Systems, IT-48, Lot: #93-21). Saporin is a ribosome inactivating protein that, upon internalization, inhibits protein biosynthesis and therefore leads to cell death. To ensure optimal cell lysis, Fab-ZAP antibody was used at least at a molarity of 6-fold that of the target antibody.

Stably transfected HEK293~CLDN18.2 cells were harvested with 0.05% trypsin/EDTA (Gibco, 25300-054) and 2.5 × ¹⁰³ cells/well were seeded in 50 μl growth medium in a 96-well culture plate. After 24 h, 25 μl Fab-ZAP and 25 μl anti-CLDN18.2 mAB or isotype control antibody diluted in the culture medium were added to the cells (Table 1). The cells were cultured for another 72 h.

Table 1. Concentration of anti-CLDN18.2 mAB and Fab-ZAP for endocytosis assay

Dilution Final concentration [ng/ml] Anti-CLDN18.2 Fab-ZAP D1 187.5 825 D2 75.0 825 D3 30.0 825 D4 12.0 825 D5 4,800 825 D6 1,920 825 D7 0.768 825 D8 0.307 825 D9 0.123 825

Molecular weight: 150 kDa for IMAB362 and 110 kDa for FabZAP.

Cell viability was analyzed as described in 6. Cells incubated without antibodies in the presence of Fab-ZAP were used as a control.

2. Epitope mapping

The antigenic epitope responsible for the specificity of CLDN18.2 was analyzed by flow cytometry (see 5) in 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. Consequently, the amino acids of CLDN18.2 were replaced by amino acids of the homologous protein CLDN18.1 at the corresponding positions. HEK293T cells were transfected with plasmids encoding the specific CLDN18.2 mutant and EGFP as a reporter gene. Purified antibodies were tested at a concentration of 5 μg/ml, while antibodies obtained from hybridoma supernatant were diluted 1:4 prior to testing. To determine whether a particular amino acid substitution impacted antibody binding, the mean fluorescence intensity (MFI) measured in the population of transfected (EGFP-positive) cells was compared between the mutant showing the highest MFI value and the mutant of interest. If the MFI of the mutant of interest was below 50%, the amino acid residue was characterized as essential for antibody binding and CLDN18.2 specificity.

3. Drug-antibody conjugates

Conjugations of DM4 and vcMMAE to the monoclonal antibody IMAB362 (lot no. p412118) and analytical characterization were performed at Piramal Healthcare (Grangemouth, UK). The methods are briefly described in the following section:

DM4 was coupled to IMAB362 via SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate). SPDB reagent is an amine and sulfhydryl reactive heterobifunctional protein crosslinker that reacts via the N-hydroxysuccinimide (NHS) ester with primary amines (as found in lysine side chains or the N-terminus of the protein) of the antibody and via the pyridinyl disulfide group with the sulfhydryl group of DM4 to form a reversible disulfide bond (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 room temperature. The modified antibody was dialyzed against 35 mM citrate buffer (pH 5.5) and the linker to antibody ratio was determined. 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 adjusted to storage buffer conditions (20 mM His, 85 mg/mL sucrose, pH 5.8) and stored at -80°C. The antibody to drug ratio was analyzed by UV spectrometry, monomer contents by SEC-HPLC and free drug contents by RP-HPLC.

vcMMAE was condensed with thiolated IMAB362. Thus, IMAB362 was initially thiolated with the heterobifunctional linker 2-IT (2-iminothiolane), which reacts with free amines of lysine residues. Then, vcMMAE containing the cathepsin-cleavable peptide linker Val-Cit (vc) was conjugated via valine to the sulfhydryl group of the thiolated antibody (Figure 1). Briefly, IMAB362 was diafiltered into PBS buffer (pH 7.2) using an ultracentrifuge filter and incubated with 2-IT at a molar ratio of 1:20 (IMAB362:2-IT) for 2 h at room temperature. The modified antibody was dialyzed into 35 mM citrate buffer (pH 5.5) and the linker to antibody ratio was determined. Subsequently, 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. Conjugated antibodies were dialyzed into storage buffer (20 mM His, 85 mg/ml sucrose, pH 5.8) and stored at -80°C. Antibody to drug ratio was analyzed by UV spectrometry, monomer contents by SEC-HPLC, and free drug contents by RP-HPLC.

4. Cell culture

Cell lines were cultured according to the suppliers' instructions and the Ganymed cell line data sheet in cell type-specific medium at 37°C in humidified incubators in the presence of 5% or 7.5% _CO2 (Table 2). Cell culture media and supplements were obtained from Invitrogen, Gibco, and Sigma.

Table 2. Cellular model systems

Cell line Types/tissue/disease Wednesday Incubation HEK293~control Human embryonic kidney DMEM/F12 (10% FCS, 1% pen/strep, 1.5 mg/ml geneticin) 7.5% _CO2 , 37°C HEK293~CLDN18.2 Human embryonic kidney (stably transfected with human CLDN18.2) DMEM/F12 (10% FCS, 1% pen/strep, 1.5 mg/ml geneticin) 7.5% _CO2 , 37°C HEK293~CLDN18.1 Human embryonic kidney (stably transfected with human CLDN18.1) DMEM/F12 (10% FCS, 1% pen/strep, 1.5 mg/ml geneticin) 7.5% _CO2 , 37°C BxPC-3 Human pancreatic adenocarcinoma RPMI (10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 10% FCS) 5% _CO2 , 37°C BxPC-3~CLDN18.2 Human pancreatic adenocarcinoma (stably transduced with human CLDN18.2) RPMI (10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1× sodium bicarbonate, 10% FCS, 0.5 μg/mL blasticidin) 5% _CO2 , 37°C NCI-N87 human gastric carcinoma RPMI (10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 10% FCS) 5% _CO2 , 37°C NCI-N87~CLDN18.2 human gastric carcinoma (stably transduced with human CLDN18.2) RPMI (10 mM HEPES+1 mM sodium pyruvate, 4.5 g/L glucose, 10% FCS, 1.5 μg/mL blasticidin) 5% _CO2 , 37°C DAN-G 1C5F2 human pancreatic adenocarcinoma RPMI (10% FCS, 1% pen/strap) 5% _CO2 , 37°C NUGC-4 10cF7-5 grade 3a human gastric adenocarcinoma RPMI (10% FCS, 1% pen/strap) 5% _CO2 , 37°C NUGC-4 10cE8 human gastric adenocarcinoma RPMI (10% FCS, 1% pen/strap) 5% _CO2 , 37°C PA-1 (Luc) human ovarian teratocarcinoma EMEM (1 mM sodium pyruvate, 0.1 mM NEAA, 1.5 g/L sodium bicarbonate, 10% FCS) 5% _CO2 , 37°C

5. Flow cytometry

Relative binding affinities and specificities of naked anti-CLDN18.2 antibodies and antibody-drug conjugates were determined by flow cytometry using CLDN18.2 positive and negative cell lines.

Cells from the exponentially growing culture were harvested with 0.05% trypsin/EDTA (Gibco, 25300-054) and counted using a Neubauer counting chamber. Cells were centrifuged for 5 min at 1500 rpm (468 μg), the supernatant was discarded and the 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 cells/ml. 100 μl of cell suspension per well (corresponding to 2 × 10 ⁵ cells/well) were transferred to a round-bottomed 96-well microplate. After centrifugation for 1 min at 1500 rpm, the supernatant was discarded and the cells were resuspended in FACS buffer containing toxin-conjugated or naked antibodies at appropriate concentrations (up to 20 μg/ml for relative affinity measurements or 50 μg/ml for expression control) and incubated for 30-45 min at 4°C (Table 3). The cells were centrifuged for 1 min at 1500 rpm and the supernatant was discarded. After the cells were washed 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, chim mAB294 assay) and incubated for 30 min at 4°C (Table 3). After incubation, 100 μl of FACS buffer was added to each sample, the cells were centrifuged for 1 min at 1500 rpm and the supernatant was discarded. The FACS buffer washing step was repeated twice. Finally, cells were resuspended in 100 µl FACS buffer and binding was determined using a BD FACS Array Bioanalyzer.

It should be noted that toxin-conjugated and naked antibodies were used in equal concentrations. Differences in molecular weight of antibodies were neglected.

Table 3. Experimental flow cytometry data

Analysis: Primary antibody Secondary antibody Comparison of IMAB362 with IMAB362-DM4 and IMAB362-vcMMAE - 100 µl
- 0.1, 0.3, 0.9, 3.1, 10.9, 38.1 133.3, 466.5, 1632.7, 5714.3, 20000 ng/ml
- 45 min. 4°C - 50 µl
- dilution 1:200
- 30 min Comparison of IMAB362 with murine or chimeric CLDN18.2 reactive antibodies - 50 µl
- 19.5, 39.1, 78.1, 156.3, 312.6, 625, 1250, 2500, 5000, 10000, 20000 ng/ml
- 30 min. 4°C - 30 µl
- dilution 1:100
- 30 min Comparison of IMAB362 with chim mAB294 - 50 µl
- 19.5, 39.1, 78.1, 156.3, 312.6, 625, 1250, 2500, 5000, 10000, 20000 ng/ml
- 30 min. 4°C - 50 µl
- 1 mcg/ml
- 30 min

6. Viability analysis

The effect of IMAB362-DM4 and IMAB362-vcMMAE on cell viability was determined using a colorimetric assay that detects cellular metabolic activities. The assay is based on the ability of metabolically active cells to reduce yellow XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) to an orange-colored formazan compound that can be detected spectrophotometrically. The intensity of the dye is proportional to the number of viable cells.

Cells were harvested using 0.05% trypsin/EDTA (Gibco, 25300-054), resuspended in culture medium (Table 2), and 50 μl of the cell suspension with the appropriate cell numbers were seeded per well in 96-well culture plates (Table 4). After 24 h, toxin-conjugated IMAB362 or control antibodies diluted in 50 μl of medium at the appropriate concentrations were added, and the cells were cultured for another 72 h.

Table 4. Overview of cell lines used in viability assays

Cell line Number of cells
for 96 holes NCI-N87~CLDN18.2 6000 NCI-N87 6000 NUGC-4 10cE8 3000 NUGC-4 10cF7-5 sort3a 3000 BxPC-3~CLDN18.2 3000 HEK293~CLDN18.2 2000 HEK293~CLDN18.1 2000 HEK293(CLDN6) 2000

Cell viability was analyzed using the AppliChem II Cell Proliferation Assay Kit (AppliChem, A8088, 1000) according to the manufacturer's instructions. After 3-5 h of incubation with XTT reagent, the optical density was measured at 480 nm (reference 630 nm) using a spectrophotometer (Tecan). The decrease in viability was calculated using the following equation:

Reduction of viability [%]: Reduction of viability [%]

blank: environment control

control: cells without antibodies

sample: cells with antibody

EC ₅₀ values were determined using GraphPad Prism 6 using nonlinear regression.

7. Analysis of the "bystander effect"

The bystander activity of the toxin-conjugated IMAB362 antibody on non-targeted cells was analyzed in vitro by co-cultivating the CLDN18.2-negative, luciferase-expressing PA-1 (Luc) cell line in the presence or absence of the CLDN18.2-positive NUGC-4 10cE8 cell line. Thus, 1.5 × ¹⁰³ PA-1(Luc) cells per well were seeded for single culture or together with 1.5 × ¹⁰³ NUGC-4 10cE8 cells per well for co-culture in RPMI medium supplemented with 10% FCS and 1% pen/strep. After 24 h, IMAB362-DM4, IMAB362-vcMMAE or unconjugated IMAB362 as a negative control were added and the cells were cultured for another 72 h. Cell viability was analyzed as described in 6. Non-target cell lysis was determined by measuring the bioluminescence of luciferase expressing PA-1(Luc) cells. Thus, 50 μl of luciferin mixture (1.92 mg/ml D-luciferin (Sigma, 50227) and 160 mM HEPES in _ddH2O ) were added per well. The plate was incubated in the dark at room temperature for 90 min and bioluminescence was measured using a luminometer (Infinite M200, TECAN). The results were expressed as integrated digital relative light units (RLU). The decrease in viability was calculated as described in 6.

8. Animal experiments

All xenotransplantation studies were performed in accordance with national regulations and ethical principles for experimental animal research. All animals were maintained under specific pathogen-free conditions in individual ventilated cages and under a 12-hour artificial light-dark cycle. Food and water were provided ad libitium. Mice were acclimatized for at least 6 days before the start of the study.

8.1. Maximum Tolerated Dose (MTD) Study

Xenograft tumors were inoculated by subcutaneous injection of 8.5 × ¹⁰⁶ BxPC-3~CLDN18.2 human pancreatic tumor cells in 200 μl PBS into the flank of a female Hsd:Athymic Nude-Foxn1nu mouse. To determine the MTD and efficacy of anti-CLDN18.2 antibody-drug conjugates in tumor-bearing mice, different doses of IMAB362-DM4 or IMAB362-vcMMAE were administered. The maximum usable dose was limited by the antibody concentration and injection volume recommended by GV-SOLAS for intravenous injection in mice (~200 μl). Both antibodies were administered at the maximal concentration as a single and repeated dose (e.g., 15 and 16 mg/kg, respectively), as well as half this concentration (e.g., 7.5 and 8 mg/kg, respectively) or vehicle blank control (group size: n=5). Antibodies were administered intravenously on day 14 post-xenografting and for a repeat dose additionally on day 21 post-xenografting. Animal body weight, health, behavior, and tumor size were monitored twice weekly using calipers, and tumor volumes were calculated using the following formula: [length x width x (width/2)]. All animals were dissected when the first tumor of the vehicle group reached a maximum of 1400 ^mm3 or when the tumor became ulcerated (day 49 post-xenografting for IMAB362-DM4, day 37 post-xenografting for IMAB362-vcMMAE). Blood samples for clinical analysis were collected under general anesthesia initiated with 250 μl of a mixture solution consisting of 1.25 ml ketamine, 1 ml xylazine (2%) and 7.75 ml _H2O . Mice were then perfused with PBS followed by 4% formalin perfusion under general anesthesia. Selected organs and tissues (stomach, esophagus, brain, heart, kidney, liver, lung, pancreas, spleen, duodenum, ileum, colon, uterus and ovaries) were dissected and fixed in 4% formalin, stored at 4°C and finally paraffin embedded. Three-micron tissue sections were cut from each FFPE (formalin-fixed, paraffin-embedded) specimen and mounted on adhesive slides (SuperFrost Ultra Plus, Thermo Fisher Scientific). After curing for 60 min at 58°C, FFPE tissue sections were deparaffinized using xylene and rehydrated using different concentrations of ethanol (2x 100%, 2x 96%, 2x 70% ethanol for 3 min each). Nuclei were stained for 5 min at room temperature with Mayer's hematoxylin, followed by rinsing under tap H ₂ O. Cytoplasm was then counterstained with aqueous 0.5% eosin for 2 min at room temperature. After dehydration using different concentrations of ethanol and xylene, sections were mounted using X-TRA Kit non-aqueous mounting medium.

8.2. Clinical biochemistry

To test for potential organ toxicity, relevant markers of pancreatic toxicity, nephrotoxicity and hepatotoxicity were analyzed in serum samples.

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

8.3. Efficacy studies

To generate human tumor xenografts, appropriate numbers of cells were suspended in 200 μl 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 doses not exceeding the MIC. Naked antibody controls were administered twice weekly by alternating IV/IP injections of ~8 mg/kg IMAB362. In early treatment studies, treatments were started 3 days after xenografting. In current treatment studies, tumors were grown to 50 to 200 ^mm3 , and mice bearing homogeneous, medium-volume tumors were reassigned to control and antibody groups prior to treatment. Animal weight, health, behavior, and tumor size were monitored twice weekly using calipers. Tumor volumes were calculated using the following formula: [length x width x (width/2)]. Termination criteria were tumor sizes >16 mm in length or width or estimated volumes >1400 mm ³ . Other termination criteria were tumor ulceration or when the animal lost more than 10% of its body weight. In the case of complete tumor growth inhibition, mice were observed for 120 days after treatment. Persistent tumors were prepared and fixed in 4% formalin for subsequent IHC studies.

9. Antibody-dependent cellular cytotoxicity (ADCC)

Antibody-dependent cellular cytotoxicity (ADCC) was determined by measuring intracellular ATP content in unlysed cells after addition of human PBMC to target cells in the presence of IMAB362 toxin conjugates. Luciferase-generated bioluminescence was used to quantify ATP.

NUGC-4 10cF7_5 sort3a p3151#10 target cells were seeded at a defined cell density (8 × ¹⁰⁶ cells) two days prior to analysis to obtain reproducible confluence.

Target cells were harvested with 0.05% trypsin/EDTA (Gibco, 25300-054) and adjusted to a concentration of 1.6 × 10 ⁵ cells/mL in growth medium containing 20 mM HEPES (Gibco, 15630-056). 8 × 10 ³ cells per well were seeded in a white 96-well PP plate and incubated for ~5 h at 37°C and 5% CO ₂ .

PBMCs were prepared from fresh buffy coats obtained from healthy donors. About 20–25 ml of blood was diluted (1:2) with PBS in 3 Falcon tubes and carefully layered onto 15 ml of Ficol-Paque Plus (GE Healthcare, 17144003) in four 50 ml Falcon tubes. The gradients were centrifuged (25 min, 700 x g, continuously). After centrifugation, PBMCs were collected from the interphase, washed in 50 ml PBS/2 mM EDTA, centrifuged (5 min, 468 x g), resuspended in 50 ml PBS/2 mM EDTA, and centrifuged again (10 min, 208 x g) to remove platelets. The pellets were resuspended in 50 ml PBS/2 mM EDTA, and the cells were counted. Then PBMC were centrifuged (5 min, 468 xg), resuspended in X-Vivo-15 culture medium (Lonza, BE04-418Q) containing 5% human serum and cultured for 1.5 h at 37°C, 5% _CO2 . PBMC were collected, centrifuged (5 min, 468 ×g) and resuspended in X-Vivo-15 culture medium (Lonza, BE04-418Q), adjusting the cell concentration (for E:T ratio of 40:1) to 1.28 × ¹⁰⁷ cells/mL. IMAB362-DM4, IMAB362-vcMMAE and IMAB362 were serially diluted (4.5-fold dilution steps) 11-fold, resulting in concentrations ranging from 160 μg/mL to 0.05 ng/mL (final concentrations from 40 μg/mL to 0.01 ng/mL). 25 μL of each dilution was added to target cells and four replicates were used for each condition. Antibody-free PBS was added to the medium and total lysis control wells. Then, 25 μL of prepared PBMCs (3.2 × 10 ⁵ cells) were added to each well to achieve an E:T ratio of 40:1 and the plates were incubated for 15 h + 1 h at 37 °C, 5% CO ₂ . After overnight incubation, 10 μl of 8% Triton X-100/PBS solution were added to the maximum lysis control wells and 10 μl of PBS to the other wells. Finally, 50 μl of freshly prepared luciferin stock solution (160 mM HEPES, 1x PBS, 3.84 mg/ml D-Luciferin (Sigma Aldrich, 50227)) was added to each well and the plates were incubated for 90 min at room temperature in the dark. Bioluminescence was measured using a luminometer (Infinite M200, TECAN). The results were expressed as integrated digital relative light units (RLU).

Specific lysis was calculated as:

specific lysis [%] - specific lysis [%]

sample - sample

total lysis - general lysis

Max viable cells - max. viable cells

(max. viable cells: 10 µl PBS, no antibodies; total lysis: 10 µl 8% (v/v) Triton X-100 in PBS, no antibodies)

All ADCC data were processed using GraphPad Prism 6 using the function "log(agonist) vs response - find EC anything". Maximum lysis was defined as the range (difference from top to bottom) of the dose-response curve, with a maximum of 100%.

10. Complement-dependent cytotoxicity (CDC)

Complement-dependent cytotoxicity (CDC) was determined by measuring intracellular ATP content in unlysed cells after addition of human complement component to target cells in the presence of toxin-conjugated IMAB362. ATP-dependent bioluminescence generated by luciferase was measured as an indicator.

NUGC-4 10cF7_5 sort3ap3151#10 or KATO-III FGF-BP#12 adM p3151#25 target cells were seeded at a defined cell number (8 × ¹⁰⁶ and 9 × ¹⁰⁶ cells, respectively) two days before the assay to obtain reproducible confluence.

Target cells were harvested with 0.05% trypsin/EDTA and adjusted to a concentration of 1.6 × 10 ⁵ cells/mL in their respective culture medium containing 10% (v/v) FCS. 8 × 10 ³ cells were seeded in a white 96-well plate and incubated at 37°C and 5% CO ₂ . After 24 h, 50 μL of antibodies serially diluted in assay medium (60% RPMI containing 20 mM HEPES, 40% human serum pooled from several healthy donors) were added (final concentration from 80 to 78.13 ng/mL) and the cells were incubated for 80 min at 37°C and 5% CO ₂ . Total lysis controls were then supplemented with 10 µl 8% (v/v) Triton X-100 in PBS, while 10 µl PBS was added to all other wells (max viable cell controls and actual samples). The luciferase reaction was started by adding 50 µl luciferin mixture (3.84 mg/ml D-luciferin, 160 mM HEPES in _ddH2O ) per well. The plate was kept in the dark at room temperature for 90 min and bioluminescence was measured using a luminometer (Infinite M200, TECAN). The results were expressed as integrated digital relative light units (RLU).

Specific lysis was calculated as:

specific lysis [%] - specific lysis [%]

sample - sample

total lysis - general lysis

max viable cells - max. viable cells

(max. viable cells: 10 µl PBS, no antibodies, total lysis: 10 µl 8% (v/v) Triton X-100 in PBS, no antibodies)/

All CDC data were processed using GraphPad 6, using the function "log(agonist) vs response - find EC anything". Maximum lysis was defined as the range (difference from top to bottom) of the dose response curve, with a maximum of 100%.

Example 2. Screening for endocytosis of CLDN18.2-specific antibodies

Although in naked antibody antitumor therapy, internalization of target-bound antibodies may reduce the amount of membrane-bound antibodies available for primary mechanisms of action such as ADCC and CDC, endocytosis is an essential feature for the development of antibody-drug conjugates (ADCs). One of the important properties of ADCs is the endocytosis of the target-ADC complex. Therefore, the rate of naked antibody endocytosis is a major factor in the development of toxin-conjugated antibodies.

Binding properties, i.e., relative affinity for CLDN18.2, cross-reactivity to CLDN18.1, and antigen epitope-mediated specificity for CLDN18.2, were determined for various murine and chimeric anti-CLDN18.2 antibodies by flow cytometric analysis (Table 5 and Table 6). Antibodies showing strong binding to CLDN18.2 were selected for further endocytosis screening.

The endocytosis efficiency of various CLDN18.2-specific and CLID18.2/CLDN18.1 reactive antibodies was tested in vitro by co-incubating antibodies with saporin-conjugated Fab fragments (Fab-ZAP) together with CLDN18.2-expressing HEK293-CLDN18.2 cells. When co-internalized with the target-bound antibody, saporin inhibits cellular protein biosynthesis, resulting in cell death that can be monitored using a cell viability assay. This method provides an indirect way to assess endocytosis of the target-antibody complex. Antibodies were tested as chimeric antibodies when available, otherwise endocytosis screening was performed with murine antibodies.

chim mAb362 (IMAB362) as well as chim mAB294 could be efficiently internalized upon CLDN18.2 binding, resulting in decreased cell viability of HEK293~CLDN18.2 cells even at very low antibody concentrations (IMAB362: EC ₅₀ = 11 ng/mL; mAB294: EC ₅₀ = 10 ng/mL). In contrast, chim mAB308 and chim mAB359 did not decrease cell viability (Figure 2). Since chim mAB294 and chim mAB359 exhibit similar relative binding affinities and show significant differences in internalization (chim mAB294: EC ₅₀ = 10 ng/ml, chim mAB359: no endocytosis), the efficiency of endocytosis appears to not only correlate with the binding affinity of the antibody but also depend on the binding epitope.

Even the internalization of mu mAB362 was superior to all other murine CLDN18.2-reactive antibodies tested, which showed no significant endocytosis in the Fab-Zap assay (Figure 3).

Thus, CLDN18.2-specific antibodies IMAB362 and chim mAB294 were efficiently internalized upon binding to CLDN18.2 and are suitable for further evaluation as drug-antibody conjugates.

Table 5. Relative binding affinity and specificity of CLDN18.2-reactive antibodies

antibody Isotype EC ₅₀
[ng/ml] EC ₅₀ normalized [%] Max.binding [MFI] Max.binding, normalized [%] CLDN18.1-reactive Identified amino acids of human CLDN18.2 recognized by antibodies IMAB
362 1 human IgG1 520 100 15797 100 No A42S, N45Q, E56Q IMAB
362 2 584 100 18496 100 IMAB
362 3 652 100 7952 100 IMAB
362 4 1242 100 5319 100 mu mAB
362 3 mouse-like
IgG1 2238 343 8826 111 A42S, N45Q, E56Q Mu
mAB
362 4 4286 345 5913 111 mu mAB
362 4 mouse IgG2a 654 53 4880 92 n.a. mu mAB
359 3 mouse IgG2b 4474 686 4297 54 No n.a. mu mAB
325 3 mouse-like
IgG1 33263 5102 3672 46 No A42S, E56Q, G65P, L69I mu mAB
62 3 mouse IgG2a 27932 4284 2800 35 No n.a. mu mAB
187 3 mouse IgG2a 26597 4079 2428 31 No N37D, N45Q, Q47E mu mAB
294 3 mice-
IgG2a 348 53 989 12 No A42S, E56Q mu mAB
370 1 mice-
IgG2a 7877 1514 1405 9 No n.a. mu mAB
330 2 mice-
ny
IgG3 11054 1894 1707 9 No n.a. mu mAB
374 1 mouse IgG2a 8262 1588 468 3 No n.a. mu mAB
385 1 mouse-like
IgG1 8534 1640 471 3 No A42S, N45Q, E56Q, G65P, L69I mu mAB
177 3 Mishi-ny
IgG1 50 8 136 2 No N37D, A42S, N45Q, Q47E, E56Q, L69I mu mAB
55 3 mouse IgG2a 1945 298 241 3 not available A42S, N45Q, E56Q, G65P, L69I mu mAB
317 3 mice-
ny
IgG1 41 6 106 1 No Q29M, A42S, N45Q, Q47E, E56Q, G65P, L69I mu mAB
279 3 mouse IgG2a 27 4 118 1 No n.a. mu mAB
363 3 mice-
ny
IgG1 15405 2363 877 11 Yes N37D mu mAB
360 1 mouse IgG2a 19319 3713 1405 9 Yes n.a. mu mAB
371 1 mouse-like
IgG1 11361 2184 747 5 Yes - mu mAB
382 2 mouse IgG2b 12424 2129 842 5 Yes - mu mAB
348 2 mice-
IgG2a 13573 2326 703 4 Yes - mu mAB
322 2 mice-
IgG2a 12770 2189 818 4 Yes - mu mAB
321 2 mice-
IgG2a 8082 1385 505 3 Yes n.a. mu mAB
339 1 mice-
IgG2a 608 117 180 1 Yes n.a. mu mAB
338 2 mice-
IgG1 3966 680 244 1 Yes N37D

CLDN18.2 binding ( _EC50 normalized [%] and normalized max binding [%], were normalized to IMAB362) and CLDN18.1 reactivity were determined by flow cytometry on HEK293 cells stably expressing the corresponding protein. The antigenic epitope responsible for CLDN18.2 specificity was analyzed by flow cytometry on HEK293T cells transiently overexpressing the corresponding CLDN18.2 mutant. na: not analyzed. Antibodies labeled with the same number (1, 2, 3, or 4) were tested in the same binding assay. 1-3: detection with APC-conjugated secondary antibodies; 4: detection with Protein L-FITC; na: not analyzed.

Table 6. Relative binding affinity and endocytosis of CLDN18.2-specific antibodies

Antibody binding Cell viability Identified amino acids of human CLDN18.2 recognized by antibodies EC ₅₀
[ng/ml] EC ₅₀ normalized [%] Max. binding [MFI] Max. binding normalized [%] EC ₅₀
[ng/ml] EC ₅₀
[%] Max. Decrease in vitality [%] IMAB362 ¹ 584 100 18496 100 11 100 100 A42S,N45Q,E56Q IMAB362 ² 1242 100 5319 100 IMAB362 ³ 13 100 100 mu muAB362 ³ (IgG2a) 4286 345 5913 111 ~30 231 66 n.a. mu muAB362 ³ (IgG1) 654 53 4880 92 2,2 17 66 A42S, N45Q, E56Q chim mAB294 ² 5701 459 3089 58 10 91 86 A42S, N45Q, E56Q chim mAB359 ¹ 2752 472 11618 63 no effect no effect no effect A42S, N45Q, E56Q chim mAB308 ¹ 18883 3236 15016 81 no effect no effect no effect N37D, A42S, E56Q chim mAB62 ¹ 32313 5538 4079 22 no effect no effect no effect n.a. chim mAB279 ¹ 60046 10291 4394 24 no effect no effect no effect n.a.

Relative binding affinity [%], max binding [%], or max viability reduction [%] normalized to IMAB362 are shown for binding and endocytosis using flow cytometry and the Fab-ZAP assay, respectively. Antibodies labeled with the same number (1 or 2) were tested in the same binding assay. Indirect assessment of internalization with antibodies labeled with 1 or 2 were performed together in one assay and with antibodies labeled with 3 in a second cell viability assay. 1: detection using APC-conjugated secondary antibodies; 2 and 3: detection with Protein L-FITC.

Example 3. conjugation of ToxinA and IMAB362

Toxin conjugation was performed at Piramal Healthcare, including final buffer exchange. Stability studies were conducted to demonstrate that the ADCs remained within specification for a defined period of time when stored under specified 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). Toxin conjugates and IMAB362 were stored in storage buffer (20 mM histidine and 85 mg/mL sucrose, pH 5.8) at 2-8°C.

Table 7. 28-day stability testing of IMAB362-DM4 and IMAB362-vcMMAE

Analysis Result IMAB362-DM4 IMAB362-vcMMAE Day 1 Day 28 Day 1 Day 28 DAR 3.2 3.2 4.5 4.0 Monomer [%] 96 95 95 93 Free drug [%] 0.4 1.0 not detected 0.3

DAR: drug-to-antibody ratio.

IMAB362-DM4 and IMAB362-vcMMAE both exhibited high monomer content ≥95% and negligible free drug (≤1%). 28-day stability testing of toxin-conjugated IMAB362 antibodies at 2-8°C storage showed only a slight decrease in monomer content and a slight increase in free drug for both ADCs. Both antibodies were efficiently conjugated and demonstrated drug-to-antibody ratios of 3.2 for IMAB362-DM4 and 4.5 for IMAB362-vcMMAE (Table 7).

Example 4. IMAB362-ADC binding

The binding properties of IMAB362 have been tested in detail previously:

• IMAB362 binds to the first extracellular loop of claudin 18 splice variant 2 (CLDN18.2).

• Affinity for CLDN18.2 is in the low nanomolecular range.

• No cross-reactivity was observed with any CLDN18.2-negative cell or tissue type.

• No cross-reactivity was observed with the closest related family member, claudin 18 splice variant 1 (CLDN18.1).

The relative binding affinities of DM4- and MMAE-conjugated IMAB362 antibodies were compared with 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).

Table 8. Overview of binding assays of IMAB362-toxin conjugates to CLDN18.2-positive cell lines

Cell line IMAB362 IMAB362-DM4 IMAB362-vcMMAE Bmax [MFI] EC ₅₀ [ng/ml] Bmax [MFI] EC ₅₀ [ng/ml] Bmax [MFI] EC ₅₀ [ng/ml] HEK293~empty - - - - - - HEK293~CLDN18.2 18318 1121 19481 1472 14159 1594 HEK293~CLDN18.1 - - - - - - DAN-G 1C5F2 - - - - - - NCI-N87 - - - - - - NCI-N87~CLDN18.2 33678 310 28605 274 16569 103 NUGC-4 10cF7-5 grade 3a 67484 1671 77500 2600 68009 2520 54277 2384 69633 3909 45597 4049 NUGC-4 10cE8 34805 1570 35799 3046 29103 2826 28336 3673 36049 7487 25785 5464 BxPC-3~CLDN18.2 51380 104 76629 507 47197 183 28664 113 35700 425 25404 261

Bmax: maximum binding, MFI: mean fluorescence intensity.

Compared with unconjugated IMAB362, DMB- and MMAE-conjugated IMAB362 showed slightly reduced relative binding affinity on cells endogenously and ectopically expressing CLDN18.2 (Figure 4, Table 8). Both toxin-conjugated antibodies had very similar EC ₅₀ values but differed slightly in maximum binding values, with IMAB362-DM4 exhibiting higher maximum binding (Table 8).

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

Binding of IMAB362-DM4 and IMAB362-vcMMAE is strictly dependent on the presence of their 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 human CLDN18.1 protein. HEK293~mock were used as negative controls (Figure 6, Table 8).

IMAB362 and the toxin-conjugated antibodies IMAB362-DM4 and IMAB362-vcMMAE bound with similar relative affinities to HEK293~CLDN18.2 cells ectopically expressing human CLDN18.2 (Table 8). Moreover, IMAB362, DM4-, and MMAE-conjugated IMAB362 did not exhibit cross-reactivity to human CLDN18.1 or to untransfected cells (Figure 6B and C).

Example 5. Efficacy and specificity of IMAB362-ADC in vitro

1. Effect on cell viability

The effects of IMAB362-DM4 and IMAB362-vcMMAE on cell viability were tested using several human gastric and pancreatic cancer cell lines endogenously and ectopically expressing CLDN18.2 using an XTT-based colorimetric assay for spectrophotometric quantification of metabolically active cells. Antitumor activity was tested at different antibody concentrations ranging from 3 to 16,875 ng/mL (Figure 7, Table 9).

IMAB362-DM4 and IMAB362-vcMMAE effectively inhibited the in vitro viability of gastric cancer cell line NUGC-4, NCI-N87~CLDN18.2 and pancreatic cancer cell line BxPC-3~CLDN18.2 (Figure 7). Both IMAB362-toxin conjugates inhibited the viability of NUGC-4 cells endogenously expressing CLDN18.2 (EC ₅₀ values: 155-631 ng/mL, maximum reduction in viability: ≥ 85%), BxPC-3-CLDN18.2 cells ectopically expressing CLDN18.2 (EC ₅₀ values: 43-54 ng/mL, maximum reduction in viability: ≥ 83%) and NCI-N87~CLDN18.2 cells (EC ₅₀ values: 75-180 ng/mL, maximum reduction in viability: 45-61%) at similar concentrations (Figure 7, Table 9).

Table 9. Overview of cell viability assays performed using IMAB362-toxin conjugates on CLDN18.2-positive cell lines

Cell line IMAB362-DM4 IMAB362-vcMMAE EC ₅₀ [ng/ml] Decreased viability [%] EC ₅₀ [ng/ml] Viability reduction [%] NCI-N87~CLDN18.2 75 49 100 45 108 61 180 56 NUGC-4 10cF7-5 grade 3a 155 88 338 86 NUGC-4 10cE8 372 69 631 67 BxPC-3~CLDN18.2 43 83 48 86 54 87 43 84

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

The specificity of the toxin-conjugated IMAB362 antibody was analyzed using HEK293 transfectants overexpressing human CLDN18.2 or the highly homologous human CLDN18.1 protein. HEK293 cells stably transfected with empty vector were used as negative controls (Figure 9). IMAB362-vcMMAE reduced cell viability on CLDN18.2-positive but not on CLDN18.2-negative cells. The effect was strictly CLDN18.2-dependent because no inhibition of cell proliferation was observed in cells expressing the homologous 18.1 protein (Figure 9).

In conclusion, IMAB362-vcMMAE and IMAB362-DM4 demonstrated similar potency in vitro, and both ADCs were highly effective in inhibiting cell viability of several human gastric and pancreatic cancer cell lines. The effect was strictly dependent on target expression.

2. "The Bystander Effect"

The bystander activity of IMAB362-DM4 and IMAB362-vcMMAE in vitro was determined using mixed tumor cell cultures consisting of CLDN18.2-positive and CLDN18.2-negative cell lines. Target-negative PA-1(Luc) cells stably expressing firefly luciferase were used as reporter cells to measure cell lysis.

Luciferase activity of co-cultures of luciferase-expressing PA-1(Luc) cells and luciferase-negative NUGC-4 cells showed that treatment with IMAB362-DM4 or IMAB362-vcMMAE effectively eliminated target-negative PA-1(Luc) cells in the presence of target-positive NUGC-4 cells. Furthermore, PA-1(Luc) cells were not affected in the absence of CLDN18.2-expressing cells (Figure 10).

Thus, IMAC362-DM4 and IMAB362-vcMMAE-ADC were able to induce a bystander effect on neighboring CLDN18.2-negative tumor cells. Both toxins were efficiently released from IMAB362 in CLDN18.2-positive tumor cells and, due to their membrane permeability, were able to exert cytotoxic activity on bystander cells.

Example 6. Antitumor efficacy of IMAB362-ADC in vivo

1. Maximum Tolerable Dose Studies

In a first in vivo experiment, the maximum tolerated dose (MTD) of IMAB362-DM4 and IMAB362-vcMMAE was determined in nude mice bearing advanced human pancreatic xenograft tumors BxPC-3~CLDN18.2. The MTD refers to the highest dose of treatment that will achieve the target effect without unacceptable toxicity.

1.1. MPD IMAB362-DM4

BxPC-3~CLDN8.2 cells ectopically expressing human CLDN18.2 were injected subcutaneously into the flank of female Hsd:Athymic Nude-Foxn1nu mice. After tumors reached a mean size of 75±13 ^mm3 (mean±SD) on day 13, mice were divided into control and antibody groups. Mice received a single dose of 7.5 or 15 mg/kg IMAB362-DM4 by bolus i.v. injection on day 14 or repeated doses of 15 mg/kg IMAB362-DM4 by bolus i.v. injection on day 14 and day 21, respectively. Control group mice received vehicle on day 14. Animals were sacrificed on day 49 post-transplantation. For toxicity testing, blood samples were collected and organs were prepared and stored for further histopathological studies.

Tumor growth

IMAB362-DM4 inhibited tumor growth in mice bearing advanced human BxPC-3~CLDN8.2 xenograft tumors. Single or repeated treatment with IMAB362-DM4 resulted in almost complete tumor regression in all treated mice during the observation period of the study (49 days) regardless of the dose. Thus, a single dose of 7 mg/kg IMAB362-DM4 may be sufficient for complete tumor remission (Figure 11).

Health status

Animal body weight, behavior, and general health were monitored twice weekly. All animals showed normal body weight throughout the experiments (Figure 12). No behavioral abnormalities were observed. However, one animal died after the second intravenous dose of 15 mg/kg IMAB362-DM4 for an unknown reason.

Clinical tests

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

In conclusion, 15 mg/kg IMAB362-DM4 (equivalent to 45 mg/ ^m2 in humans) as a single dose was well tolerated in mice and demonstrated high antitumor efficacy in CLDN18.2-positive xenografts. Due to limitations in concentration and injection volume, intravenous injection of higher doses was not possible, and the maximum tolerated single dose has not been determined.

Histological analysis

For histological analysis, paraffin sections of brain, heart, kidney, liver, pancreas, spleen, and stomach were stained with hematoxylin and eosin and examined microscopy for IMAB362-vcMMAE-mediated morphological changes. No morphological changes were observed in tissue sections from IMAB362-DM4-treated animals compared to vehicle-treated mice. Notably, stomach, the only tissue expressing murine Cldn18.2, did not show antibody-mediated tissue damage (Figure 14).

1.2. MPD IMAB362-vcMMAE

The MTD of IMAB362-vcMMAE was tested using the same mouse model as for IMAB362-DM4 (1.1). Mice were randomized to groups after tumors reached a mean of 111±27 ^mm3 (mean±SD) on day 13 and treated with a single bolus i.v. injection of 8 or 16 mg/kg IMAB362-vcMMAE (equivalent to 24 and 48 mg/ ^m2 in humans) on day 14 or repeated doses of bolus i.v. injections of 16 mg/kg IMAB362-vcMMAE on days 14 and 21. Control mice received vehicle control on day 14. Animals were sacrificed on day 37. Clinical biochemical analysis was performed and organs were collected and stored for further histopathological studies.

Tumor growth

IMAB362-vcMMAE treatment induced tumor regression and further inhibited tumor growth in mice bearing advanced human BxPC-3~CLDN8.2 xenograft tumors. At the end of the study (37 days), single or repeated IMAB362-vcMMAE treatment resulted in nearly complete tumor regression in all treated mice regardless of dose. Thus, a single dose of 8 mg/kg IMAB362-vcMMAE may be sufficient to induce complete tumor remission (Figure 15).

Health status

The body weight, behavior, and general health of the animals were monitored twice a week. All animals showed normal body weight throughout the experiments (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, such abnormal behavior was not observed in any other animal or after the second administration of IMAB362-vcMMAE.

Clinical Chemistry

We determined serum levels of surrogate markers for hepato-, nephro-, or pancreatic toxicity (alanine transaminase (GPT), aspartate transaminase (GOT), glutamate dehydrogenase (GLDH), alpha-amylase, cholinesterase, creatinine kinase (CK), lactate dehydrogenase (LDH), lipase, urea, glucose, total protein, and albumin). No significant abnormalities in surrogate markers were observed in the serum of IMAB362-vcMMAE-treated animals compared with the vehicle control group (Figure 17). Creatinine and gamma-glutamyl transferase were below the detection limit in all groups. Thus, no evidence of liver, pancreas, or nephrotoxicity was observed in clinical chemistry assays across the dose range evaluated.

Histological analysis

For histological analysis, paraffin sections of brain, heart, kidney, liver, pancreas, spleen, and stomach were stained with hematoxylin and eosin and examined microscopy for IMAB362-vcMMAE-mediated morphological changes.

No IMAB362-vcMMAE-associated morphological changes were observed in tissue sections from IMAB362-vcMMAE-treated animals compared to vehicle-treated mice, indicating that IMAB362-vcMMAE does not induce tissue injury or inflammation. Notably, even the stomach, the only tissue expressing murine Cldn18.2, did not show antibody-mediated tissue injury (Figure 22).

2. Efficiency studies

The antitumor effects of IMAB362-DM4 and IMAB362-vcMMAE were further analyzed in vivo in athymic nude mice Nude-Foxn1 ^nu mice subcutaneously transplanted with human carcinoma cells endogenously or ectopically expressing CLDN18.2. The optimal therapeutic dose of IMAB362-DM4 and IMAB362-vcMMAE in animal tumor models was determined in dose-ranging studies (Figure 18 and Figure 20, respectively). Further efficacy studies in human xenograft tumors were performed with the optimal dose of IMAB362-DM4 and IMAB362-vcMMAE (Figure 19 and Figure 21, respectively).

IMAB362-DM4 and IMAB362-vcMMAE potently inhibited tumor growth and improved survival of tumor-bearing mice in different early xenograft models (initiation of therapy 3 days after tumor implantation) as well as in advanced solid tumor treatments (initiation of therapy at tumor size ~100 mm ³ ).

Treatment of advanced xenografted human gastric tumors NCI-N87~CLDN18.2

In a dose-ranging study, the antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE was analyzed in mice bearing advanced CLDN18.2-positive NCI-N87~CLDN18.2 xenograft tumors. Thirteen days after transplantation, animals were treated with 15.2, 7.6, or 3.8 mg/kg IMAB362-DM4 or 16, 8, or 4 mg/kg IMAB362-vcMMAE or vehicle control, administered as single bolus i.v. injections. Control animals received 8 mg/kg unconjugated IMAB362 (twice weekly, i.v./i.b.).

IMAB362-DM4 and IMAB362-vcMMAE potently inhibited tumor growth, mediated tumor regression and prolonged survival of tumor-bearing mice in a dose-dependent manner, while naked IMAB362 antibody did not exhibit statistically significant antitumor effects in this preferred treatment model (Figure 18). Both toxin-conjugated IMAB362 antibodies prolonged survival of tumor-bearing mice (median survival: 73 days in the vehicle group versus 143 days in the IMAB362-vcMMAE 16 mg/kg group and 136 days in the IMAB362-DM4 15.2 mg/kg group) (Figure 18).

Treatment of early xenotransplanted human gastric tumors NUGC-4 10cF7-5 sort3a

The antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE was analyzed in mice subcutaneously transplanted with NUGC-4 10cF7-5 sort3a gastric carcinoma cells endogenously expressing CLDN18.2. Animals were treated on day 3 post-transplantation with a single i.v. injection of 15.2 mg/kg IMAB362-DM4, 16 mg/kg IMAB362-vcMMAE, or vehicle control.

IMAB362-DM4 and IMAB362-vcMMAE prevented tumor growth in treated animals, whereas all control mice developed tumors (p<0.0001) (Figure 19). After a predetermined observation time of 120 days, 9 out of 10 animals that received IMAB362-DM4 or IMAB362-vcMMAE were alive and tumor-free, whereas all animals in the vehicle control group had to be euthanized due to termination criteria no later than 41 days after transplantation (median survival 34 days, p<0.0003) (Figure 19).

Treatment of advanced tumors of xenografted human pancreatic tumors with BxPC-3~CLDN18.2

The dose-dependent antitumor activity of IMAB362-DM4 and IMAB362-vcMMAE in vivo was analyzed in a dose-ranging study in mice bearing advanced BxPC-3~CLDN18.2 xenografted pancreatic 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, vehicle, administered as single bolus i.v. injections or repeated doses of 8 mg/kg IMAB362 (twice weekly, i.v./i.b.).

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

Treatment of early xenografted human pancreatic tumors with DAN-G 1C5F2

The in vivo antitumor activity of IMAB362-DM4 and IMAB362-vcMMAE was tested in mice subcutaneously transplanted with DAN-G 1C5F2 pancreatic carcinoma cells endogenously expressing CLDN18.2. DAN-G 1C5F2 cells contain exceptionally low amounts of CLDN18.2 on the cell surface; however, significant amounts of protein or RNA are detectable by immunoblot or qRT-PCR. IHC analyses of DAN-G 1C5F2 xenograft tumors showed that only a subset of tumor cells exhibited moderate to strong staining for membrane-associated CLDN18.2. Thus, DAN-G 1C5F2 xenograft tumors may be amenable to treatment with antibody-drug conjugates that exhibit a bystander killing effect. Animals were treated on day 3 post-transplantation with a single i.v. injection of 15.2 mg/kg IMAB362-DM4, 16 mg/kg IMAB362-vcMMAE, or vehicle control.

IMAB362-DM4 and IMAB362-vcMMAE significantly inhibited tumor growth and prolonged survival of tumor-bearing mice compared to vehicle control (Figure 21). In the majority of mice (>50%), tumor growth was completely prevented. After an observation period of 120 days, 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 vehicle group animals had to be euthanized within 31 days due to withdrawal criteria such as cancer cachexia (median survival 24 days) (Figure 21). Both conjugates, IMAB362-DM4 and IMAB362-vcMMAE, significantly inhibited tumor growth and prolonged survival of mice bearing xenografted tumors exhibiting heterogeneous expression of CLDN18.2.

Thus, tumors with low and/or heterogeneous CLDN18.2 expression (e.g., NUGC-4 and DAN-G xenograft tumors) can be effectively treated with IMAB362-DM4 or IMAB362-vcMMAE, and a large proportion of tumor-bearing animals were cured. The antitumor activity of both ADCs can be explained based on the observed effect: the release of cell membrane-permeable forms of DM4 and MMAE after cellular processing facilitates the killing of adjacent tumor cells, even if they are target-negative. Thus, both ADCs are highly effective in killing tumors containing only fractions of CLDN18.2-positive cells.

Example 7 Induction of Apoptosis

The cytotoxicity of toxin-conjugated IMAB362 was assessed by apoptosis assays measuring caspase 3/7 activity and phosphatidylserine externalization. Caspase activation is one of the earliest measurable markers of apoptosis and 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 specific caspase 3/7 proluminogenic 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 was translocated from the inner leaflet of the plasma membrane to the outer leaflet immediately after the induction of apoptosis. To distinguish between living and dead cells, cells were stained with the DNA dye propidium iodide (PI).

To analyze apoptosis induction, CLDN18.2-positive NUGC4 cells were treated with a single dose of IMAB362-toxin conjugates 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 test the induction of apoptosis by IMAB362 toxin-conjugated antibodies. At 4 days post-treatment, ~50% of IMAB362-DM4- or IMAB362-vcMMAE-treated cells were found to be annexin V or annexin V/PI positive, indicating that cell death occurred via apoptosis induction. In contrast, naked IMAB362 without cross-linkers did not induce apoptosis over the concentration range used (Figure 23B).

Thus, treatment of CLDN18.2-positive tumor cells with IMAB362 conjugated to DM4 or vcMMAE induces apoptosis.

Example 8. Treatment of ADVANCED xenografted human gastric tumors with NUGC-4 10cF7-5

The antitumor efficacy of IMAB362-DM4 and IMAB362-vcMMAE was analyzed in mice subcutaneously transplanted with NUGC-4 10cF7-5 sort3a gastric carcinoma cells endogenously expressing CLDN18.2. Animals bearing advanced tumors (tumor size ~200 mm3) were treated on day 10 post-transplantation with intravenous injection of 15.2 mg/kg IMAB362-DM4, 16 mg/kg IMAB362-vcMMAE, or vehicle control and after tumor relapse in the IMAB362-conjugated antibody treatment group with a second injection of the corresponding drug (day 38).

IMAB362-DM4 and IMAB362-vcMMAE significantly inhibited tumor growth and mediated tumor regression in all treated animals (day 52), whereas all control mice developed tumors (IMAB362-DM4: p<0.05, IMAB362-vcMMAE: p<0.001) (Figure 24). At 28 days post-therapy (day 38 post-transplantation), recurrent tumor growth (tumor size ≥~100 ^mm3 ) was observed in 50% of IMAB362-DM4-treated animals. A second injection of respective IMAB362-DM4 and IMAB362-vcMMAE again resulted in partial or complete tumor remission. Tumor recurrence was finally observed in 7 of 8 animals in the IMAB362-DM4 group and 4 of 8 animals in the IMAB362-vcMMAE group. After the predetermined treatment end time (day 108 post-transplantation), 4 of 8 animals in the IMAB362-vcMMAE group and one animal in both the IMAB362-DM4 and IMAB362 groups were alive, while all vehicle group animals were the last to die at day 52 post-transplantation (median survival 32.5 days for vehicle, 90 days for IMAB362-DM4 (p<0.0003 vs. vehicle) and not determined for IMAB362-vcMMAE (p<0.0003 vs. vehicle)) (Figure 24).

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 with unconjugated IMAB362 using human gastric carcinoma NGGC-4 10cF7_5 sort3a p3151#10 cells endogenously expressing CLDN18.2 (Figure 25, Table 10).

Table 10. ADCC activity of IMAB362-DM4 and IMAB362-vcMMAE on NUGC-4 10cF7_5 sort3a p3151 #10 cells

Donor IMAB362 IMAB362-DM4 IMAB362-vcMMAE Max.lysis [%] EC ₅₀ [ng/ml] Max.lysis [%] EC ₅₀ [ng/ml] Max.lysis [%] EC ₅₀ [ng/ml] Donor 1 75 1228 66 1151 73 1085 Donor 2 82 142 92 217 92 275

Both toxin-conjugated antibodies, IMAB362-DM4 and IMAB362-vcMMAE, demonstrated similar EC50 and maximum lysis values compared to unconjugated IMAB362 antibody, indicating that ADCC activity was retained after drug conjugation.

Complement dependent cytotoxicity (CDC)

The CDC activity of IMAB362-DM4 and IMAB362-vcMMAE was analyzed in human gastric carcinoma cells endogenously expressing CLDN18.2, NGGC-4 10cF7_5 sort3A p3151#10, and KATO-III FGF-BP#12 adM p3151#25 (Figure 26, Table 11).

Table 11. CDC activity of IMAB362-DM4 and IMAB362-vcMMAE on endogenously CLDN18.2-expressing carcinoma cells.

Cell line IMAB362 IMAB362-DM4 IMAB362-vcMMAE Max.lysis [%] EC ₅₀ [ng/ml] Max.lysis [%] EC ₅₀ [ng/ml] Max.lysis [%] EC ₅₀ [ng/ml] KATO-III FGF-BP #12 adM p3151 #25 43 19292 56 6439 51 7244 NUGC-4 10cF7_5 sort3a p3151 #10 54 28043 48 15759 49 28338

CDC activity was not affected by toxin conjugation with the IMAB362 antibody. Both toxin-conjugated antibodies, IMAB362-DM4 and IMAB362-vcMMAE, demonstrated at least similar EC ₅₀ values and maximal lysis compared to unconjugated IMAB362 antibody.

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

LIST OF SEQUENCES

<110> Ganymed Pharmaceuticals AG

<120> DRUG CONJUGATES COMPRISING ANTIBODIES AGAINST CLAUDIN 18.2

<130> 342-84 PCT

<150> PCT/EP2015/058206

<151> 2015-04-15

<160> 51

<170> PatentIn version 3.5

<210> 1

<211> 261

<212> PRT

<213> Homo sapiens

<400> 1

Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile

1 5 10 15

Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr

20 25 30

Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly

35 40 45

Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg

50 55 60

Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg

65 70 75 80

Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val

85 90 95

Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser

100 105 110

Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser

115 120 125

Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val

130 135 140

Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly

145 150 155 160

Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe

165 170 175

Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met

180 185 190

Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala

195 200 205

Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly

210 215 220

Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile

225 230 235 240

Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser

245 250 255

Lys His Asp Tyr Val

260

<210> 2

<211> 261

<212> PRT

<213> Homo sapiens

<400> 2

Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu

1 5 10 15

Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr

20 25 30

Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly

35 40 45

Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg

50 55 60

Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg

65 70 75 80

Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val

85 90 95

Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser

100 105 110

Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser

115 120 125

Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val

130 135 140

Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly

145 150 155 160

Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe

165 170 175

Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met

180 185 190

Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala

195 200 205

Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly

210 215 220

Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile

225 230 235 240

Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser

245 250 255

Lys His Asp Tyr Val

260

<210> 3

<211> 10

<212> PRT

<213> Homo sapiens

<400> 3

Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Homo sapiens

<400> 4

Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln

1 5 10

<210> 5

<211> 14

<212> PRT

<213> Homo sapiens

<400> 5

Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe

1 5 10

<210> 6

<211> 13

<212> PRT

<213> Homo sapiens

<400> 6

Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly

1 5 10

<210> 7

<211> 13

<212> PRT

<213> Homo sapiens

<400> 7

Asp Ser Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile

1 5 10

<210> 8

<211> 55

<212> PRT

<213> Homo sapiens

<400> 8

Met Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala

1 5 10 15

Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser

20 25 30

Gly Phe Thr Glu Cys Arg Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala

35 40 45

Met Leu Gln Ala Val Arg Ala

50 55

<210> 9

<211> 24

<212> PRT

<213> Homo sapiens

<400> 9

Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser Ala Lys

1 5 10 15

Ala Asn Met Thr Leu Thr Ser Gly

20

<210> 10

<211> 40

<212> PRT

<213> Homo sapiens

<400> 10

Ala Asn Met Leu Val Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr

1 5 10 15

Thr Gly Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe

20 25 30

Gly Ala Ala Leu Phe Val Gly Trp

35 40

<210> 11

<211> 153

<212> PRT

<213> Homo sapiens

<400> 11

Met Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala

1 5 10 15

Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser

20 25 30

Gly Phe Thr Glu Cys Arg Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala

35 40 45

Met Leu Gln Ala Val Arg Ala Leu Met Ile Val Gly Ile Val Leu Gly

50 55 60

Ala Ile Gly Leu Leu Val Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile

65 70 75 80

Gly Ser Met Glu Asp Ser Ala Lys Ala Asn Met Thr Leu Thr Ser Gly

85 90 95

Ile Met Phe Ile Val Ser Gly Leu Cys Ala Ile Ala Gly Val Ser Val

100 105 110

Phe Ala Asn Met Leu Val Thr Asn Phe Trp Met Ser Thr Ala Asn Met

115 120 125

Tyr Thr Gly Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr

130 135 140

Phe Gly Ala Ala Leu Phe Val Gly Trp

145 150

<210> 12

<211> 107

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 12

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 13

<211> 326

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 13

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

1 5 10 15

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

20 25 30

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

35 40 45

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

50 55 60

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

65 70 75 80

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

85 90 95

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

100 105 110

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

165 170 175

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 14

<211> 466

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 14

Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe

35 40 45

Ser Ser Tyr Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu

50 55 60

Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Tyr Asp Tyr Pro Trp Phe Ala Tyr Trp Gly Gln

115 120 125

Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val

130 135 140

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

145 150 155 160

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

165 170 175

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

195 200 205

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

210 215 220

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

225 230 235 240

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Lys

465

<210> 15

<211> 467

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 15

Met Asp Trp Leu Trp Asn Leu Leu Phe Leu Met Ala Ala Ala Gln Ser

1 5 10 15

Ile Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

50 55 60

Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala

65 70 75 80

Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys

465

<210> 16

<211> 465

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 16

Met Glu Trp Ile Trp Ile Phe Leu Phe Ile Leu Ser Gly Thr Ala Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asp Tyr Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Phe Cys Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly

115 120 125

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

130 135 140

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

145 150 155 160

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

165 170 175

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

180 185 190

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

195 200 205

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

210 215 220

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

225 230 235 240

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

245 250 255

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

260 265 270

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

275 280 285

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

290 295 300

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

305 310 315 320

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

325 330 335

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

340 345 350

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

355 360 365

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

370 375 380

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

385 390 395 400

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

405 410 415

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

420 425 430

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

435 440 445

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

Lys

465

<210> 17

<211> 467

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 17

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys

465

<210> 18

<211> 466

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 18

Met Glu Trp Arg Ile Phe Leu Phe Ile Leu Ser Gly Thr Ala Gly Val

1 5 10 15

His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro

20 25 30

Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

35 40 45

Asp Tyr Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu

50 55 60

Trp Ile Gly Glu Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu

65 70 75 80

Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr

85 90 95

Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr

100 105 110

Phe Cys Ala Arg Gly Val Leu Leu Arg Ala Met Asp Tyr Trp Gly Gln

115 120 125

Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

130 135 140

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

145 150 155 160

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

165 170 175

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

195 200 205

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

210 215 220

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

225 230 235 240

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Lys

465

<210> 19

<211> 469

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 19

Met Asp Trp Ile Trp Ile Met Leu His Leu Leu Ala Ala Ala Thr Gly

1 5 10 15

Ile Gln Ser Gln Val His Leu Gln Gln Ser Gly Ser Glu Leu Arg Ser

20 25 30

Pro Gly Ser Ser Val Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Val

35 40 45

Phe Pro Phe Ala Tyr Met Ser Trp Ile Arg Gln Lys Pro Gly His Gly

50 55 60

Phe Glu Trp Ile Gly Asp Ile Leu Pro Ser Ile Gly Arg Thr Ile Tyr

65 70 75 80

Gly Glu Lys Phe Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Val Ser

85 90 95

Asn Thr Ala Tyr Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala

100 105 110

Ile Tyr Tyr Cys Ala Arg Gly Glu Gly Tyr Gly Ala Trp Phe Ala Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly

130 135 140

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

245 250 255

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

260 265 270

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

305 310 315 320

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

325 330 335

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

340 345 350

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

355 360 365

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

370 375 380

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

385 390 395 400

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405 410 415

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

420 425 430

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

435 440 445

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

450 455 460

Leu Ser Pro Gly Lys

465

<210> 20

<211> 240

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 20

Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val Ser

1 5 10 15

Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr

20 25 30

Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg

65 70 75 80

Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr

100 105 110

Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr

115 120 125

Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

130 135 140

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

145 150 155 160

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

165 170 175

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

180 185 190

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

195 200 205

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

210 215 220

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235 240

<210> 21

<211> 235

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 21

Met His Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile

20 25 30

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Ser

35 40 45

Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser

50 55 60

Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

85 90 95

Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg

100 105 110

Ser Ser Tyr Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

130 135 140

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

145 150 155 160

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

165 170 175

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

180 185 190

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

195 200 205

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

210 215 220

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 22

<211> 234

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 22

Met Glu Phe Gln Thr Gln Val Phe Val Phe Val Leu Leu Trp Leu Ser

1 5 10 15

Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser

20 25 30

Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn

35 40 45

Val Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

50 55 60

Lys Ala Leu Ile Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp

65 70 75 80

Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp

100 105 110

Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 23

<211> 240

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 23

Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser

1 5 10 15

Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala

20 25 30

Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg

65 70 75 80

Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr

100 105 110

Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr

115 120 125

Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

130 135 140

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

145 150 155 160

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

165 170 175

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

180 185 190

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

195 200 205

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

210 215 220

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235 240

<210> 24

<211> 240

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 24

Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val Ser

1 5 10 15

Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr

20 25 30

Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg

65 70 75 80

Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr

100 105 110

Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr

115 120 125

Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

130 135 140

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

145 150 155 160

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

165 170 175

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

180 185 190

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

195 200 205

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

210 215 220

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235 240

<210> 25

<211> 239

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 25

Met Asp Ser Gln Ala Gln Val Leu Ile Leu Leu Leu Leu Trp Val Ser

1 5 10 15

Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala

20 25 30

Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg

65 70 75 80

Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr

100 105 110

Tyr Cys Lys Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 26

<211> 240

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 26

Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser

1 5 10 15

Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala

20 25 30

Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg

65 70 75 80

Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp

85 90 95

Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr

100 105 110

His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr

115 120 125

Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

130 135 140

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

145 150 155 160

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

165 170 175

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

180 185 190

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

195 200 205

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

210 215 220

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235 240

<210> 27

<211> 240

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 27

Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser

1 5 10 15

Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala

20 25 30

Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg

65 70 75 80

Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr

100 105 110

Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr

115 120 125

Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

130 135 140

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

145 150 155 160

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

165 170 175

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

180 185 190

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

195 200 205

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

210 215 220

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235 240

<210> 28

<211> 234

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 28

Met Glu Ser Gln Thr Leu Val Phe Ile Ser Ile Leu Leu Trp Leu Tyr

1 5 10 15

Gly Ala Asp Gly Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser

20 25 30

Met Ser Val Gly Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn

35 40 45

Val Val Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro

50 55 60

Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp

65 70 75 80

Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr

100 105 110

Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 29

<211> 117

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 29

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115

<210> 30

<211> 118

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 30

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 31

<211> 116

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 31

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 32

<211> 118

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 32

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 33

<211> 118

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 33

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Val Leu Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 34

<211> 120

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 34

Gln Val His Leu Gln Gln Ser Gly Ser Glu Leu Arg Ser Pro Gly Ser

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Val Phe Pro Phe

20 25 30

Ala Tyr Met Ser Trp Ile Arg Gln Lys Pro Gly His Gly Phe Glu Trp

35 40 45

Ile Gly Asp Ile Leu Pro Ser Ile Gly Arg Thr Ile Tyr Gly Glu Lys

50 55 60

Phe Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Val Ser Asn Thr Ala

65 70 75 80

Tyr Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr

85 90 95

Cys Ala Arg Gly Glu Gly Tyr Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 35

<211> 113

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 35

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 36

<211> 106

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 36

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 37

<211> 107

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 37

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 38

<211> 113

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 38

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 39

<211> 113

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 39

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 40

<211> 112

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 40

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 41

<211> 113

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 41

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr His Cys Gly Gln

85 90 95

Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 42

<211> 113

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 42

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 43

<211> 107

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: Translation of PCR product

<400> 43

Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly

1 5 10 15

Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 44

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Epitope

<400> 44

Met Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr

1 5 10 15

<210> 45

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Epitope

<400> 45

Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn

1 5 10 15

<210> 46

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Epitope

<400> 46

Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly Leu

1 5 10 15

<210> 47

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Epitope

<400> 47

Pro Val Thr Ala Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys

1 5 10 15

<210> 48

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Epitope

<400> 48

Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser

1 5 10 15

<210> 49

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Epitope

<400> 49

Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr

1 5 10 15

<210> 50

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Epitope

<400> 50

Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg Gly

1 5 10 15

<210> 51

<211> 467

<212> PRT

<213> Artificial Sequence

<220>

<223> chimeric monoclonal antibody

<400> 51

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys

465

Claims (39)

1. A conjugate of an antibody that binds to CLDN18.2 and a therapeutic moiety for the treatment or prevention of an oncological disease characterized by malignant tumor cells expressing CLDN18.2, where the antibody comprises: (a) a heavy chain comprising a variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence represented by SEQ ID NO: 32, and a light chain comprising a variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence represented by SEQ ID NO: 39; or (b) a heavy chain comprising a variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence represented by SEQ ID NO: 30, and a light chain comprising a variable region comprising CDR1, CDR2 and CDR3 of the amino acid sequence represented by SEQ ID NO: 35; and wherein the therapeutic moiety is a cytotoxic or cytostatic agent covalently linked to the antibody. 2. The conjugate according to claim 1, characterized in that the antibody comprises: (a) a heavy chain variable region of an antibody as defined in SEQ ID NO: 32 and a light chain variable region of an antibody as defined in SEQ ID NO: 39; or (b) a heavy chain variable region of an antibody as defined in SEQ ID NO: 30 and a light chain variable region of an antibody as defined in SEQ ID NO: 35. 3. The conjugate according to claim 1 or 2, characterized in that the antibody comprises: (a) an antibody heavy chain sequence comprising the amino acid sequence represented by SEQ ID NO: 17 and an antibody light chain sequence comprising the amino acid sequence represented by SEQ ID NO: 24; or (b) an antibody heavy chain sequence comprising the amino acid sequence represented by SEQ ID NO: 51 and an antibody light chain sequence comprising the amino acid sequence represented by SEQ ID NO: 24. 4. A conjugate according to any one of claims 1-3, characterized in that the cytotoxic or cytostatic agent is a maytansinoid or auristatin. 5. The conjugate of claim 4, wherein the maytansinoid is selected from the group consisting of DM1 and DM4, and wherein the auristatin is selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). 6. A conjugate according to any one of claims 1-5, characterized in that the therapeutic fragment is covalently attached to the antibody by a linker. 7. The conjugate according to claim 6, characterized in that the linker is a cleavable linker. 8. The conjugate according to claim 6, characterized in that the linker is cleaved under intracellular conditions. 9. The conjugate according to claim 6, characterized in that the linker is hydrolyzed at a pH of less than 5.5. 10. The conjugate according to claim 6, characterized in that the linker is cleaved by an intracellular protease. 11. The conjugate according to claim 6, characterized in that the linker is a linker cleavable by cathepsin. 12. The conjugate according to claim 6, characterized in that the linker contains a dipeptide. 13. The conjugate according to claim 12, characterized in that the dipeptide is val-cit or phe-lys. 14. A method for treating or preventing a malignant neoplasm expressing CLDN18.2, comprising administering a conjugate according to any one of claims 1-13 to an oncological patient, wherein the conjugate is administered in an amount effective for treating or preventing a malignant neoplasm expressing CLDN18.2. 15. The method according to claim 14, characterized in that the conjugate is administered in a dose of 3 to 30 mg/kg of body weight. 16. The method according to claim 14 or 15, characterized in that the conjugate is administered in a dose of 9 to 90 mg/ ^m2 of the body surface of a human patient. 17. The method according to any one of paragraphs 14-16, characterized in that a single dose of the conjugate or two or more doses of the conjugate are administered. 18. The method according to any one of paragraphs 14-17, characterized in that the conjugate is administered by intravenous injection. 19. The method according to any one of paragraphs. 14-18, which further comprises performing surgery, chemotherapy and/or radiation therapy. 20. The method according to any one of claims 14-19, characterized in that the expression of CLDN18.2 occurs on the cell surface of malignant tumor cells. 21. The method according to any one of paragraphs 14-20, characterized in that the malignant neoplasm is an adenocarcinoma, in particular an adenocarcinoma at a late stage. 22. The method according to any one of claims 14-21, characterized in that the malignant neoplasm is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastases, Kruckenberg tumor, peritoneal metastases and/or lymph node metastases. 23. The method according to any one of claims 14-22, characterized in that the malignant neoplasm is selected from the group consisting of gastric cancer, esophageal cancer, particularly lower esophageal cancer, esophagogastric junction cancer, and gastroesophageal cancer. 24. The method according to any one of claims 14-23, characterized in that the patient is a HER2/neu negative patient or a HER2/neu positive patient who is not eligible for trastuzumab therapy. 25. The method according to any one of claims 14-24, characterized in that CLDN18.2 has an amino acid sequence corresponding to SEQ ID NO: 1. 26. A pharmaceutical composition for treating or preventing an oncological disease characterized by malignant tumor cells expressing CLDN18.2, wherein said pharmaceutical composition comprises a therapeutically effective amount of a conjugate according to any one of claims 1-13 and a pharmaceutically acceptable diluent, carrier or excipient. 27. A medicinal product for the treatment or prevention of an oncological disease characterized by malignant tumor cells expressing CLDN18.2, wherein said medicinal product comprises a therapeutically effective amount of a drug-antibody conjugate according to any one of claims 1-12. 28. The medicinal product according to claim 27, which is present in the form of a kit containing a container that includes a conjugate. 29. The conjugate of any one of claims 1-13, the pharmaceutical composition of claim 26 or the medicinal product of claim 27 or 28 for use in therapy, in particular for use in a method for treating or preventing a malignant neoplasm, in particular a CLDN18.2-expressing malignant neoplasm. 30. A medicinal product according to any one of claims 27-29, further comprising printed instructions for using the product in a method for treating or preventing a malignant neoplasm, in particular a CLDN18.2-expressing malignant neoplasm. 31. A conjugate or pharmaceutical composition according to claim 29, or a medical preparation according to claim 27 or 28, wherein the method for treating or preventing an oncological disease is a method according to any one of claims 14-25.