Bispecific binding agents binding to CLDN18.2 and CD3

Abstract

To provide novel agents and methods for treatment of cancer diseases.SOLUTION: The present invention provides binding agents comprising at least two binding domains, wherein a first binding domain has specificity for CLDN18.2 and a second binding domain has specificity for CD3, and methods of using these binding agents or nucleic acids encoding therefor for treating cancer.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Cancer of the stomach and esophagus (gastroesophageal; GE) is one of the malignancies with the highest unmet medical need. Gastric cancer is the third leading cause of cancer death worldwide (Prz Gastroenterol. 2019;14(1):26-38). The incidence of esophageal cancer has increased in recent decades, which is consistent with changes in histology and location of the primary tumor. Esophageal adenocarcinoma is now more prevalent than squamous cell carcinoma in the United States and Western Europe, and most tumors are located in the distal esophagus. Despite the aggressiveness of established standard treatments with significant side effects, the overall 5-year survival rate for GE cancer is 20-25%.

[0002] The majority of patients suffer from locally advanced or metastatic disease and must undergo first-line chemotherapy. Treatment regimens are based on platinum and fluoropyrimidine derivatives, often in combination with a third compound (e.g., taxane or anthracycline). Nevertheless, a median progression-free survival of 5 to 7 months and a median overall survival of 9 to 11 months is the best that can be expected.

[0003] The lack of significant efficacy of various new generation chemotherapy combination regimens for these cancers has stimulated research into the use of targeted agents. Trastuzumab has recently been approved for Her2 / neu positive gastroesophageal cancer. However, there remains a high medical need, as only about 20% of patients express this target and are eligible for this treatment.

[0004] The tight junction molecule claudin 18 (CLDN18) is a fully transmembrane protein (tetraspanin) with four transmembrane hydrophobic regions and two extracellular loops (loop 1 is surrounded by hydrophobic regions 1 and 2, and loop 2 is surrounded by hydrophobic regions 3 and 4). CLDN18 exists in two different splice variants described in mouse and human (Niimi, Mol. Cell. Biol. 21:7380-90, 2001). The splice variants (Genbank accession numbers: splice variant 1 (CLDN18.1): NP_057453, NM_016369, and splice variant 2 (CLDN18.2): NM_001002026, NP_001002026) have molecular weights of approximately 27.9 / 27.72 kD. The splice variants CLDN18.1 and CLDN18.2 differ in the N-terminal portion including the first transmembrane (TM) domain and loop 1, but the major protein sequences at the C-terminus are identical.

[0005] In normal tissues, there is no detectable expression of CLDN18.2, except in the stomach, where it is expressed only on short-lived differentiated gastric epithelial cells. CLDN18.2 is maintained during the malignant transformation process and is therefore frequently presented on the surface of human gastric cancer cells. Moreover, this pan-tumor antigen is ectopically activated at significant levels in adenocarcinomas of the esophagus, pancreas, and lung. CLDN18.2 protein has also been localized in lymph node metastases of gastric adenocarcinomas and in distant metastases, especially to the ovaries (so-called Krukenberg tumors).

[0006] The differential expression of claudins such as CLDN18.2 between cancer and normal cells, their membrane localization, and their absence in the majority of normal tissues associated with toxicity make these molecules attractive targets for cancer immunotherapy, and the use of antibody-based therapies targeting CLDN18.2 in cancer treatment promises a high level of therapeutic specificity.

[0007] IMAB362 (Zolbetuximab, formerly known as Claudiximab), a chimeric IgG1 antibody against CLDN18.2, was developed by Ganymed Pharmaceuticals AG. IMAB362 recognizes the first extracellular domain (ECD1) of CLDN18.2 with high affinity and specificity. IMAB362 does not bind to any other members of the claudin family, including the closely related splice variant 1 of claudin 18 (CLDN18.1). IMAB362 exhibits precise tumor cell specificity and bundles two independent and highly potent mechanisms of action. Upon target binding, IMAB362 mediates cell killing primarily by ADCC and CDC. Therefore, IMAB362 efficiently lyses CLDN18.2-positive cells, including human gastric cancer cell lines, in vitro and in vivo. The antitumor effect of IMAB362 was demonstrated in mice bearing xenograft tumors inoculated with CLDN18.2-positive cancer cell lines.

[0008] IgG1 antibodies typically participate in the cellular immune system through the interaction of their Fc domain with Fcγ receptors (FcγRs) expressed on various immune cells, including natural killer cells, which are key drivers of ADCC. However, IgG1 monoclonal antibodies (mAbs) that induce ADCC face several limitations, such as the widespread distribution of low affinity Fc receptor variants in the population (up to 80%) and reduced mAb potency due to IgG1 modifications in vivo (Chames et al., (2009) Br J Pharmacol, 157(2):220-233). Therapeutic antibodies must also compete with the patient's IgG, resulting in the need for high doses of mAbs in vivo. In addition, therapeutic antibodies can interact with FcγRIIb, an inhibitory FcγR expressed by B cells, macrophages, dendritic cells, and neutrophils, resulting in negative signaling that reduces efficacy. Summary of the Invention

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

[0010] The solution to this problem, which is the basis of the present invention, is based on the concept of generating a binding agent that contains at least one binding domain specific for CLDN18.2 (i.e. cancer cells). This binding agent also contains a binding domain specific for the T cell specific antigen CD3, which allows it to bind to T cells and recruit them into a complex, thus allowing the cytotoxic effect of T cells to be targeted to cancer cells. The formation of this complex can induce signaling in the cytotoxic T cells, alone or in combination with accessory cells, resulting in the release of cytotoxic mediators.

[0011] The inventors report for the first time that a binding agent comprising at least one binding domain in Fab format targeting CLDN18.2 and another binding domain in scFv format targeting a T cell-specific antigen such as CD3 can induce potent T cell-mediated lysis and is effective in treating tumor diseases.

[0012] The present invention generally provides binding agents that bind to CLDN18.2, and in particular provides bispecific binding agents that bind to CLDN18.2 and CD3.

[0013] The present invention relates to a binding agent comprising a binding domain having specificity for CLDN18.2 and a binding domain having specificity for CD3, the binding agent comprising at least three polypeptide chains, a first polypeptide chain comprising a variable region of a heavy chain (VH) derived from an immunoglobulin having specificity for CLDN18.2 (VH(CLDN18.2)), a second polypeptide chain comprising a variable region of a VH derived from an immunoglobulin having specificity for CD3 (VH(CD3)) and a variable region of a light chain (VL) derived from an immunoglobulin having specificity for CD3 (VL(CD3)), and a third polypeptide chain comprising a binding domain having specificity for CLDN18.2. A binding agent is provided that comprises a VL derived from an immunoglobulin with specificity for 18.2 (VL(CLDN18.2)).

[0014] In one embodiment, the binding agent of the invention is a bispecific trimeric binding agent.

[0015] In one embodiment, the first polypeptide chain comprises a heavy chain constant region 1 (CH1) derived from an immunoglobulin or a functional variant thereof.

[0016] In one embodiment, the first polypeptide chain and the second polypeptide chain comprise a heavy chain constant region 2 (CH2) derived from an immunoglobulin or a functional variant thereof and a heavy chain constant region 3 (CH3) derived from an immunoglobulin or a functional variant thereof.

[0017] In one embodiment, the third polypeptide chain comprises a light chain constant region (CL) derived from an immunoglobulin or a functional variant thereof.

[0018] In one embodiment, the VH and the CH1, CH2, and CH3 of the first polypeptide chain are arranged from N-terminus to C-terminus in the following order: VH(CLDN18.2)-CH1-CH2-CH3.

[0019] In one embodiment, the VH, VL, and CH2, and CH3 of the second polypeptide chain are arranged from N-terminus to C-terminus in the following order: VH(CD3)-VL(CD3)-CH2-CH3, or VL(CD3)-VH(CD3)-CH2-CH3.

[0020] In one embodiment, the first polypeptide chain interacts with the second polypeptide chain and the third polypeptide chain, In one embodiment, VH(CLDN18.2) and VL(CLDN18.2) interact to form a binding domain with specificity for CLDN18.2, and VH(CD3) and VL(CD3) interact to form a binding domain with specificity for CD3.

[0021] In one embodiment, CH2 of the first polypeptide chain interacts with CH2 of the second polypeptide chain and / or CH3 of the first polypeptide chain interacts with CH3 of the second polypeptide chain, In one embodiment, CH1 of the first polypeptide chain interacts with CL of the third polypeptide chain.

[0022] In one embodiment, the binding agent of the invention comprises an additional binding domain having specificity for CLDN18.2, the second polypeptide chain further comprises VH(CLDN18.2), and the binding agent comprises a fourth polypeptide chain identical to the third polypeptide chain.

[0023] In one embodiment, the binding agent of the invention is a bispecific trimeric binding agent.

[0024] In one embodiment, the second polypeptide chain further comprises a CH1 derived from an immunoglobulin or a functional variant thereof.

[0025] In one embodiment, the immunoglobulin is IgG1, preferably human IgG1. In one embodiment, in the binding agents described herein, VH(CLDN18.2) and / or VL(CLDN18.2) are derived from IgG1, VH(CD3) and / or VL(CD3) are derived from IgG1, and / or CH1, CH2, CH3, and / or CL are derived from IgG1, with IgG1 being preferably human IgG1.

[0026] In one embodiment, the VH, VL, and CH1, CH2, and CH3 of the second polypeptide chain are arranged from N-terminus to C-terminus in the following order: VH(CLDN18.2)-CH1-VH(CD3)-VL(CD3)-CH2-CH3, or VH(CLDN18.2)-CH1-VL(CD3)-VH(CD3)-CH2-CH3.

[0027] In one embodiment, the second polypeptide chain interacts with a fourth polypeptide chain.

[0028] In one embodiment, the VH(CLDN18.2) of the second polypeptide chain interacts with the VL(CLDN18.2) of a fourth polypeptide chain to form an additional binding domain with specificity for CLDN18.2.

[0029] In one embodiment, the CH1 of the second polypeptide chain interacts with the CL of the fourth polypeptide chain.

[0030] In one embodiment, the VH(CD3) comprises the CDR1, CDR2, and CDR3 of an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 58, and 61.

[0031] In one embodiment, the VH(CD3) is (i) comprises a CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 43) or a functional variant thereof, a CDR2 comprising the amino acid sequence RIRSKANNYATYYADSVKG (SEQ ID NO: 50) or a functional variant thereof, and a CDR3 comprising the amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO: 45) or a functional variant thereof; (ii) comprises a CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 43) or a functional variant thereof, a CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKG (SEQ ID NO: 44) or a functional variant thereof, and a CDR3 comprising the amino acid sequence HGNFGDEYVSWFAY (SEQ ID NO: 51) or a functional variant thereof; or (iii) comprises a CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 43) or a functional variant thereof, a CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKG (SEQ ID NO: 44) or a functional variant thereof, and a CDR3 comprising the amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO: 45) or a functional variant thereof.

[0032] In one embodiment, the VL(CD3) comprises CDR1, CDR2, and CDR3 of SEQ ID NO:55.

[0033] In one embodiment, the VL(CD3) comprises a CDR1 comprising the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 46) or a functional variant thereof, a CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 47) or a functional variant thereof, and a CDR3 comprising the amino acid sequence ALWYSNHWV (SEQ ID NO: 48) or a functional variant thereof.

[0034] In one embodiment, VH(CLDN18.2) comprises CDR1, CDR2, and CDR3 of SEQ ID NO:39.

[0035] In one embodiment, VH(CLDN18.2) comprises a CDR1 comprising the amino acid sequence SYWIN (SEQ ID NO: 32) or a functional variant thereof, a CDR2 comprising the amino acid sequence NIYPSDSYTNYNQKFQG (SEQ ID NO: 33) or a functional variant thereof, and a CDR3 comprising the amino acid sequence SWRGNSFDY (SEQ ID NO: 34) or a functional variant thereof.

[0036] In one embodiment, the VL(CLDN18.2) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 42. 2, and CDR3.

[0037] In one embodiment, VL(CLDN18.2) comprises a CDR1 comprising the amino acid sequence KSSQSLLNSGNQKNYLT (SEQ ID NO: 35) or a functional variant thereof, a CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO: 36) or a functional variant thereof, and a CDR3 comprising the amino acid sequence QNDYSYPFT (SEQ ID NO: 37) or a functional variant thereof.

[0038] In one embodiment, (i) VH(CD3) comprises CDR1, CDR2, and CDR3 of SEQ ID NO:58, VL(CD3) comprises CDR1, CDR2, and CDR3 of SEQ ID NO:55, VH(CLDN18.2) comprises CDR1, CDR2, and CDR3 of SEQ ID NO:39, and VL(CLDN18.2) comprises CDR1, CDR2, and CDR3 of SEQ ID NO:42; or (ii) VH(CD3) comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 61, VL(CD3) comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 55, VH(CLDN18.2) comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 39, and VL(CLDN18.2) comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 42, or (iii) VH(CD3) comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 54, VL(CD3) comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 55, VH(CLDN18.2) comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 39, and VL(CLDN18.2) comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 42.

[0039] In one embodiment, VH(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 58 or a functional variant thereof, VL(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 55 or a functional variant thereof, VH(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 39 or a functional variant thereof, and / or VL(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 42 or a functional variant thereof.

[0040] In one embodiment, VH(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 61 or a functional variant thereof, VL(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 55 or a functional variant thereof, VH(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 39 or a functional variant thereof, and / or VL(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 42 or a functional variant thereof.

[0041] In one embodiment, VH(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 54 or a functional variant thereof, VL(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 55 or a functional variant thereof, VH(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 39 or a functional variant thereof, and / or VL(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 42 or a functional variant thereof.

[0042] In one embodiment, in the first polypeptide chain, CH1 is linked to CH2 by a peptide linker. In one embodiment, the peptide linker comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 27) or a functional variant thereof.

[0043] In one embodiment, the VH(CD3) or VL(CD3) is linked to CH2 by a peptide linker. In one embodiment, the VH(CD3) or VL(CD3) is linked to CH1 by a peptide linker. In one embodiment, the peptide linker is 4 S) x or a functional variant thereof, wherein X is 2, 3, 4, 5, or 6. In one embodiment, the peptide linker comprises the amino acid sequence (G 4 S) 2 (SEQ ID NO: 26) or a functional variant thereof. In one embodiment, the peptide linker connecting the VH(CD3) or VL(CD3) and CH2 comprises the amino acid sequence KTHTCPPCP (SEQ ID NO: 21) or a functional variant thereof. In one embodiment, the peptide linker comprises the amino acid sequence (G 4 S) 2 KTHTCPPCP (SEQ ID NO: 23) or a functional variant thereof. In one embodiment, the peptide linker comprises the amino acid sequence EPKSSDKTHTCPPCP (SEQ ID NO: 22) or a functional variant thereof.

[0044] In one embodiment, the VH(CD3) and VL(CD3) are linked together by a peptide linker. In one embodiment, the peptide linker has the amino acid sequence (GKPGS) x or a functional variant thereof, wherein x is 2, 3, 4, 5, or 6. In one embodiment, the peptide linker comprises the amino acid sequence (GKPGS): 4 (SEQ ID NO: 11) or a functional variant thereof.

[0045] In certain embodiments, the CH1 domain, CH2 domain, and / or CH3 domain of the binding agent of the invention comprise one or more amino acid modifications, in particular substitutions and / or deletions, at positions corresponding to positions in human IgG1 according to EU numbering. In one embodiment, the CH1 of the first polypeptide chain and / or the second polypeptide chain comprises an amino acid sequence that comprises an aspartic acid residue at position 208 according to EU numbering. In one embodiment, the CH1 of the first polypeptide chain comprises an amino acid sequence that comprises an aspartic acid residue at position 208 according to EU numbering, and the CH1 of the second polypeptide chain comprises an amino acid sequence that comprises an asparagine residue at position 208 according to EU numbering.

[0046] Moreover, in certain embodiments, a binding agent of the invention does not substantially bind, e.g., does not detectably bind, human FcγRI, IIa, IIb, and / or IIIa. In one embodiment, CH2 of the first polypeptide chain and / or the second polypeptide chain comprises an amino acid sequence that includes one or more of the following: a proline residue at position 233, a valine residue at position 234, an alanine residue at position 235, a deletion at position 236, a lysine residue at position 267, and a glutamic acid residue at position 295, according to EU numbering. In one embodiment, CH2 of the first polypeptide chain and the second polypeptide chain comprise an amino acid sequence comprising a proline residue at position 233, a valine residue at position 234, an alanine residue at position 235, a deletion at position 236, and a lysine residue at position 267 according to EU numbering, and CH2 of the first polypeptide chain further comprises a glutamine residue at position 295 according to EU numbering, and CH2 of the second polypeptide chain further comprises a glutamine residue at position 295 according to EU numbering.

[0047] In one embodiment, CH3 of the first polypeptide chain and / or the second polypeptide chain comprises an amino acid sequence comprising one or more of the following: a glutamine residue at position 357, a lysine residue at position 364, an aspartic acid residue at position 368, a serine residue at position 370, an aspartic acid residue at position 384, a glutamic acid residue at position 418, and an aspartic acid residue at position 421 according to EU numbering. In one embodiment, CH3 of the first polypeptide chain comprises one or more of the following: a glutamine residue at position 357, a serine residue at position 364, an aspartic acid residue at position 368, a serine residue at position 370, an aspartic acid residue at position 384, a glutamic acid residue at position 418, and an aspartic acid residue at position 421 according to EU numbering. and CH3 of the second polypeptide chain comprises an amino acid sequence comprising a glutamine residue at position 357, a lysine residue at position 364, a leucine residue at position 368, a lysine residue at position 370, an asparagine residue at position 384, a glutamine residue at position 418, and an asparagine residue at position 421 according to EU numbering.

[0048] In one embodiment, the first polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 28, or a functional variant thereof. In one embodiment, the second polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 30, or a functional variant thereof. In one embodiment, the second polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 29, or a functional variant thereof. In one embodiment, the third polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 31, or a functional variant thereof.

[0049] In one embodiment, the first polypeptide chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73, or a functional variant thereof. In one embodiment, the second polypeptide chain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 75, 76, and 77, or a functional variant thereof. In one embodiment, the third polypeptide chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 78, or a functional variant thereof.

[0050] Preferably, in one embodiment, (i) the first polypeptide chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 73 or a functional variant thereof; (ii) the second polypeptide chain comprises or consists of an amino acid sequence represented by SEQ ID NO: 75 or a functional variant thereof; (iii) the third polypeptide chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 78 or a functional variant thereof.

[0051] Preferably, in one embodiment, (i) the first polypeptide chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 73 or a functional variant thereof; (ii) the second polypeptide chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 74 or a functional variant thereof; (iii) the third polypeptide chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 78 or a functional variant thereof.

[0052] Preferably, in one embodiment, (i) the first polypeptide chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 73 or a functional variant thereof; (ii) the second polypeptide chain comprises or consists of an amino acid sequence represented by SEQ ID NO: 76 or a functional variant thereof; (iii) the third polypeptide chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 78 or a functional variant thereof.

[0053] Preferably, in one embodiment, (i) the first polypeptide chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 73 or a functional variant thereof; (ii) the second polypeptide chain comprises or consists of an amino acid sequence represented by SEQ ID NO: 77 or a functional variant thereof; (iii) the third polypeptide chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 78 or a functional variant thereof.

[0054] The present invention also relates to a binding agent comprising a binding domain having specificity for CLDN18.2, the binding domain comprising a variable region of a heavy chain (VH) (VH(CLDN18.2)) derived from an immunoglobulin having specificity for CLDN18.2 and a variable region of a light chain (VL) (VL(CLDN18.2)) derived from an immunoglobulin having specificity for CLDN18.2, wherein VH(CLDN18.2) comprises a CDR1 comprising the amino acid sequence SYWIN (SEQ ID NO: 32) or a functional variant thereof, an amino acid sequence of NIYPSDSYTNYNQKFQG (SEQ ID NO: 33) or a functional variant thereof, and a CDR3 comprising the amino acid sequence SWRGNSFDY (SEQ ID NO: 34) or a functional variant thereof, wherein VL(CLDN18.2) comprises a CDR1 comprising the amino acid sequence KSSQSLLNSGNQKNYLT (SEQ ID NO: 35) or a functional variant thereof, a CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO: 36) or a functional variant thereof, and a CDR3 comprising the amino acid sequence QNDYSYPFT (SEQ ID NO: 37) or a functional variant thereof. In one embodiment, the binding agent further comprises a binding domain with specificity for CD3. In one embodiment, a binding domain with specificity for CD3 comprises a VH (VH(CD3)) derived from an immunoglobulin with specificity for CD3 and a VL (VL(CD3)) derived from an immunoglobulin with specificity for CD3, wherein VH(CD3) comprises CDR1, CDR2, and CDR3 of amino acid sequences selected from the group consisting of SEQ ID NOs: 54, 58, and 61, and VL(CD3) comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 55.

[0055] In one embodiment, a functional variant of the amino acid sequence NIYPSDSYTNYNQKFQG (SEQ ID NO: 33) comprises or retains the amino acid residues QG.

[0056] In one embodiment, the binding agent is in the form of a full-length antibody or an antibody fragment. In one embodiment, the binding domain with specificity for CLDN18.2 is a Fab fragment, a Fab' fragment, a F(ab') 2In one embodiment, the binding domain with specificity for CD3 is in the form of a Fab fragment, a Fab' fragment, a F(ab') fragment, an Fv fragment, or an scFv fragment. 2 The antibody may be in the form of a fragment, an Fv fragment, or an scFv fragment.

[0057] In one embodiment, the binding agent is a bispecific molecule, such as a bispecific antibody. In one embodiment, the binding agent is a bispecific single chain antibody. In one embodiment, the binding agent is capable of monovalent or bivalent binding to CLDN18.2. In one embodiment, the binding agent comprises two binding domains with specificity for CLDN18.2. In one embodiment, the binding domain with specificity for CLDN18.2 is in the form of a Fab fragment and the binding domain with specificity for CD3 is in the form of a scFcV fragment. In one embodiment, VH(CLDN18.2) is linked to VL(CLDN18.2) by a peptide linker and / or VH(CD3) is linked to VL(CD3) by a peptide linker, such as a peptide linker selected from the group consisting of SEQ ID NOs: 2-20.

[0058] The invention also provides a binding agent comprising two binding domains with specificity for CLDN18.2 and a binding domain with specificity for CD3, the binding agent comprising at least four polypeptide chains, a first polypeptide chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 73 or a functional variant thereof, a second polypeptide chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 74 or a functional variant thereof, a third polypeptide chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 78 or a functional variant thereof, and a fourth polypeptide chain being identical to the third polypeptide chain. In one embodiment, the polypeptide chains of the binding agent and / or the domains of the polypeptide chains of the binding agent interact with each other as described herein. In one embodiment, the first polypeptide chain interacts with a second polypeptide chain and a third polypeptide chain, and the second polypeptide chain interacts with a fourth polypeptide chain.

[0059] The invention also provides a binding agent comprising a binding domain having specificity for CLDN18.2 and a binding domain having specificity for CD3, the binding agent comprising at least three polypeptide chains, a first polypeptide chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 73 or a functional variant thereof, a second polypeptide chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 75 or a functional variant thereof, and a third polypeptide chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 78 or a functional variant thereof. In one embodiment, the polypeptide chains of the binding agent and / or domains of the polypeptide chains of the binding agent interact with each other as described herein. In one embodiment, the first polypeptide chain interacts with the second polypeptide chain and the third polypeptide chain.

[0060] The invention also provides a binding agent comprising two binding domains with specificity for CLDN18.2 and a binding domain with specificity for CD3, the binding agent comprising at least four polypeptide chains, the first polypeptide chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 73 or a functional variant thereof, the second polypeptide chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 76 or a functional variant thereof, the third polypeptide chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 78 or a functional variant thereof, and the fourth polypeptide chain being identical to the third polypeptide chain. In one embodiment, the polypeptide chains of the binding agent and / or the domains of the polypeptide chains of the binding agent interact with each other as described herein. In one embodiment, the first polypeptide chain interacts with the second polypeptide chain and the third polypeptide chain, and the second polypeptide chain interacts with the fourth polypeptide chain.

[0061] The invention also provides a binding agent comprising two binding domains with specificity for CLDN18.2 and a binding domain with specificity for CD3, the binding agent comprising at least four polypeptide chains, the first polypeptide chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 73 or a functional variant thereof, the second polypeptide chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 77 or a functional variant thereof, the third polypeptide chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 78 or a functional variant thereof, and the fourth polypeptide chain being identical to the third polypeptide chain. In one embodiment, the polypeptide chains of the binding agent and / or the domains of the polypeptide chains of the binding agent interact with each other as described herein. In one embodiment, the first polypeptide chain interacts with the second polypeptide chain and the third polypeptide chain, and the second polypeptide chain interacts with the fourth polypeptide chain.

[0062] In one embodiment, VH(CLDN18.2), VL(CLDN18.2), VH(CD3), and / or VL(CD3) are humanized.

[0063] In one embodiment, CD3 is expressed on the surface of T cells. In one embodiment, the binding agent of the invention binds to the epsilon chain of CD3. In one embodiment, binding of the binding agent to CD3 on T cells results in proliferation and / or activation of the T cells. In one embodiment, proliferation and / or activation of T cells induces proliferation and / or activation of CD4 T cells and / or CD8 T cells (preferably CD107a+ T cells). In one embodiment, the proliferated and / or activated T cells are capable of degranulation. In one embodiment, the activated T cells release cytotoxic factors (e.g., perforin and granzymes) and initiate cytolysis and / or apoptosis of cancer cells.

[0064] In one embodiment, CLDN18.2 is expressed in cancer cells. In one embodiment, CLDN18.2 is expressed on the surface of cancer cells. In one embodiment, the binding agent binds to the extracellular portion of CLDN18.2. In one embodiment, the binding agent induces T cell-mediated cytotoxicity against cancer cells expressing CLDN18.2.

[0065] In one embodiment, the cancer cells are from a cancer selected from the group consisting of gastric cancer, esophageal cancer, cancer of the gastroesophageal junction (GEJ), pancreatic cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, rectal cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer and metastases thereof, Krukenberg tumors, peritoneal metastases, and / or lymph node metastases.

[0066] In some embodiments, the binding agent of the invention, or one or more of the polypeptide chains of the binding agent of the invention, may or may not comprise a secretion signal (e.g. an N-terminal secretion signal, particularly an immunoglobulin such as an IgG secretion signal, e.g. the sequence MGWSCIILFLVATATGVHS) and / or a tag (particularly a C-terminal tag, e.g. a His tag, particularly the sequence Gly-Gly-Ser-(His) 6 Or (His) 6 , or Strep tag).

[0067] In some embodiments, the binding agent of the present invention comprises one or more post-translational modifications. The present invention also provides a binding agent derived from one or more post-translational modifications of the binding agent described herein. In one embodiment, the one or more post-translational modifications are selected from pyroglutamation at the N-terminus of one or more VH(CLDN18.2), pyroglutamation at the N-terminus of VH(CD3), deletion of a lysine at the C-terminus of the first polypeptide chain, and deletion of a lysine at the C-terminus of the second polypeptide chain.

[0068] The invention also provides nucleic acids encoding the polypeptide chains of the binding agents of the invention.The invention also provides nucleic acids encoding the binding agents of the invention.The invention also provides sets of nucleic acids which together encode the binding agents of the invention.

[0069] The present invention also provides a vector comprising a nucleic acid or set of nucleic acids of the present invention. The present invention also provides a set of vectors comprising a set of nucleic acids of the present invention. In one embodiment, each nucleic acid of the set of nucleic acids is comprised in a vector of the set of vectors. In one embodiment, the vector or set of vectors is capable of expressing the binding agent.

[0070] In one embodiment, the nucleic acid is operably linked to any number of regulatory elements (promoter, origin of replication, selectable marker, ribosome binding site, inducer, etc.). The vector can be an expression vector, and can be an extrachromosomal vector or an integrating vector. In some embodiments, the nucleic acids encoding the polypeptide chains of the binding agents of the invention are each contained in a single expression vector. These nucleic acids can be under the control of different promoters or the same promoter. In such embodiments, various vector ratios can be used to drive the formation of the binding agents of the invention.

[0071] The present invention also provides a host cell comprising the nucleic acid, set of nucleic acids, vector, or set of vectors. In one embodiment, the host cell is a mammalian cell, preferably a CHO cell, a BHK cell, a HeLa cell, a COS cell, a HEK293 cell, a HEK293 The host cell is a mammalian cell selected from the group consisting of: T cells, and the like. In one embodiment, the host cell is a bacterial cell, a yeast cell, a fungal cell, a plant cell, or an insect cell.

[0072] The present invention also provides a pharmaceutical composition comprising the binding agent of the present invention, the nucleic acid of the present invention, the set of nucleic acids of the present invention, the vector of the present invention, the set of vectors of the present invention, or the host cell of the present invention. In one embodiment, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier and / or excipient.

[0073] The present invention also provides a binding agent of the invention, a nucleic acid of the invention, a set of nucleic acids of the invention, a vector of the invention, a set of vectors of the invention, a host cell of the invention, or a pharmaceutical composition of the invention for use in therapy.

[0074] The present invention also provides a binding agent of the present invention, a nucleic acid of the present invention, a set of nucleic acids of the present invention, a vector of the present invention, a set of vectors of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention for use in the treatment or prevention of cancer.

[0075] The present invention also provides a method for treating or preventing cancer, the method comprising administering to a subject in need thereof a binding agent of the present invention, a nucleic acid of the present invention, a set of nucleic acids of the present invention, a vector of the present invention, a set of vectors of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention. In one embodiment, the cancer involves cancer cells that express CLDN18.2.

[0076] The present invention also provides use of the binding agent of the present invention, the nucleic acid of the present invention, the set of nucleic acids of the present invention, the vector of the present invention, the set of vectors of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention for the preparation of a medicament. In one embodiment, the medicament is for the treatment or prevention of cancer. In one embodiment, the cancer involves cancer cells expressing CLDN18.2.

[0077] In one embodiment, the cancer is selected from the group consisting of gastric cancer, esophageal cancer, cancer of the gastroesophageal junction (GEJ), pancreatic cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, rectal cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer and metastases thereof, Krukenberg tumor, peritoneal metastasis, and / or lymph node metastasis.

[0078] The present invention also provides a binding agent, a nucleic acid, a set of nucleic acids, a vector, a set of vectors, a host cell, or a pharmaceutical composition as described herein for use in a method of treatment as described herein. In one embodiment, provided is a method of treating or preventing a disease, such as cancer, comprising administering to a subject having a disease, such as a subject having cancer, a binding agent of the present invention, a nucleic acid of the present invention, a set of nucleic acids of the present invention, a vector of the present invention, a set of vectors of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention. Preferably, the disease involves cells, such as disease cells, that express CLDN18.2. Preferably, the disease is cancer, and the cancer involves cancer cells that express CLDN18.2.

[0079] According to the present invention, CLDN18.2 preferably has the amino acid sequence according to SEQ ID NO:1.

[0080] In some embodiments, the first polypeptide chain does not comprise a VL(CLDN18.2). In some embodiments, the third polypeptide chain does not comprise a VH(CLDN18.2).

[0081] In some embodiments, the binding domain of the binding agent with specificity for CLDN18.2 is in the form of a Fab fragment. The binding domain of the binding agent of the invention having isomerism is in the form of an scFv portion.

[0082] In some embodiments, the binding agent of the invention does not bind to CLDN18.1. Preferably, the binding agent does not bind to human, mouse, or cynomolgus CLDN18.1. In some embodiments, the binding agent of the invention does not bind to CLDN9, such as human CLDN9.

[0083] In some embodiments, the binding agents of the invention bind to CLDN18.2 from multiple species, such as human, mouse, and cynomolgus monkey CLDN18.2.

[0084] In some embodiments, treating a patient with a binding agent described herein extends the survival of the patient. In some embodiments, the binding agent described herein exhibits one or more immune effector functions. In some embodiments, the one or more immune effector functions are selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis, and inhibition of proliferation. The binding agent described herein is also preferably capable of redirecting T cells to attack cancer cells, thus acting via redirected T cell cytotoxicity (RTCC). In some embodiments, the binding agent is not capable of inducing, or is substantially not capable of inducing, ADCC. In some embodiments, the binding agent is not capable of inducing, or is substantially not capable of inducing, CDC. In some embodiments, the binding agent is not capable of inducing, or is substantially not capable of inducing, ADCP. In some embodiments, the binding agent is capable of reducing, preferably significantly reducing, tumor growth and / or volume in a subject, such as a patient.

[0085] The present invention also provides a method for producing a binding agent of the present invention. In one embodiment, the method for producing a binding agent of the present invention comprises transfecting a host cell with a nucleic acid of the present invention, a set of nucleic acids of the present invention, a vector of the present invention, or a set of vectors of the present invention. In one embodiment, the host cell expresses a nucleic acid encoding a binding agent of the present invention. In one embodiment, the host cell co-expresses a nucleic acid encoding a first polypeptide chain of a binding agent of the present invention and a nucleic acid encoding a second polypeptide chain of a binding agent of the present invention. In one embodiment, the host cell further expresses a nucleic acid encoding a third polypeptide chain of a binding agent of the present invention. In one embodiment, the host cell further expresses a nucleic acid encoding a fourth polypeptide chain of a binding agent of the present invention. In one embodiment, the nucleic acid is included in a vector or a set of vectors. In one embodiment, the host cell expresses all polypeptide chains of a binding agent of the present invention. In one embodiment, the host cell after transfection preferably produces a binding agent of the present invention when grown under conditions suitable for binding agent production, such as those described herein or known in the art. In one embodiment, the binding agent of the present invention can be obtained from the host cell.

[0086] Thus, in one embodiment, a method for producing a binding agent of the invention comprises the steps of transfecting a host cell with a nucleic acid encoding a first polypeptide chain of a binding agent of the invention, a nucleic acid encoding a second polypeptide chain of a binding agent of the invention, a nucleic acid encoding a third polypeptide chain of a binding agent of the invention, and optionally a fourth polypeptide chain of a binding agent of the invention, expressing said nucleic acids in the host cell, and obtaining a binding agent of the invention. In one embodiment, the host cell is a mammalian cell, preferably a CHO cell, a BHK cell, a HeLa cell, a COS cell, a HEK293 cell, a HEK293 In one embodiment, the host cell is a mammalian cell selected from the group consisting of a bacterial cell, a yeast cell, a fungal cell, a plant cell, or an insect cell. In one embodiment, the binding agent is produced in vitro. In one embodiment, the binding agent is produced in vivo and is used to treat, for example, a disease associated with a disease, particularly a cell expressing CLDN18.2. In one embodiment, the first and / or second polypeptide chains of the binding agent of the invention are produced in a subject to be treated, such as a subject having a disease associated with a particular disease, e.g., cancer. In one embodiment, the first and / or second polypeptide chains of the binding agent of the invention are produced in, e.g., one host cell, and the third polypeptide chain is produced in, e.g., another host cell. In one embodiment, all polypeptide chains of the binding agent of the invention are produced in the same host cell.

[0087] In one embodiment, the polypeptide chains of the binding agent of the present invention are linked together, e.g., covalently linked. In one embodiment, the polypeptide chains of the binding agent are produced as one polypeptide including all the polypeptide chains of the binding agent. In one embodiment, at least two polypeptide chains of the binding agent are linked together and produced as one polypeptide. In one embodiment, the first polypeptide chain and the second polypeptide chain are linked together and produced as one polypeptide, and the third polypeptide chain is produced as a separate polypeptide. In one embodiment, the third polypeptide chain and the first polypeptide chain are linked together and produced as one polypeptide, and the second polypeptide chain is produced as a separate polypeptide. In one embodiment, the second polypeptide chain and the third polypeptide chain are linked together and produced as one polypeptide, and the first polypeptide chain is produced as a separate polypeptide or is linked to a fourth polypeptide chain, both of which are produced together as one polypeptide. Preferably, the polypeptide chains of the binding agent of the invention are produced separately, i.e., as separate polypeptides, e.g., in the same or different cells, and interact during or after production, e.g., intracellularly or extracellularly, to form the binding agent. Preferably, the polypeptide chains are produced as separate polypeptides, i.e., the first polypeptide chain is produced as one polypeptide, the second polypeptide chain is produced as one polypeptide, the third polypeptide chain is produced as one polypeptide, and optionally, the fourth polypeptide chain is produced as one polypeptide, and these polypeptide chains of the invention interact to form the binding agent of the invention. In one embodiment, administering the binding agent of the invention comprises administering the first, second, third, and optionally fourth polypeptide chains formulated in a carrier, such as lipid nanoparticles, liposomes, lipoplexes, etc. In one embodiment, the first and second polypeptide chains are administered together, e.g., formulated in the same or different carriers, and the third polypeptide chain is administered, e.g., formulated separately in a carrier.In one embodiment, a fourth polypeptide chain is further administered, e.g., formulated separately in a carrier. In one embodiment, a third polypeptide chain and a fourth polypeptide chain are administered together, e.g., formulated in the same or different carriers. In one embodiment, all peptide chains of the binding agent of the invention are formulated together in the same carrier. In one embodiment, each polypeptide chain of the binding agent of the invention is formulated in a separate carrier, where the carriers may be the same or different.

[0088] Other features and advantages of the invention will be apparent from the following detailed description and claims. [Brief description of the drawings]

[0089] [Figure 1] Figure 1A shows a "Fab-scFv" format that includes a VH (first polypeptide chain as described herein) recombinantly fused to one side of a heterodimeric Fc, a single chain Fv ("scFv") (second polypeptide chain as described herein) recombinantly fused to the other side of the heterodimeric Fc, and a light chain (LC; third polypeptide chain as described herein) transfected separately to form a Fab domain with the VH of the first polypeptide chain. Figure 1B shows a "Fab2-scFv" format that includes a VH (first polypeptide chain as described herein) recombinantly fused to one side of a heterodimeric Fc, a VH (second polypeptide chain as described herein) recombinantly fused to the scFv fused to the other side of the heterodimeric Fc, and a LC (third and fourth polypeptide chains as described herein) transfected separately to form a Fab domain with the VH of the first polypeptide chain and additional VHs of the second polypeptide chain. [Figure 2A]Binding of anti-CLDN18.2 x anti-CD3 bispecific antibodies with mouse CLDN18.2 ABD to A) KP-4 cells and B) NUGC-4 cells. Controls included anti-RSV x anti-CD3 bsAb, cells only, and secondary antibody only. The data show that anti-CLDN18.2 x anti-CD3 bsAb bound to NUGC-4 cells in a dose-dependent manner, with little binding to KP-4 cells at all concentrations tested. Notably, the bsAbs in the "Fab2-scFv" format (i.e., XENP24647, XENP24648, and XENP24649) bound much more strongly to NUGC-4 cells compared to the bsAbs in the "Fab-scFv" format (i.e., XENP24645 and XENP24646), likely due to the special binding activity conveyed by the "Fab2-scFv" format. [Figure 2B] Binding of anti-CLDN18.2 x anti-CD3 bispecific antibodies with mouse CLDN18.2 ABD to A) KP-4 cells and B) NUGC-4 cells. Controls included anti-RSV x anti-CD3 bsAb, cells only, and secondary antibody only. The data show that anti-CLDN18.2 x anti-CD3 bsAb bound to NUGC-4 cells in a dose-dependent manner, with little binding to KP-4 cells at all concentrations tested. Notably, the bsAbs in the "Fab2-scFv" format (i.e., XENP24647, XENP24648, and XENP24649) bound much more strongly to NUGC-4 cells compared to the bsAbs in the "Fab-scFv" format (i.e., XENP24645 and XENP24646), likely due to the special binding activity conveyed by the "Fab2-scFv" format. [Figure 3A] Figure 1 shows the induction of RTCC in A) KP-4 and B) NUGC-4 cells by anti-CLDN18.2 x anti-CD3 bsAb with murine CLDN18.2 ABD. The data show that the prototypic anti-CLDN18.2 x anti-CD3 bsAb induced RTCC in NUGC-4 but not KP-4 cells in a dose-dependent manner. [Figure 3B] Figure 1 shows the induction of RTCC in A) KP-4 and B) NUGC-4 cells by anti-CLDN18.2 x anti-CD3 bsAb with murine CLDN18.2 ABD. The data show that the prototypic anti-CLDN18.2 x anti-CD3 bsAb induced RTCC in NUGC-4 but not KP-4 cells in a dose-dependent manner. [Figure 4A] Figure 1 shows expression levels in A) NUGC-4 cells and B) SNU-601 cells as determined by flow cytometry. The data show that SNU-601 expresses more CLDN18.2 than NUGC-4. [Figure 4B] Figure 1 shows expression levels in A) NUGC-4 cells and B) SNU-601 cells as determined by flow cytometry. The data show that SNU-601 expresses more CLDN18.2 than NUGC-4. [Figure 5A] Binding of anti-CLDN18.2 x anti-CD3 bispecific antibodies with murine CLDN18.2 ABD to A) NUGC-4 cells and B) SNU-601 cells. Controls included anti-RSV x anti-CD3 bispecific antibodies, cells only, and secondary antibody only. The data show that each of the bispecific antibodies bound to both NUGC-4 and SNU-601 in a dose-dependent manner, with higher maximal binding to SNU-601 cells compared to NUGC-4, consistent with the respective CLDN18.2 expression levels in each cell line. [Figure 5B] Binding of anti-CLDN18.2 x anti-CD3 bispecific antibodies with murine CLDN18.2 ABD to A) NUGC-4 cells and B) SNU-601 cells. Controls included anti-RSV x anti-CD3 bispecific antibodies, cells only, and secondary antibody only. The data show that each of the bispecific antibodies bound to both NUGC-4 and SNU-601 in a dose-dependent manner, with higher maximal binding to SNU-601 cells compared to NUGC-4, consistent with the respective CLDN18.2 expression levels in each cell line. [Figure 6] Figure 1 shows induction of RTCC (as indicated by a reduction in viable target cells) in A) NUGC-4 cells and B) SNU-601 cells after 24 hours of incubation with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data shows that the bispecific antibody enhances killing of target cells (i.e., NUGC-4 and SNU-601 cells) as indicated by a reduction in viable cells. [Figure 7A] Figure 1 shows induction of RTCC (as indicated by an increase in killed target cells) in A) NUGC-4 cells and B) SNU-601 cells after 24 hours of incubation with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show that the bispecific antibody enhances killing of target cells (i.e., NUGC-4 and SNU-601 cells) as indicated by an increase in dead / dying cells. [Figure 7B] Figure 1 shows induction of RTCC (as indicated by an increase in killed target cells) in A) NUGC-4 cells and B) SNU-601 cells after 24 hours of incubation with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show that the bispecific antibody enhances killing of target cells (i.e., NUGC-4 and SNU-601 cells) as indicated by an increase in dead / dying cells. [Figure 8A]Figure 1 shows induction of RTCC (as indicated by a reduction in viable target cells) in A) NUGC-4 cells and B) SNU-601 cells after 48 hours of incubation with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data shows that the bispecific antibody enhances killing of target cells (i.e., NUGC-4 and SNU-601 cells) as indicated by a reduction in viable cells. [Figure 8B] Figure 1 shows induction of RTCC (as indicated by a reduction in viable target cells) in A) NUGC-4 cells and B) SNU-601 cells after 48 hours of incubation with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data shows that the bispecific antibody enhances killing of target cells (i.e., NUGC-4 and SNU-601 cells) as indicated by a reduction in viable cells. [Figure 9A] Figure 1 shows induction of RTCC (as indicated by an increase in killed target cells) in A) NUGC-4 cells and B) SNU-601 cells after 48 hours of incubation with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data shows that the bispecific antibody enhances killing of target cells (i.e., NUGC-4 and SNU-601 cells) as indicated by an increase in dead / dying cells. [Figure 9B]Figure 1 shows induction of RTCC (as indicated by an increase in killed target cells) in A) NUGC-4 cells and B) SNU-601 cells after 48 hours of incubation with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data shows that the bispecific antibody enhances killing of target cells (i.e., NUGC-4 and SNU-601 cells) as indicated by an increase in dead / dying cells. [Figure 10A] Figure 1 shows the percentage of CD4+ T cells expressing A) CD69, B) CD25, and C) CD107a after incubation of NUGC-4 cells with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD for 48 hours. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show trends consistent with RTCC, i.e., higher affinity CD3 binding and / or bivalent CLDN18.2 binding enhances T cell activation and degranulation. [Figure 10B] Figure 1 shows the percentage of CD4+ T cells expressing A) CD69, B) CD25, and C) CD107a after incubation of NUGC-4 cells with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD for 48 hours. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show trends consistent with RTCC, i.e., higher affinity CD3 binding and / or bivalent CLDN18.2 binding enhances T cell activation and degranulation. [Figure 10C]Figure 1 shows the percentage of CD4+ T cells expressing A) CD69, B) CD25, and C) CD107a after incubation of NUGC-4 cells with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD for 48 hours. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show trends consistent with RTCC, i.e., higher affinity CD3 binding and / or bivalent CLDN18.2 binding enhances T cell activation and degranulation. [Figure 11A] Figure 1 shows the percentage of CD8+ T cells expressing A) CD69, B) CD25, and C) CD107a after incubation of NUGC-4 cells with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD for 48 hours. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show trends consistent with RTCC, i.e., higher affinity CD3 binding and / or bivalent CLDN18.2 binding enhances T cell activation and degranulation. [Figure 11B] Figure 1 shows the percentage of CD8+ T cells expressing A) CD69, B) CD25, and C) CD107a after incubation of NUGC-4 cells with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD for 48 hours. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show trends consistent with RTCC, i.e., higher affinity CD3 binding and / or bivalent CLDN18.2 binding enhances T cell activation and degranulation. [Figure 11C]Figure 1 shows the percentage of CD8+ T cells expressing A) CD69, B) CD25, and C) CD107a after incubation of NUGC-4 cells with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD for 48 hours. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show trends consistent with RTCC, i.e., higher affinity CD3 binding and / or bivalent CLDN18.2 binding enhances T cell activation and degranulation. [Figure 12A] Figure 1 shows the percentage of CD4+ T cells expressing A) CD69, B) CD25, and C) CD107a after incubation of SNU-601 cells with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD for 48 hours. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show trends consistent with RTCC, i.e., higher affinity CD3 binding and / or bivalent CLDN18.2 binding enhances T cell activation and degranulation. [Figure 12B] Figure 1 shows the percentage of CD4+ T cells expressing A) CD69, B) CD25, and C) CD107a after incubation of SNU-601 cells with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD for 48 hours. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show trends consistent with RTCC, i.e., higher affinity CD3 binding and / or bivalent CLDN18.2 binding enhances T cell activation and degranulation. [Figure 12C]Figure 1 shows the percentage of CD4+ T cells expressing A) CD69, B) CD25, and C) CD107a after incubation of SNU-601 cells with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD for 48 hours. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show trends consistent with RTCC, i.e., higher affinity CD3 binding and / or bivalent CLDN18.2 binding enhances T cell activation and degranulation. [Figure 13A] Figure 1 shows the percentage of CD8+ T cells expressing A) CD69, B) CD25, and C) CD107a after incubation of SNU-601 cells with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD for 48 hours. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show trends consistent with RTCC, i.e., higher affinity CD3 binding and / or bivalent CLDN18.2 binding enhances T cell activation and degranulation. [Figure 13B] Figure 1 shows the percentage of CD8+ T cells expressing A) CD69, B) CD25, and C) CD107a after incubation of SNU-601 cells with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD for 48 hours. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show trends consistent with RTCC, i.e., higher affinity CD3 binding and / or bivalent CLDN18.2 binding enhances T cell activation and degranulation. [Figure 13C]Figure 1 shows the percentage of CD8+ T cells expressing A) CD69, B) CD25, and C) CD107a after incubation of SNU-601 cells with human PBMCs (effector to target cell ratio of 20:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with mouse CLDN18.2 ABD for 48 hours. Controls included anti-RSV x anti-CD3 bispecific antibody, target cells only, and target cells and effector cells only. The data show trends consistent with RTCC, i.e., higher affinity CD3 binding and / or bivalent CLDN18.2 binding enhances T cell activation and degranulation. [Figure 14] Figure 1 shows expression levels in A) SNU-601 cells and B) SNU-601(2E4) cells (enriched for CLDN18.2 expressing population) as determined by flow cytometry. The data show that SNU-601(2E4) contains a substantially higher population of CLDN18.2+ cells. The experiments in this section were performed using SNU-601(2E4) cells. [Figure 15]Binding to SNU-601(2E4) cells by anti-CLDN18.2 x anti-CD3 bispecific antibodies with humanized CLDN18.2 ABD. Controls used were XENP24644 (H0L0 CLDN18.2; bivalent mAb), XENP29470 (H1L1 CLDN18.2; bivalent mAb), XENP29471 (H2L1 CLDN18.2; bivalent mAb), XENP24645 (Fab-scFv), XENP24647 (Fab2-scFv), cells only, and secondary antibody only. The data show that bispecific antibodies with humanized CLDN18.2 ABD bound to SNU-601(2E4) cells similarly to bispecific antibodies with murine CLDN18.2 ABD, indicating that humanization maintained the binding potency of the antibodies. In particular, bispecific antibodies in the "Fab2-scFv" format showed similar binding to bivalent anti-CLDN18.2 mAbs. In addition, bispecific antibodies based on H1L1 humanized variants (e.g., XENP29472, XENP29474, XENP29476, and XENP29478) maintained better binding than bispecific antibodies based on H2L1 humanized variants (e.g., XENP29473, XENP29475, XENP29477, and XENP29479). [Figure 16A]Figure 1 shows induction of RTCC in SNU-601(2E4) cells as shown by A) the reduction in the number of CFSE+ SNU-601(2E4), B) the percentage of CFSE+ SNU-601(2E4) cells stained with Zombie Aqua, and C) Zombie Aqua MFI in CFSE+ SNU-601(2E4) cells after incubation of SNU-601(2E4) cells with human PBMCs (effector to target cell ratio of 10:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD for 24 hours. Controls used were XENP24645 (Fab-scFv), XENP24647 (Fab2-scFv), cells only, and secondary antibody only. Consistent with the binding data, humanization maintained the induction of RTCC by bsAb with the humanized CLDN18.2 ABD. [Figure 16B] Figure 1 shows induction of RTCC in SNU-601(2E4) cells as shown by A) the reduction in the number of CFSE+ SNU-601(2E4), B) the percentage of CFSE+ SNU-601(2E4) cells stained with Zombie Aqua, and C) Zombie Aqua MFI in CFSE+ SNU-601(2E4) cells after incubation of SNU-601(2E4) cells with human PBMCs (effector to target cell ratio of 10:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD for 24 hours. Controls used were XENP24645 (Fab-scFv), XENP24647 (Fab2-scFv), cells only, and secondary antibody only. Consistent with the binding data, humanization maintained the induction of RTCC by bsAb with the humanized CLDN18.2 ABD. [Figure 16C]Figure 1 shows induction of RTCC in SNU-601(2E4) cells as shown by A) the reduction in the number of CFSE+ SNU-601(2E4), B) the percentage of CFSE+ SNU-601(2E4) cells stained with Zombie Aqua, and C) Zombie Aqua MFI in CFSE+ SNU-601(2E4) cells after incubation of SNU-601(2E4) cells with human PBMCs (effector to target cell ratio of 10:1) and anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD for 24 hours. Controls used were XENP24645 (Fab-scFv), XENP24647 (Fab2-scFv), cells only, and secondary antibody only. Consistent with the binding data, humanization maintained the induction of RTCC by bsAb with the humanized CLDN18.2 ABD. [Figure 17A] Activated CD4+ T cells as indicated by A) CD69 MFI on CD4+ T cells and B) percentage of CD4+ T cells expressing CD69 after incubation of human PBMCs (10:1 effector to target cell ratio) and SNU-601 (2E4) with anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD for 24 hours. Controls used were XENP24645 (Fab-scFv-Fc), XENP24647 (Fab2-scFv), cells only, and secondary antibody only. Consistent with the binding data, humanization maintained activation of T cells by bsAb with humanized CLDN18.2 ABD. [Figure 17B]Activated CD4+ T cells as indicated by A) CD69 MFI on CD4+ T cells and B) percentage of CD4+ T cells expressing CD69 after incubation of human PBMCs (10:1 effector to target cell ratio) and SNU-601 (2E4) with anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD for 24 hours. Controls used were XENP24645 (Fab-scFv-Fc), XENP24647 (Fab2-scFv), cells only, and secondary antibody only. Consistent with the binding data, humanization maintained activation of T cells by bsAb with humanized CLDN18.2 ABD. [Figure 18A] CD4+ T cell degranulation as indicated by A) CD107a MFI on CD4+ T cells and B) percentage of CD4+ T cells expressing CD107a after incubation of human PBMCs (effector to target cell ratio of 10:1) and SNU-601 (2E4) with anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD for 24 hours. Controls used were XENP24645 (Fab-scFv-Fc), XENP24647 (Fab2-scFv), cells only, and secondary antibody only. [Figure 18B] CD4+ T cell degranulation as indicated by A) CD107a MFI on CD4+ T cells and B) percentage of CD4+ T cells expressing CD107a after incubation of human PBMCs (effector to target cell ratio of 10:1) and SNU-601 (2E4) with anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD for 24 hours. Controls used were XENP24645 (Fab-scFv-Fc), XENP24647 (Fab2-scFv), cells only, and secondary antibody only. [Figure 19A]Activated CD8+ T cells as shown by A) CD69 MFI on CD8+ T cells and B) percentage of CD8+ T cells expressing CD69 after incubation of human PBMCs (10:1 effector to target cell ratio) and SNU-601 (2E4) with anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD for 24 hours. Controls used were XENP24645 (Fab-scFv-Fc), XENP24647 (Fab2-scFv), cells only, and secondary antibody only. Consistent with the binding data, humanization maintained activation of T cells by bsAb with humanized CLDN18.2 ABD. [Figure 19B] Activated CD8+ T cells as shown by A) CD69 MFI on CD8+ T cells and B) percentage of CD8+ T cells expressing CD69 after incubation of human PBMCs (10:1 effector to target cell ratio) and SNU-601 (2E4) with anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD for 24 hours. Controls used were XENP24645 (Fab-scFv-Fc), XENP24647 (Fab2-scFv), cells only, and secondary antibody only. Consistent with the binding data, humanization maintained activation of T cells by bsAb with humanized CLDN18.2 ABD. [Figure 20A] CD8+ T cell degranulation as indicated by A) CD107a MFI on CD8+ T cells and B) percentage of CD8+ T cells expressing CD107a after incubation of human PBMCs (effector to target cell ratio of 10:1) and SNU-601 (2E4) with anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD for 24 hours. Controls used were XENP24645 (Fab-scFv-Fc), XENP24647 (Fab2-scFv), cells only, and secondary antibody only. [Figure 20B]CD8+ T cell degranulation as indicated by A) CD107a MFI on CD8+ T cells and B) percentage of CD8+ T cells expressing CD107a after incubation of human PBMCs (effector to target cell ratio of 10:1) and SNU-601 (2E4) with anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD for 24 hours. Controls used were XENP24645 (Fab-scFv-Fc), XENP24647 (Fab2-scFv), cells only, and secondary antibody only. [Figure 21A] Figure 1 shows secretion of A) IFNγ and B) TNFα by T cells after incubation with human PBMCs (effector to target cell ratio of 10:1) and SNU-601 (2E4) for 24 hours with anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD. Controls used were XENP24645 (Fab-scFv-Fc) and XENP24647 (Fab2-scFv). Consistent with the binding data, humanization maintained induction of cytokine secretion by bsAb with humanized CLDN18.2 ABD. [Figure 21B] Figure 1 shows secretion of A) IFNγ and B) TNFα by T cells after incubation with human PBMCs (effector to target cell ratio of 10:1) and SNU-601 (2E4) for 24 hours with anti-CLDN18.2 x anti-CD3 bispecific antibody with humanized CLDN18.2 ABD. Controls used were XENP24645 (Fab-scFv-Fc) and XENP24647 (Fab2-scFv). Consistent with the binding data, humanization maintained induction of cytokine secretion by bsAb with humanized CLDN18.2 ABD. [Figure 22]Binding of anti-CLDN18.2 x anti-CD3 bispecific antibodies with mouse and humanized (variant H1L1) CLDN18.2 ABDs to HEK293 cells transiently transfected to express A) human CLDN18.2, B) cynomolgus CLDN18.2, and C) mouse CLDN18.2. The data show that anti-CLDN18.2 x anti-CD3 bispecific antibodies bound in a dose-dependent manner to cells transfected with human CLDN18.2, cynomolgus CLDN18.2, and mouse CLDN18.2, respectively. In particular, bispecific antibodies in the "Fab2-scFv" format (i.e., XENP24647 and XENP29476) bind to CLDN18.2 transfected cells with similar potency as the much more potent bivalent mAb format (i.e., XENP24644) compared to bispecific antibodies in the "Fab-scFv" format (i.e., XENP24645 and XENP29472). [Figure 23] 1 shows the EC50 of binding of anti-CLDN18.2 x anti-CD3 bispecific antibodies with mouse and humanized (variant H1L1) CLDN18.2 ABD to HEK293 cells transiently transfected to express human CLDN18.2, cynomolgus CLDN18.2, and mouse CLDN18.2. [Figure 24] Binding of anti-CLDN18.2 x anti-CD3 bispecific antibodies with mouse and humanized (variant H1L1) CLDN18.2 ABDs to HEK293 cells transiently transfected to express A) human CLDN18.1, B) cynomolgus CLDN18.1, C) mouse CLDN18.1, and D) human CLDN9 is shown. The data show that none of the anti-CLDN18.2 x anti-CD3 bispecific antibodies exhibited off-target binding. [Diagram 25]Figure 1 shows induction of RTCC in NUGC-4 cells by bispecific antibodies and a comparative anti-CLDN18.2 x anti-CD3 bispecific antibody (AMG 910) using PBMCs from A) a first donor and B) a second donor. The data show that XENP32461 induced RTCC with similar potency as the comparative bispecific antibody, and XENP31726 induced RTCC with enhanced potency compared to the comparative bispecific antibody. [Figure 26] Shown are A) induction of RTCC, B) induction of CD4 T cell activation (shown as percentage of cells expressing CD107a), and C) induction of CD8 T cell activation (shown as percentage of cells expressing CD107a) by anti-CLDN18.2 x anti-CD3 bispecific antibody in the presence of BxPC3 cells (containing 600k CLDN18.2 binding sites) and effector cells at an E:T ratio of 10:1. [Figure 27] Shows induction of A) IFNγ, B) TNFα, and C) IL2 secretion by anti-CLDN18.2 x anti-CD3 bispecific antibody in the presence of BxPC3 cells (containing 600k CLDN18.2 binding sites) and effector cells at an E:T ratio of 10:1. [Figure 28] Shown are A) induction of RTCC, B) induction of CD4 T cell activation (shown as percentage of cells expressing CD107a), and C) induction of CD8 T cell activation (shown as percentage of cells expressing CD107a) by anti-CLDN18.2 x anti-CD3 bispecific antibody in the presence of GSU cells (containing 70k CLDN18.2 binding sites) and effector cells at an E:T ratio of 10:1. [Figure 29] Shows induction of A) IFNγ, B) TNFα, and C) IL2 secretion by anti-CLDN18.2 x anti-CD3 bispecific antibody in the presence of GSU cells (containing 70k CLDN18.2 binding sites) and effector cells at an E:T ratio of 10:1. [Diagram 30]Shown are A) induction of RTCC, B) induction of CD4 T cell activation (shown as percentage of cells expressing CD107a), and C) induction of CD8 T cell activation (shown as percentage of cells expressing CD107a) by anti-CLDN18.2 x anti-CD3 bispecific antibody in the presence of NUGC4 cells (containing 50k CLDN18.2 binding sites) and effector cells at an E:T ratio of 10:1. [Diagram 31] Shows induction of A) IFNγ, B) TNFα, and C) IL2 secretion by anti-CLDN18.2 x anti-CD3 bispecific antibody in the presence of NUGC4 cells (containing 50k CLDN18.2 binding sites) and effector cells at an E:T ratio of 10:1. [Diagram 32] Shown are A) induction of RTCC, B) induction of CD4 T cell activation (shown as percentage of cells expressing CD107a), and C) induction of CD8 T cell activation (shown as percentage of cells expressing CD107a) by anti-CLDN18.2 x anti-CD3 bispecific antibody in the presence of KatoIII cells (containing 30k CLDN18.2 binding sites) and effector cells at an E:T ratio of 10:1. [Diagram 33] Shows induction of A) TNFα and B) IL2 secretion by anti-CLDN18.2 x anti-CD3 bispecific antibody in the presence of KatoIII cells (containing 30k CLDN18.2 binding sites) and effector cells at an E:T ratio of 10:1. [Diagram 34] Figure 1 shows A) induction of RTCC, B) induction of CD8 T cell activation (shown as the percentage of cells expressing CD107a), and C) induction of TNFα secretion by anti-CLDN18.2 x anti-CD3 bispecific antibody in the presence of KatoIII cells (containing 30k CLDN18.2 binding sites) and effector cells at an E:T ratio of 3:1. [Diagram 35]Shown are A) induction of RTCC, B) induction of CD4 T cell activation (shown as the percentage of cells expressing CD107a), and C) induction of CD8 T cell activation (shown as the percentage of cells expressing CD107a) by anti-CLDN18.2 x anti-CD3 bispecific antibody in the presence of HGC27 cells (containing 20k CLDN18.2 binding sites) and effector cells at an E:T ratio of 3:1. [Diagram 36] Shows induction of A) IFNγ, B) TNFα, and C) IL2 secretion by anti-CLDN18.2 x anti-CD3 bispecific antibody in the presence of HGC27 cells (containing 20k CLDN18.2 binding sites) and effector cells at an E:T ratio of 3:1. [Figure 37] Shown is A) induction of RTCC and B) induction of TNFα secretion by anti-CLDN18.2 x anti-CD3 bispecific antibody in the presence of KatoIII cells (containing 30k CLDN18.2 binding sites) and effector cells at an E:T ratio of 1:1. [Figure 38A] Figure 2 shows the change in tumor volume and body weight in NOG mice bearing NUGC-4 10cF7_5_3E10 tumors and transplanted with human PBMCs treated with ASP2138 (XENP31726). Human PBMCs were injected intravenously at 5x106 cells. One week after human PBMC injection, on day -8, NUGC-4 10cF7_5_3E10 cells were inoculated subcutaneously in the flank of mice at 1x106 cells. NOG mice were intraperitoneally administered PBS or ASP2138 once a week on days 0 and 7. A) Tumor volume and B) body weight for each group were plotted at each time point as mean ± SEM (n=10). C) Plot shows individual tumor volumes on day 14, with mean ± SEM represented by short horizontal lines and error bars. Statistical analysis was performed on the values ​​on day 14. ns: not significant, **: p less than 0.01 compared with the value of the PBS group (Dunnett's multiple comparison test). [Figure 38B]Figure 2 shows the change in tumor volume and body weight in NOG mice bearing NUGC-4 10cF7_5_3E10 tumors and transplanted with human PBMCs treated with ASP2138 (XENP31726). Human PBMCs were injected intravenously at 5x106 cells. One week after human PBMC injection, on day -8, NUGC-4 10cF7_5_3E10 cells were inoculated subcutaneously in the flank of mice at 1x106 cells. NOG mice were intraperitoneally administered PBS or ASP2138 once a week on days 0 and 7. A) Tumor volume and B) body weight for each group were plotted at each time point as mean ± SEM (n=10). C) Plot shows individual tumor volumes on day 14, with mean ± SEM represented by short horizontal lines and error bars. Statistical analysis was performed on the values ​​on day 14. ns: not significant, **: p less than 0.01 compared with the value of the PBS group (Dunnett's multiple comparison test). [Figure 38C] Figure 2 shows the change in tumor volume and body weight in NOG mice bearing NUGC-4 10cF7_5_3E10 tumors and transplanted with human PBMCs treated with ASP2138 (XENP31726). Human PBMCs were injected intravenously at 5x106 cells. One week after human PBMC injection, on day -8, NUGC-4 10cF7_5_3E10 cells were inoculated subcutaneously in the flank of mice at 1x106 cells. NOG mice were intraperitoneally administered PBS or ASP2138 once a week on days 0 and 7. A) Tumor volume and B) body weight for each group were plotted at each time point as mean ± SEM (n=10). C) Plot shows individual tumor volumes on day 14, with mean ± SEM represented by short horizontal lines and error bars. Statistical analysis was performed on the values ​​on day 14. ns: not significant, **: p less than 0.01 compared with the value of the PBS group (Dunnett's multiple comparison test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] The present invention is described in detail below, but it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, which may be modified. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0091] Below are described elements of the present invention that, although listed with specific embodiments, can be combined in any manner and in any number to create additional embodiments. It should be understood that the variously described examples and preferred embodiments should not be construed as limiting the invention to only those embodiments explicitly described. The description should be understood to support and encompass the explicitly described embodiments and embodiments combining any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application unless the context indicates otherwise.

[0092] Preferably, the terms used herein are those defined in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Defined as explained in Acta, CH-4010 Basel, Switzerland, (1995).

[0093] The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2002). nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0094] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated member, integer, or step, or group of members, integers, or steps, and not the exclusion of any other member, integer, or step, or group of members, integers, or steps, although in some embodiments such other members, integers, or steps, or group of members, integers, or steps, may be excluded, i.e., the subject matter consists of a stated member, integer, or step, or group of members, integers, or steps. However, for specific embodiments of the present disclosure, the term "comprising" is intended to encompass the possibility that no additional members are present, i.e., for purposes of this embodiment, "comprising" should be understood to have the meaning of "consisting of" or "consisting essentially of".

[0095] The terms "a," "an," and "the," and similar references used in the context of describing the present invention (especially in the context of the claims) are intended to cover both the singular and the plural, unless otherwise indicated herein or the context clearly contradicts. The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or the context clearly contradicts. The use of any and all examples or illustrative language (e.g., "etc.") provided herein is intended merely to better illustrate the present invention and does not impose limitations on the scope of the invention as otherwise claimed. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0096] The term "about" means approximately or near, and in one embodiment, in the context of any numerical value or range described herein, any numerical value or range that is recited or claimed. means ±20%, ±10%, ±5%, or ±3% of the range.

[0097] Throughout the text of this specification, several documents are cited. Each of the documents cited herein (including, for example, all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing in this specification is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0098] The present invention relates to bispecific binding agents which bind a target molecule monovalently via a single antigen-binding domain or bivalently via two antigen-binding domains, each of which binds an antigen independently.

[0099] The primary target molecule for the binding agents described herein is CLDN18.2.

[0100] Claudins are a family of proteins that are the most important components of tight junctions, which constitute a paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. Claudins are transmembrane proteins that span the membrane four times, with both the N- and C-termini located in the cytoplasm. The first extracellular loop, called EC1 or ECL1, consists of an average of 53 amino acids, and the second extracellular loop, called EC2 or ECL2, consists of approximately 24 amino acids. Cell surface proteins of the claudin family, such as CLDN18.2, are expressed in tumors of various origins and, due to their selective expression (not expressed in normal tissues associated with toxicity) and localization to the cell membrane, are particularly suitable as target structures in the context of antibody-mediated cancer immunotherapy.

[0101] CLDN18.2 has been identified as being differentially expressed in tumor tissues, and the only normal tissue expressing CLDN18.2 is the stomach. In normal tissues, CLDN18.2 is selectively expressed in differentiated epithelial cells of the gastric mucosa. CLDN18.2 is expressed in cancers of various origins, such as pancreatic cancer, esophageal cancer, gastric cancer, bronchial cancer, breast cancer, and ENT tumors. CLDN18.2 is a useful target for the prevention and / or treatment of primary tumors, such as gastric cancer, esophageal cancer, cancer of the gastroesophageal junction (GEJ), pancreatic cancer, lung cancer, including non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, rectal cancer, colorectal cancer, liver cancer, head and neck cancer, and gallbladder cancer, as well as their metastases, particularly gastric cancer metastases, including Krukenberg tumors, peritoneal metastases, and lymph node metastases. The term "claudin 18" or "CLDN18" refers to claudin 18 and includes all variants, including claudin 18 splice variant 1 (claudin 18.1 (CLDN18.1)) and claudin 18 splice variant 2 (claudin 18.2 (CLDN18.2)).

[0102] The term "claudin 18.2" or "CLDN18.2" preferably relates to human CLDN18.2, specifically to a protein comprising, and preferably consisting of, the amino acid sequence according to SEQ ID NO: 1 in the sequence listing or a variant of said amino acid sequence. The first extracellular loop of CLDN18.2 preferably comprises amino acids 27 to 81, more preferably amino acids 29 to 78 of the amino acid sequence shown in SEQ ID NO: 1. The second extracellular loop of CLDN18.2 preferably comprises amino acids 140 to 180 or 144 to 167 of the amino acid sequence shown in SEQ ID NO: 1. The first and second extracellular loops preferably form the extracellular portion of CLDN18.2.

[0103] A second target molecule for the binding agents described herein is CD3 (cluster of differentiation 3). The CD3 complex represents an antigen expressed on mature human T cells, thymocytes, and a subset of natural killer cells as part of the multi-molecular T cell receptor (TCR) complex. The T cell coreceptor is a protein complex and is composed of four different chains. In mammals, This complex contains the CD3 gamma, CD3 delta, and two CD3 epsilon chains. These chains associate with a molecule known as the T cell receptor (TCR) and the zeta chain to generate an activation signal in T lymphocytes. Together, the TCR, zeta chain, and CD3 molecules make up the TCR complex.

[0104] Human CD3ε is designated GenBank Accession No. NM_000733. Human CD3γ is designated GenBank Accession No. NM_000073. Human CD3δ is designated GenBank Accession No. NM_000732. CD3 is involved in TCR signal transduction. As described in Lin and Weiss, Journal of Cell Science 114, 243-244 (2001), activation of the TCR complex by binding of an MHC-presented specific antigen epitope results in phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) by Src family kinases, triggering the recruitment of additional kinases and Ca 2+ For example, clustering of CD3 on T cells by immobilized anti-CD3 antibodies results in T cell activation similar to T cell receptor engagement, but independent of the typical specificity of that clone.

[0105] As used herein, "CD3" refers to an antigen that includes human CD3 and is expressed on human T cells as part of the multimolecular T cell receptor complex. With respect to CD3, the binding agents of the invention preferably recognize the epsilon chain of CD3. In some embodiments, they recognize an epitope corresponding to the first 27 N-terminal amino acids of CD epsilon or a functional fragment of this 27 amino acid stretch.

[0106] The term "variant" according to the present invention refers in particular to mutants, splice variants, conformations, isoforms, allelic variants, species variants, and species homologs, especially those that occur naturally. Allelic variants refer to changes in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies multiple allelic variants for a given gene. Species homologs are nucleic acid or amino acid sequences that have different species of origin from the origin of the given nucleic acid or amino acid sequence. The term "variant" is intended to encompass any post-translational modification variants and conformational variants.

[0107] According to the present invention, the term "CLDN18.2 positive cancer" or similar terms refers to cancers with cancer cells that express CLDN18.2, preferably on their surface.

[0108] "Cell surface" is used according to its normal meaning in the art, and thus includes the outside of a cell that is available for binding by proteins and other molecules.

[0109] CLDN18.2 is located on the surface of a cell and is expressed on the surface of the cell if it can be bound by a CLDN18.2-specific antibody added to the cell.

[0110] CD3 is located on the surface of a cell and is expressed on the surface of said cell if it can be bound by a CD3-specific antibody added to the cell.

[0111] The term "extracellular portion" in the context of the present invention refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from the outside of said cell (e.g. by an antigen-binding molecule, such as an antibody, that is located extracellularly). Preferably, the term refers to one or more extracellular loops or domains, or fragments thereof.

[0112] The terms "portion" and "fragment" are used interchangeably herein and refer to a contiguous element. For example, a portion of a structure, such as an amino acid sequence or a protein, refers to a continuous element of said structure. A portion, part, or fragment of a structure preferably comprises one or more functional properties of said structure. For example, a portion, part, or fragment of an epitope or peptide is preferably immunologically equivalent to the epitope or peptide from which it is derived. A portion or fragment of an amino acid sequence preferably comprises a sequence of at least 4, particularly at least 6, at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids of a protein sequence.

[0113] A "fragment" in relation to an amino acid sequence (peptide or protein) refers to a part of the amino acid sequence, i.e. to a sequence that represents an amino acid sequence truncated at the N-terminus and / or C-terminus. A fragment truncated at the C-terminus (N-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 3' end of the open reading frame. A fragment truncated at the N-terminus (C-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, as long as this contains an initiation codon that serves to start the translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence.

[0114] A "variant" or similar expression of an amino acid sequence herein refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or may be a modified version of the wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, for example, 1 to about 20 amino acid modifications compared to the parent, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0115] "Wild-type," or "WT," or "native," as used herein with respect to an amino acid sequence, refers to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0116] For the purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translational modification variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that occur naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0117] Amino acid insertion variants include the insertion of one, two or more amino acids in a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertions are also possible, with appropriate screening of the resulting products.

[0118] 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.

[0119] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence (e.g., the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids). The deletion may be at any position in the protein. Amino acid deletion variants, including deletions at the N-terminus and / or C-terminus of the protein, are also referred to as N-terminal and / or C-terminal truncated variants.

[0120] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another in its place. Preference is given to modifications at positions of the amino acid sequence that are not conserved between homologous proteins or peptides and / or to replace amino acids with other amino acids with similar properties. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e. the substitution of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally classified into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

[0121] In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids shown in the table below.

[0122] [Table A]

[0123] Preferably, the degree of similarity (preferably identity) between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 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 over 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, in some embodiments consecutive amino acids. In some 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) is performed using tools known in the art, preferably using best sequence alignment, e.g., using Align, using standard settings. This can be done preferably using EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0124] "Sequence similarity" refers to the percentage of amino acids that are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between these sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between these sequences.

[0125] The term "identical %", "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences being compared. Said percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is usually carried out by comparing the sequences over a segment or "window of comparison" after optimal alignment to identify local regions of corresponding sequences. The alignment of the best fit for comparison may be performed manually, with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is calculated using the method of the United States National Center for Biotechnology Information. (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq) are used to determine the sequence of sequences that are most likely to be aligned. In some embodiments, the algorithm parameters used in the BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Maximum Sequencing set to 0; matches in a query range; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap set to Linear In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.

[0126] The percentage of identity is obtained by determining the number of identical positions where the compared sequences correspond, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0127] In some embodiments, the degree of similarity or identity is given over a region of 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 sequence. For example, if the reference nucleic acid sequence is 200 nucleotides, the degree of identity is given over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, consecutive nucleotides. In some embodiments, the degree of similarity or identity is given over the entire length of the reference sequence.

[0128] Homologous amino acid sequences, according to the present disclosure, exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98% or at least 99% of the amino acid residues.

[0129] The amino acid sequence variants described herein can be readily prepared by one skilled in the art, for example by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions, or deletions is detailed, for example, in Sambrook et al. (1989). Furthermore, the peptide and amino acid variants described herein can be readily prepared with the aid of known peptide synthesis techniques, for example by solid phase synthesis and similar methods.

[0130] In one embodiment, the fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties identical or similar to the amino acid sequence from which it is derived (i.e. functionally equivalent). For the antigen-binding domains constituting functional VH and VL variants, one particular function is to retain the binding of said binding domain. The term "functional fragment" or "functional variant", as used herein, refers in particular to variant molecules or variant sequences that contain an amino acid sequence in which one or more amino acids have been altered compared to the amino acid sequence of the parent molecule or parent sequence, and that can still exert one or more, or all, of the functions of the parent molecule or parent sequence (e.g. forming a binding domain with specificity for a particular antigen). For example, the binding domains constituting functional VH and VL variants, or functional CDR variant sequences, have the same or similar binding properties compared to the parent molecule. In one embodiment, the modification in the amino acid sequence of the parent molecule or sequence does not significantly affect or change the properties of this molecule or sequence. In another embodiment, the properties of the molecule constituting the functional fragment or functional variant (e.g., binding properties such as binding strength of the binding domain) may be reduced but still significantly present, for example, the binding properties such as binding strength of the binding domain constituting the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. For example, the functional variant may contain 1, 2, 3, 4, 5, or more amino acid insertions, additions, substitutions, and / or deletions compared to the parent molecule. However, in other embodiments, the properties of the molecule constituting the functional variant or functional fragment (e.g., binding properties of the binding domain constituting the functional fragment or functional variant) may be enhanced compared to the parent molecule.In some embodiments, a "functional variant" is a "functional fragment", e.g., an amino acid sequence that is truncated at the N-terminus and / or C-terminus compared to the parent molecule, but retains or essentially retains one or more or all of the functions of the parent molecule as described above, in particular an amino acid sequence that is functionally equivalent to the parent molecule.

[0131] The term "functional variant" of an amino acid sequence refers to a "functional" fragment of said amino acid sequence. Includes pieces.

[0132] The properties (eg, binding properties) of the functional variants can be analyzed by known methods, for example, using the ELISA assays described herein for binding agents that compete with each other.

[0133] An amino acid sequence (peptide, protein, or polypeptide, e.g., VH, VL, CH1, CH2, or CH3) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide, e.g., VH, VL, CH1, CH2, or CH3) refers to the origin of the initial amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to this particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of this particular sequence or a fragment thereof, preferably a functional variant (e.g., a functional fragment) as described herein. For example, it will be understood by those skilled in the art that an amino acid sequence suitable for use herein may be altered (e.g., amino acid insertion, amino acid deletion, amino acid addition, and / or amino acid substitution) such that the sequence differs from the naturally occurring or native sequence from which the amino acid sequence is derived, while retaining or essentially retaining the desired activity of the native sequence. For example, the amino acid sequences of the VH, VL, CH1, CH2, and / or CH3 domains on the peptide chains of the binding agents of the invention are derived from the amino acid sequences of the VH, VL, CH1, CH2, and / or CH3 domains of an immunoglobulin, but may be altered compared to the domains from which they are derived. For example, according to the invention, a VH or VL derived from an immunoglobulin comprises an amino acid sequence that may be identical to the amino acid sequence of the VH or VL, respectively, that is derived from the respective parent VH or VL sequence or that may differ at one or more amino acid positions compared to the respective parent VH or VL sequence. For example, the VH domain of the binding agent of the invention may comprise an amino acid sequence that comprises one or more amino acid insertions, additions, deletions, and / or substitutions compared to the amino acid sequence of the VH domain from which it is derived. For example, the VL domain of the binding agent of the invention may comprise an amino acid sequence that comprises one or more amino acid insertions, additions, deletions, and / or substitutions compared to the amino acid sequence of the VL domain from which it is derived.Preferably, a VH or VL having an amino acid sequence that is a functional variant of a parent VH or VL amino acid sequence provides the same or essentially the same function as the parent VH or VL amino acid sequence, e.g., with respect to binding specificity, binding strength, etc. However, as the skilled artisan will recognize, in some embodiments it may be preferable to provide a functional variant of an amino acid sequence (e.g., a VH or VL) that has altered properties compared to the amino acid sequence of the parent molecule. The same considerations apply, for example, to the amino acid sequences of the CDRs, and to other amino acid sequences (e.g., the amino acid sequences of the CH1, CH2, CH3, and / or CL domains).

[0134] When a bispecific binding agent is described as comprising a VH "derived" from an immunoglobulin and a VL "derived" from the same or a different immunoglobulin, the term "derived" indicates that the bispecific binding agent was generated by recombining, by any known method, the VH and VL from said immunoglobulin into the resulting bispecific binding agent. In this context, "recombining" is not intended to be limited by any particular recombination method, and therefore includes all of the methods of producing bispecific binding agents described herein below or known in the art, including, for example, recombination at the nucleic acid level and / or by co-expression of different molecules in the same cell. The term "bispecific antibody" or "bsAb" refers to a binding agent having two different antigen-binding domains defined by different amino acid sequences. In some embodiments, the different antigen-binding domains bind to different epitopes on the same antigen. However, in preferred embodiments, the different antigen-binding domains bind to different target antigens. Bispecific binding agents can be in any format, including any of the bispecific formats described herein. A binding agent can bind to each of the different antigens or epitopes with one, two or more binding domains, i.e., can bind to each of the different antigens or epitopes monovalently, divalently (or bivalently), trivalently, tetravalently or even higher valency.

[0135] According to the present invention, CLDN18.2 is not substantially expressed in cells when the expression level is low compared to the expression in gastric cells or gastric tissue. Preferably, this expression level is less than 10% of the expression in gastric cells or gastric tissue, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% or even lower. Preferably, CLDN18.2 is not substantially expressed in cells when the expression level exceeds the expression level in non-cancerous tissue other than the stomach by no more than 2-fold, preferably by 1.5-fold, preferably not exceeding the expression level in said non-cancerous tissue. Preferably, CLDN18.2 is not substantially expressed in cells when the expression level is below the detection limit and / or when the expression level is too low to be bound by a CLDN18.2-specific antibody added to the cells.

[0136] According to the present invention, CLDN18.2 is expressed in a cell when the expression level exceeds the expression level in non-cancerous tissue other than the stomach, preferably more than 2-fold, preferably 10-fold, 100-fold, 1000-fold, or 10000-fold. Preferably, CLDN18.2 is expressed in a cell when the expression level is above the detection limit and / or is high enough to be bound by a CLDN18.2-specific antibody added to the cell. Preferably, CLDN18.2 expressed in a cell is expressed or exposed on the surface of the cell.

[0137] According to the present invention, the term "disease" refers to any pathological condition, including cancer, particularly the forms of cancer described herein. Any reference herein to cancer or a particular form of cancer also includes metastasis of this cancer. In a preferred embodiment, the disease treated according to the present teachings involves cells that express CLDN18.2.

[0138] "Diseases associated with cells expressing CLDN18.2" or similar expressions means, according to the present invention, that CLDN18.2 is expressed in cells of diseased tissues or organs. In one embodiment, the expression of CLDN18.2 in cells of diseased tissues or organs is increased compared to the state in healthy tissues or organs. 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, expression is found only in diseased tissues, while expression in healthy tissues is suppressed. According to the present invention, diseases associated with cells expressing CLDN18.2 include cancer diseases. According to the present invention, cancer diseases are preferably diseases in which cancer cells express CLDN18.2.

[0139] As used herein, "cancer disease" or "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. "Cancer cells" refers to abnormal cells that grow by rapid and uncontrolled cell proliferation and continue to grow after the stimulus that initiated the new growth has ceased. Preferably, the "cancer disease" is characterized by cells that express CLDN18.2, and the cancer cells express CLDN18.2. The cells that express CLDN18.2 are preferably cancer cells, preferably cancer cells of a cancer described herein.

[0140] The term "cancer" according to the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, Cancer types include glioma, rectal cancer, endometrial cancer, renal cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, gastroesophageal junction (GEJ) cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, and lung cancer, as well as metastases thereof. Examples of this are lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer, or metastases of the above cancer types or tumors. The term cancer according to the present invention also includes cancer metastases.

[0141] According to the present invention, "carcinoma" refers to malignant tumors derived from epithelial cells. This group represents the most common cancers, including common forms of breast, prostate, lung and colon cancer.

[0142] "Adenocarcinoma" refers to cancer that arises in glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissue includes skin, glands, and various other tissues that line body cavities and organs. Epithelium is embryologically derived from ectoderm, endoderm, and mesoderm. To be classified as an adenocarcinoma, a cell does not necessarily have to be part of a gland, as long as it has secretory properties. This type 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 adenocarcinomas may not. By staining the cells from the biopsy, the pathologist determines whether the tumor is an adenocarcinoma or another type of cancer. Adenocarcinoma can arise in many tissues of the body due to the ubiquitous distribution of glands in the body. Although not every gland can secrete the same substances, as long as the cell has an exocrine function, it is considered glandular, and therefore the malignant form is named adenocarcinoma. Malignant adenocarcinomas often invade other tissues and metastasize if given enough time. Ovarian adenocarcinoma is the most common type of ovarian cancer and includes serous, mucinous, clear cell, and endometrioid adenocarcinoma.

[0143] "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastases is a very complex process that depends on the detachment of malignant cells from the primary tumor, their invasion into the extracellular matrix, their penetration through the endothelial basement membrane into the body cavities and blood vessels, and then their invasion into the target organ after being carried by the blood. Finally, the growth of new tumors at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, because cells or components of the tumor may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the present invention relates to "distant metastasis", which refers to metastasis away from the primary tumor and the regional lymph node system. In one embodiment, the term "metastasis" according to the present invention relates to lymph node metastasis. One particular form of metastasis treatable using the therapeutic or treatment method of the present invention is metastasis originating from gastric cancer as a primary site. In a preferred embodiment, such gastric cancer metastasis is Krukenberg tumor, peritoneal metastasis, and / or lymph node metastasis.

[0144] Krukenberg tumor is a rare metastatic tumor of the ovary that accounts for 1%-2% of all ovarian tumors. The prognosis of Krukenberg tumor remains very poor, and there is no established treatment for Krukenberg tumor. Krukenberg tumor is a metastatic signet ring cell adenocarcinoma of the ovary. The stomach is the primary site in most Krukenberg tumor cases (70%). Carcinomas of the colon, appendix, and breast (mainly invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg tumor arising from carcinomas of the gallbladder, biliary tract, pancreas, small intestine, ampulla of Vater, cervix, and bladder / urachus have been reported.

[0145] "Treating" refers to administering a therapeutic agent to a subject, such as to prevent or eliminate a disease (e.g., to reduce the size or number of tumors in a subject); to halt or lessen the progression of a disease in a subject; "In some embodiments, the term "compound" refers to administering a compound or composition, or a combination of compounds or compositions, to a subject in order to inhibit or delay the onset of disease in a subject; to inhibit or delay the onset of new disease in a subject; to reduce the frequency or severity of symptoms and / or recurrences in a subject currently suffering from or previously suffering from a disease; and / or to prolong, i.e., increase, the longevity of the subject.

[0146] Specifically, the term "treatment of a disease" includes curing, shortening the duration, amelioration, prevention, slowing or inhibiting the progression or deterioration, or preventing or delaying the onset of a disease or a symptom thereof.

[0147] In the context of the present invention, terms such as "protect", "prevent", "prophylactic", "preventive" or "protective" relate to the prevention or treatment, or both, of the occurrence and / or spread of disease in a subject, and in particular to minimizing the likelihood that a subject will develop a disease or to delay the onset of a disease. For example, a person at risk of cancer would be a candidate for treatment to prevent cancer.

[0148] "At risk" refers to a subject that is identified as having a higher than normal chance of developing a disease (especially cancer) compared to the general population.In addition, a subject that has had or is currently suffering from a disease (especially cancer) is at high risk of developing the disease, and therefore the subject may continue to develop the disease.Subjects that currently have or have had cancer are also at high risk of cancer metastasis.

[0149] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may or may not be afflicted with a disease or disorder (e.g., cancer). In many embodiments, the individual is a human. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age and thus include adults, elderly, children, and newborns. In an embodiment of the present disclosure, an "individual" or "subject" is a "patient."

[0150] The term "patient" according to the present invention means a subject of treatment (especially a diseased subject), for example a human, a non-human primate, or another animal, in particular a mammal, such as a cow, horse, pig, sheep, goat, dog, cat, or rodent, such as a mouse or rat. In a particularly preferred embodiment, the patient is a human.

[0151] "Target cell" is intended to mean any unwanted cell, such as a cancer cell, etc. In a preferred embodiment, the target cell expresses CLDN18.2.

[0152] The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effect, e.g., with respect to the type of immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or properties of the antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to the immune system of a subject, it induces an immune response (in particular the stimulation, priming, and / or expansion of T cells) with specificity that reacts with the reference amino acid sequence. Thus, a molecule that is immunologically equivalent to an antigen exhibits the same or essentially the same properties and / or exerts the same or essentially the same effect as the antigen targeted by the T cells with respect to the stimulation, priming, and / or expansion of T cells.

[0153] "Activated" or "stimulated" as used herein refers to a state of immune effector cells, such as T cells, that have been stimulated sufficiently to induce detectable cell proliferation. Activation can also be associated with the initiation of signal transduction pathways, induced cytokine production, and detectable effector functions. The term "activated immune effector cells" refers specifically to immune effector cells that are undergoing cell division.

[0154] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen and differentiates into an effector cell, such as an effector T cell.

[0155] The term "clonal expansion" or "expansion" refers to the process by which a specific entity multiplies. In the context of the present disclosure, the term is preferably used in reference to an immunological reaction in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cells that recognize said antigen are amplified. Preferably, clonal expansion leads to differentiation of immune effector cells.

[0156] The term "antigen" preferably relates to a molecule, such as a protein or peptide, that contains an epitope against which an agent is directed and / or should be directed to induce an immune response. An antigen, such as a T cell epitope, or a processing product thereof, in one embodiment, binds to a T cell receptor or a B cell receptor or binds to an immunoglobulin molecule, such as an antibody. Thus, the antigen or a processing product thereof may specifically react with an antibody or a T lymphocyte (T cell). In a preferred embodiment, the antigen is a tumor-associated antigen, such as CLDN18.2 (i.e. a component of cancer cells that may originate from the cytoplasm, cell surface and cell nucleus), in particular an antigen that is produced in large amounts, preferably intracellularly or as a surface antigen of cancer cells.

[0157] In the context of the present invention, the term "tumor-associated antigen" or "cancer-associated antigen" preferably relates to a protein which, under normal conditions, is specifically expressed in a limited number of tissues and / or organs or at a particular developmental stage and which is expressed or aberrantly expressed in one or more tumor or cancer tissues. In the context of the present invention, tumor-associated antigens are preferably associated with the cell surface of cancer cells and are preferably not or barely expressed in normal tissues.

[0158] The term "epitope" refers to an antigenic determinant in a molecule, e.g., a portion of a molecule that is recognized by the immune system (e.g., by an antibody). For example, an epitope is a distinct, three-dimensional site on an antigen that is recognized by the immune system. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids in length, for example, the epitope 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.

[0159] The term "binding agent" as used herein refers to any agent capable of binding to a desired antigen. In certain embodiments of the invention, the binding agent is an antibody, an antibody fragment, or a construct thereof. The binding agent may also include synthetic, modified, or non-naturally occurring moieties, particularly non-peptide moieties. Such moieties may link the desired antigen-binding functionality or region of, for example, an antibody or antibody fragment. In one embodiment, the binding agent comprises an antigen-binding CDR or variable It is a synthetic construct containing the region.

[0160] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably to antibodies or antigen receptors such as B-cell receptors (BCR). Immunoglobulins are characterized by structural domains (i.e. immunoglobulin domains) with a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound and soluble immunoglobulins. Soluble immunoglobulins are commonly called antibodies. Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains linked via disulfide bonds. These chains are mainly composed of immunoglobulin domains such as VL (variable light chain) domain, CL (constant light chain) domain, VH (variable heavy chain) domain, and CH (constant heavy chain) domains CH1, CH2, CH3, and CH4. There are five types of immunoglobulin heavy chains in mammals: α, δ, ε, γ, and μ, which constitute the various classes of immunoglobulins (i.e., IgA, IgD, IgE, IgG, and IgM). Immunoglobulin classes are also referred to as "isotypes" (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM), which refer to the class of immunoglobulins encoded by the heavy chain constant region genes. When a particular isotype (e.g., IgG1) is referred to herein, the term is not intended to be limited to a particular isotype sequence (e.g., a particular IgG1 sequence), but is used to indicate that the antibody is closer in sequence to this isotype (e.g., IgG1) than to other isotypes. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at the carboxy terminus. In mammals, there are two types of light chains (i.e., lambda and kappa). Immunoglobulin chains contain a variable region and a constant region. The constant regions are essentially conserved among the various isotypes of immunoglobulins, while the variable parts are highly diversified and are responsible for antigen recognition.

[0161] The term "antibody" refers to an immunoglobulin, or an antigen-binding portion thereof, comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and chimeric antibodies. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) comprising a CH1 domain, a CH2 domain, and a CH3 domain, and a heavy chain constant region (amino acid residues 118-447 of human IgG1 according to EU numbering), with CH1 typically being linked to CH2-CH3 by a peptide linker (also called a "hinge"). Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The terms "region" and "domain" are used interchangeably herein. The VH and VL domains can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions of high conservation, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk, J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to the Kabat numbering system and references to amino acid positions of constant regions herein are according to EU numbering (Edelman et al., 1999). et al., (1969) Proc. Natl. Acad. Sci. USA 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edit. 1991 NIH Publication No. 91-3242). The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody bind to various cells of the immune system (e.g., effector cells) and host tissues, including the first component (C1q) of the classical complement system. or may mediate the binding of immunoglobulin to the factor.

[0162] The term "amino acid corresponding to position ..." as used herein refers to the number of the amino acid position in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence that "corresponds" to an amino acid or segment in another sequence is one that is aligned with the other amino acid or segment, typically using a standard sequence alignment program such as ALIGN, ClustalW, or the like with default settings, and has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. Methods for aligning sequences, or segments in sequences, and thereby determining corresponding positions in the sequences as amino acid positions according to the invention, are deemed to be known in the art.

[0163] The term "IgG Fc ligand" as used herein refers to a molecule (preferably a polypeptide) that binds to the Fc region of an IgG immunoglobulin to form an Fc / Fc ligand complex. Fc ligands include, but are not limited to, FcγRI, FcγRII, FcγRIII, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), a family of Fc receptors that are homologous to FcγR (Davis et al., (2002) Immunol. Rev. 190:123-136). Particular IgG Fc ligands are FcRn and Fc gamma receptors. "Fc ligand" as used herein can be from any organism, such as mouse, human, and cynomolgus monkey.

[0164] "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to any member of a family of proteins that bind to the IgG Fc region and are encoded by the FcγR genes. In humans, this family includes, but is not limited to, FcγRI (CD64), e.g., isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), e.g., isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), e.g., isoforms FcγRIIIa (including allotypes V158 and F158), and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., (2002) Immunol. Lett. 82:57-65). FcγR can be from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2).

[0165] "FcRn" or "neonatal Fc receptor" as used herein refers to a protein that binds to the IgG Fc region and is at least partially encoded by the FcRn gene. FcRn can be from any organism, including but not limited to human, mouse, rat, rabbit, and monkey. A functional FcRn protein comprises two polypeptides, often referred to as a heavy chain (encoded by the FcRn gene) and a light chain (beta2-microglobulin). Unless otherwise specified, "FcRn" or "FcRn protein" refers to the complex of FcRn heavy chain and beta-2-microglobulin. "FcRn variants" as used herein are those that have increased binding to the FcRn receptor and may also have increased serum half-life.

[0166] "Fc" or "Fc region" or "Fc domain" as used herein means It refers to a polypeptide comprising the CH2 and CH3 domains of an IgG molecule, and optionally a peptide linker such as a hinge. The CH2-CH3 domain of human IgG1 comprises amino acids 231-447 and the hinge comprises positions 216-230 according to EU numbering. Thus, with respect to IgG, the term "Fc domain", as used herein, includes amino acids 231-447 (CH2-CH3) and 216-447 (hinge-CH2-CH3) according to EU numbering, as well as functional variants thereof, including functional fragments. An "Fc fragment" may contain fewer amino acids (e.g., an N-terminal or C-terminal truncation variant) but still retain the ability to form a dimer with another Fc domain or Fc fragment, as can be detected using standard methods (e.g., size-based methods (e.g., non-denaturing chromatography, size exclusion chromatography, etc.), and thus is a functional variant. According to the present invention, the IgG Fc domain is preferably a human IgG Fc domain, such as an Fc domain from human IgG1, IgG2, or IgG4.

[0167] The terms "hinge", "hinge region", "antibody hinge region", or "hinge domain", as used herein, refer to a peptide linker comprising the amino acids between CH1 and CH2 of an immunoglobulin (e.g., IgG). Structurally, in a naturally occurring IgG (e.g., IgG1) molecule, CH1 ends at amino acid position 215 according to EU numbering, and CH2 begins at amino acid position 231 according to EU numbering. Thus, in the case of IgG, the hinge comprises amino acids 216-230 according to EU numbering.

[0168] A "variant Fc domain" comprises amino acid modifications compared to a parent Fc domain. Thus, a "variant IgG1 Fc domain" (e.g. a variant human IgG1 Fc domain) comprises amino acid modifications (e.g. amino acid substitutions and / or deletions) at positions corresponding to positions of an IgG1 Fc domain (e.g. a human IgG1 Fc domain) and is preferably a functional variant of the parent Fc domain. Such a variant IgG Fc domain retains at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding parent human IgG Fc domain. Optionally, the variant Fc domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the parent Fc domain. Optionally, the variant Fc domain may have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the parent Fc domain. Preferably, the variant Fc domain retains the ability to form a dimer with another Fc domain, as measured using techniques described herein or known in the art (e.g., non-denaturing gel electrophoresis).

[0169] Fc modification The binding agents described herein comprise at least three different polypeptide chains, where two polypeptide chains (e.g., a first polypeptide chain and a second polypeptide chain) comprise a CH2-CH3 region, preferably derived from an IgG (e.g., IgG1, in particular human IgG1). Preferably, said first and second polypeptide chains comprising a CH2-CH3 region can interact, e.g., dimerize, thereby forming a heterodimer comprising said first and second polypeptide chains of the binding agents described herein. Preferably, the CH2-CH3 region of the binding agents of the invention is derived from IgG1 (more preferably human IgG1), although CH2 and CH3 from other serotypes may also be used as described herein. Furthermore, as discussed herein, the binding agents of the invention self-assemble, e.g., within a production host cell. For example, the CH2 of the first polypeptide chain interacts with the CH2 of the second polypeptide chain, and / or the CH3 of the first polypeptide chain interacts with the CH2 of the second polypeptide chain. It is envisioned that the CH1 domain interacts with the CH3 of the first and second polypeptide chain, thereby forming an Fc fragment. This Fc fragment may include one or more amino acid modifications or "Fc modifications" discussed herein for human IgG1 to promote interaction between the CH2 of the first and second polypeptide chains and / or between the CH3 of the first and second polypeptide chains, and / or to facilitate purification of heteromultimers (e.g., heterodimers) comprising first and second polypeptide chains that interact with each other more than homomultimers comprising only one type of polypeptide chain, and / or to confer additional beneficial functionality as discussed herein. The amino acid modifications discussed herein may also be included in the CH1 domain, e.g., in the first and / or second polypeptide chains of the binding agent of the invention. In addition, peptide linkers, e.g., peptide linkers in the scFv portion, or peptide linkers connecting additional domains of the binding agent, e.g., CH1 and scFv, or CH2 and scFv, or CH1 and CH2, may include one or more amino acid modifications as described herein. For example, a peptide linker connecting a CH2-CH3 region to another region or domain (e.g., VH(CD3), VL(CD3), or CH1) may have a serine at the amino acid position corresponding to position 220 (where cysteine ​​would normally be found) according to EU numbering of naturally occurring IgG1. Using a peptide linker that contains a serine at said position corresponding to position 220 of human IgG1 reduces disulfide formation between the two chains that comprise the CH2-CH3 regions.

[0170] Thus, the formation of the binding agents of the present invention is based on the use of various monomers (e.g., first and second polypeptide chains as described herein) that contain amino acid substitutions including CH1, CH2, and / or CH3, such as those that "bias" the formation of heterodimers formed by said monomers over homodimers, preferably in conjunction with "pI modifications" that allow simple purification of heterodimers away from homodimers, and optionally in combination with "ablation modifications" as discussed herein and additional Fc modifications that "bias" the formation of heterodimers formed by said monomers over homodimers as described herein. Those skilled in the art will understand that any of the amino acid modifications discussed herein for the CH1, CH2, and / or CH3 domains and peptide linkers (e.g., hinge variants) may be combined with additional amino acid modifications discussed herein or known in the art. For example, any of the skew modifications and pI modifications may be combined independently with ablation modifications and other Fc modifications. The Fc modifications of the present invention may be amino acid insertions, additions, deletions, or substitutions.

[0171] The Fc modifications discussed herein are defined according to the amino acid modifications that make them up. For example, N434S is an Fc modification with a substitution of serine to asparagine at position 434 relative to the parent human IgG1 Fc polypeptide and according to EU numbering. The identity of the parent amino acid may not be specified, in which case the variant is referred to as 434S, i.e., the CH2-CH3 region that makes up the Fc modification contains a serine at the amino acid position corresponding to position 434 of human IgG1 according to EU numbering.

[0172] pI engineering pI modifications increase the isoelectric point (pI) difference between monomers and allow for isoelectric purification of homo- and heteromultimeric proteins. In general, pI modifications increase the pI of a polypeptide chain (basic change) or decrease the pI of a polypeptide chain (acidic change).

[0173] As discussed herein, a pI difference of at least 0.1, e.g., 0.2, 0.3, 0.4, or 0.5 between two polypeptide chains may allow separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point known in the art. Thus, each of the polypeptide chains that interact with each other (e.g., as discussed herein) may have a pI difference of at least 0.1, e.g., 0.2, 0.3, 0.4, or 0.5 between the two polypeptide chains. Inclusion of pI modifications that alter the pI of the first and second polypeptide chains described, such that each of the polypeptide chains has a different pI, and the heteromultimers formed by the polypeptide chains also have different pIs, facilitating isoelectric purification of binding agents containing the heteromultimers. These substitutions also aid in determining and monitoring the formation of any contaminating, unwanted homo- or heteromultimers.

[0174] pI modifications can be included in one or both chains of a heteromultimer comprising a heavy chain constant region (e.g., the first and second polypeptide chains of a binding agent of the invention), as well as in CH1, CH2, and / or CH3, and / or in a peptide linker, such as a linker connecting the VH and VL domains of an scFv portion. For example, pI variants can be included in the first and / or second polypeptide chains of a binding agent of the invention to reduce or prevent homomultimer formation. Typically, pI variants are used that, when included in both the first and second polypeptide chains, increase the pI of one polypeptide chain and decrease the pI of the second polypeptide chain. This can be done, for example, by replacing a neutral amino acid residue with a positively or negatively charged amino acid residue or vice versa, or by changing a charged amino acid residue from a positive charge to a negative charge or vice versa, as discussed herein. Thus, in certain embodiments of the invention, a sufficient change in pI in at least one of the polypeptide chains of the binders described herein is provided such that, for example, heteromultimers of the first and second polypeptide chains can be purified away from homomultimers. In certain embodiments, the invention uses a pI difference of only 0.1, 0.2, 0.3, 0.4, or 0.5 pH units, or higher. As will be appreciated by those of skill in the art, the amount of pI modification included in one or more polypeptide chains of the binders of the invention to obtain good separation will depend in part on the starting pI of the polypeptide chain, the pI of the CH region, the Fv scaffold region, and the like. The change in pI can be calculated by any method known in the art, for example, based on the CH region, using the method described by Sillero and Maldonado (Sillero, Maldonado, (2006) Comput. Biol. Med. 36(2):157-166). Alternatively, the pI of each polypeptide chain can be compared. In addition, the heteromultimers can be separated according to their size.

[0175] In certain embodiments, no pI modifications, asymmetric modifications, additional Fc modifications, or ablation modifications, etc. are included in the variable region of the binding agent of the invention.

[0176] In certain embodiments, the pI modifications are derived from different IgG isotypes, so that the pI of each polypeptide chain is changed without introducing immunogenicity (see US Patent Application Publication No. 2014 / 0370013). Preferably, the pI modifications are derived from human IgG isotypes, so that the risk of introducing immunogenicity is reduced. Although the modifications discussed herein are described in relation to human IgG1, all IgG isotypes can be modified in this manner, as well as isotype hybrids. R133E and R133Q can also be used when the heavy chain constant domain is derived from IgG2-4.

[0177] IgG1 is a common isotype of therapeutic antibodies for various reasons, including high effector function. However, the CH region of IgG1 has a higher pI compared to that of IgG2. By introducing IgG2-derived residues into the IgG1 backbone at specific positions, the pI of the resulting monomer can be lowered or increased, as well as the serum half-life increased. For example, human IgG1 has a glycine at position 137 according to EU numbering (pI approx. 5.97) and human IgG2 has a glutamic acid at the corresponding position (pI approx. 3.22); by replacing the glutamic acid residue with a glycine residue, the pI of the resulting polypeptide is affected. Lowering the pI of the antibody constant region also increases the serum half-life in vivo. (See U.S. Patent Application Serial No. 13 / 194,904; Ghetie and Ward, 1997, Immunol Today. 18(12):592-598). Similarly, variable regions with low pI can extend serum half-life (See Igawa et al., (2010) PEDS 23(5):385-392).

[0178] In a preferred combination of pI modifications, one polypeptide chain (e.g., a first polypeptide chain of a binding agent of the invention, comprising, for example, VH(CLDN18.2), CH1, CH2, and CH3) comprises an aspartic acid residue at position 208, a glutamic acid residue at position 295, an aspartic acid residue at position 384, a glutamic acid residue at position 418, and an aspartic acid residue at position 421 according to EU numbering (i.e., N208D / Q295E / N384D / Q418E / N421D for human IgG1), and another polypeptide chain (e.g., a second polypeptide chain of said binding agent, comprising, for example, VH(CD3) and VL(CD3), CH2 and CH3, and optionally VH(CLDN18.2) and CH1) comprises a positively charged peptide linker ("scFv linker") linking VH(CD3) and VL(CD3), for example having the amino acid sequence (GKPGS) 4 or a functional variant thereof.

[0179] In polypeptide chains that do not contain CH1 and therefore do not contain the amino acid position corresponding to position 208 according to EU numbering, the following negative pI modifications may be used: a glutamic acid residue at position 295, an aspartic acid residue at position 384, a glutamic acid residue at position 418, and an aspartic acid residue at position 421 according to EU numbering (human IgG1: Q295E / N384D / Q418E / N421D).

[0180] In some embodiments, a polypeptide chain (e.g., a first polypeptide chain) comprises a set of variants discussed herein and the polypeptide chain that interacts with it (e.g., a second polypeptide chain) comprises a charged scFv linker, e.g., a positively charged scFv linker selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or a functional variant thereof, or a negatively charged scFv linker selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, and 20, or a functional variant thereof.

[0181] Asymmetric modification "Asymmetric modification" refers to a steric modification that promotes the interaction of polypeptide chains that contain such modifications.One strategy using steric modification is called "knob and hole" in the art, which refers to amino acid manipulation, and typically introduces a protrusion into a first heavy chain polypeptide in the Fc region (CH2-CH3) and a corresponding cavity into a second heavy chain polypeptide in the Fc region (CH2-CH3), so that the protrusion is located in the cavity at the interface of these two heavy chains, promoting heterodimer formation and preventing homodimer formation (see US Patent Application No. 61 / 596,846, Ridgway et al., (1996) Protein Engineering 9(7):617; Atwell et al., (1997) J. Mol. Biol. 270:26; US Patent No. 8,216,805). The "protuberances" are constructed by replacing small amino acid side chains from the interface of the first heavy chain polypeptide with larger side chains. In the interface of the second heavy chain polypeptide, compensatory "cavities" of the same or similar size as the protuberances are formed by replacing large amino acid side chains with smaller ones (U.S. Pat. No. 5,731,168). "Knob and hole" modifications can be combined with disulfide bonds to bias the formation of the first and second heavy chain polypeptides toward heteromultimerization, e.g., heterodimerization (see Merchant et al., (1998) Nature Biotech. 16:677).

[0182] Useful asymmetric modifications include, but are not limited to, the following pairs of double modifications, where one part of each pair of double modifications is present in one polypeptide chain of a binding agent of the invention (e.g., a first polypeptide chain described herein) and another part is present in another polypeptide chain of a binding agent of the invention (e.g., a second polypeptide chain described herein): S364K / E357Q:L for human IgG1 according to EU numbering 368D / K370S;L368D / K370S:S364K;L368D / K370S:S364K / E357Q;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L368D / K370S:S364K / E357L;K370S:S364K / E357Q;T366S / L368A / Y407V:T366W, and T366S / L368A / Y407V / Y349C:T366W / S354C. Preferably, in the binder of the present invention, L368D / K370S:S364K / E357Q is used.

[0183] The asymmetric modifications described herein may also affect pI (see Gunasekaran et al., (2010) J. Biol. Chem. 285(25):19637) and therefore purification, and therefore may also be considered pI variants.

[0184] Ablation modification "Ablation" as used herein means reducing or eliminating activity. "Abolishing FcγR binding" means that an Fc region comprising one or more ablation modifications has greater than 50% loss of FcγR binding activity compared to an Fc region that does not contain this particular modification. Preferably, an Fc region comprising one or more ablation modifications has greater than 70%, 80%, 90%, 95%, 98% or even more loss of FcγR binding activity. Preferably, the FcγR binding activity of an Fc region comprising one or more ablation modifications compared to an Fc region that does not contain this particular modification is below the level of binding detectable in a Biacore, SPR, or BLI assay.

[0185] As is known, the Fc domain of human IgG1 has the highest binding to Fcγ receptors, so ablation modifications may be used when the constant domain of the binder is derived from IgG1. Alternatively, or in addition to ablation modifications, mutation of glycosylation position 297 (typically to A or S) may, for example, significantly eliminate binding to FcγRIIIa. Human IgG2 and IgG4 naturally have low binding to Fcγ receptors (Parren et al., 1992, J. Clin Invest. 90:1537-1546; Bruhns et al., 2009, Blood 113:3716-3725), so CH1, CH2, and CH3 domains derived from IgG2 or IgG4 may be used with or without ablation modifications in the binder of the present invention. Amino acid modifications that eliminate FcγR binding are described, for example, in Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.; 75(24):12161-12168 (2001).

[0186] Thus, the Fc portion of the binding agents of the invention may comprise one or more "FcγR ablation modifications" or "Fc knockout (FcKO or KO) modifications." In certain embodiments, it is desirable to reduce or eliminate binding of the Fc domain to one or more or all of the Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, etc.). In some embodiments, it is desirable to eliminate FcγRIIIa binding of binding agents that bind monovalently to CD3, such as the binding agents of the invention, in order to eliminate or significantly reduce ADCC activity. Thus, in the binding agents of the invention In one embodiment, one or more of the polypeptide chains (e.g., the first and second polypeptide chains) of the binding agents of the invention comprise one or more FcγR ablation variants. In a preferred embodiment, the one or more ablation variants comprise the following amino acids for human IgG1 according to EU numbering: G236R, S239G, S239K, S239Q, S239R, V266D, S267K, S267R, H268K, E269R, 299R, 299K, K322A, A327G, A327L, A327R, ...Q, S239R, V266D, S267K, S267R, H268K, E2 N, A327Q, L328E, L328R, P329A, P329H, P329K, A330L, A330S / P331S, I332K, I332R, V266D / A327Q, V266D / P329K, S267R / A327Q, S267R / P329K, G236R / L328R, E233P / L234V / L235A / G23 6_ / S267K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236del, S23 9K / S267K, 267K / P329K, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236d el / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del, where "del" represents an amino acid deletion at the indicated position. Preferably, the modification E233P / L234V / L235A / G236_ / S267K for human IgG1 is used according to EU numbering in both the first and second polypeptide chains of the binding agent of the invention. It should be noted that the ablation modifications disclosed herein eliminate FcγR binding but generally do not eliminate FcRn binding.However, techniques are known and can be used to decrease or increase binding to FcRn in order to decrease or increase the serum half-life of the binding agent (see, e.g., Dall'Acqua et al. 2006, J. Biol. Chem., 281:23514-24; Hinton et al. 2006, J. Immunol., 176:346-56; and Zalevsky et al. 2010 Nat. Biotechnol., 28:157-9).

[0187] For example, in a binding agent of the invention, the first polypeptide chain comprises an amino acid sequence according to SEQ ID NO:28, and the second polypeptide chain comprises an amino acid sequence according to SEQ ID NO:30, and the first and second polypeptide chains comprise an asymmetric modification of the L368D / K370S:S364K / E357Q set, and the first polypeptide chain further comprises a pI modification of the N208D / Q295E / N384D / Q418E / N421D set, and the first and second polypeptide chains both further comprise an ablation modification of the E233P / L234V / L235A / G236del / S267K set, e.g., the first polypeptide chain comprises a VH(CLDN18.2) and a CH1, and the second polypeptide chain comprises a scFv(CD3), optionally comprising a VH(CLDN18.2) and a CH1. Of course, further modifications may be included in the amino acid sequence of each binding agent, for example, a binding agent may include further amino acid modifications, such as substitutions, in addition to the modifications discussed above.

[0188] Further Fc modifications In addition to other modifications described herein, such as pI modifications, asymmetric modifications, and ablation modifications, many useful modifications may be used, such as those that alter binding to one or more FcγR receptors, those that alter binding to the FcRn receptor, etc.

[0189] Thus, there are many useful amino acid substitutions that can be made to alter the binding of the binding agents of the invention to one or more FcγR receptors. Substitutions that increase binding, as well as substitutions that decrease binding, can be useful. For example, it is known that increasing binding to FcγRIIIa increases ADCC. Similarly, decreasing binding to FcγRIIb can be beneficial. Amino acid substitutions that can be used in the present invention include those described in U.S. Patent Application Serial No. 11 / 124, 620, 11 / 174,287, 11 / 396,495, and 11 / 538,406, all of which are incorporated herein by reference in their entirety. Particularly useful amino acid substitutions that can be incorporated into the binding agents of the present invention include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D / 332E / 330L, 243A, 243L, 264A, 264V, and 299T.

[0190] Additionally, as disclosed in U.S. Patent Application Serial No. 12 / 341,769, which is incorporated herein by reference in its entirety, there are further modifications useful for increasing binding to FcRn and increasing serum half-life, including, but not limited to, 434S, 434A, 428L, 308F, 259I, 428L / 434S, 259I / 308F, 436I / 428L, 436I / 434S, 436V / 434S, 436V / 428L, and 259I / 308F / 428L according to EU numbering.

[0191] In addition, the CH3 of one or both polypeptide chains (preferably both polypeptide chains) forming the Fc heterodimer may contain the modifications M428L / N434S, which confers a longer serum half-life.

[0192] As will be understood by those skilled in the art, the modifications discussed herein may be combined independently with other modifications. In one embodiment, one polypeptide chain (e.g., a first polypeptide chain) of the binding agent of the present invention comprises N208D, Q295E, N384D, Q418E, and N481D according to EU numbering, and another polypeptide chain (e.g., a second polypeptide chain) of the binding agent comprises a positively charged scFv linker as described herein. Preferably, the first polypeptide chain of the binding agent further comprises K370S and L368D according to EU numbering, and the second polypeptide chain of the binding agent further comprises E357Q and S364K according to EU numbering. In addition, in a preferred embodiment, both the first polypeptide chain and the second polypeptide chain further comprise E233P, L234V, L235A, G236del, and S267K according to EU numbering. Most preferably, the first polypeptide chain of a binding agent of the invention comprises N208D, E233P, L234V, L235A, G236del, S267K, Q295E, L368D, K370S, N384D, Q418E, and N481D, and the second polypeptide chain of the binding agent comprises E233P, L234V, L235A, G236del, S267K, E357Q, and S364K, and optionally C220S to remove the cysteine ​​that typically pairs with the light chain.

[0193] The term "monoclonal binding agent" as used herein includes "monoclonal antibody" and refers to a preparation of binding agent molecules of single molecular composition. A monoclonal binding agent composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to a binding agent exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal binding agents may be produced by hybridomas (e.g., B cells) obtained from transgenic or transchromosomal non-human animals (e.g., transgenic mice) having genomes containing human heavy and light chain transgenes fused with immortalized cells.

[0194] The term "recombinant binding agent," as used herein, includes "recombinant antibody," and includes all binding agents prepared, expressed, produced, or isolated by recombinant means, such as, for example, (a) binding agents derived from animals (e.g., mice) that are transgenic or transchromosomal for immunoglobulin genes, or hybrids prepared therefrom. (b) binding agents isolated from a host cell (e.g., a transfectoma) that has been transformed to express the binding agent; (c) binding agents isolated from a recombinant, combinatorial antibody library; and (d) binding agents prepared, expressed, produced or isolated by any other means involving splicing of immunoglobulin gene sequences into other DNA sequences.

[0195] The term "human binding agent," as used herein, includes "human antibodies," and is intended to include binding agents having variable and constant regions derived from human germline immunoglobulin sequences. Human binding agents may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0196] The term "humanized binding agent", as used herein, includes "humanized antibodies", and refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, with the remaining immunoglobulin structure of the molecule being based on the structure and / or sequence of a human immunoglobulin. This can be achieved, for example, by grafting the six non-human antibody complementarity determining regions (CDRs) that together form the antigen-binding site into homologous human acceptor framework regions (FRs) (see WO 92 / 22653 and EP 0629240). The antigen-binding site may comprise a complete variable domain fused to a constant domain, or it may comprise only the complementarity determining regions (CDRs) grafted into the appropriate framework regions of the variable domain. Substitution of framework residues of the parent binding agent (i.e., a non-human binding agent, e.g., a murine antibody) into human framework regions (backmutation) may be required to fully reconstitute the binding affinity and specificity of the parent binding agent. Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of the binding agent. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions, e.g., to more closely resemble human immunoglobulins. Some forms of humanized binding agents preserve all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs of the mouse antibody). Other forms have one or more CDRs that are altered with respect to the original binding agent (e.g., an antibody). Thus, a humanized binding agent may include non-human CDR sequences, primarily human framework regions optionally containing one or more amino acid backmutations to the non-human amino acid sequences, and fully human constant regions.

[0197] The term "chimeric binding agent" as used herein includes "chimeric antibody" and refers to a binding agent in which a portion of each of the amino acid sequences of the heavy and light chains is homologous to the corresponding sequence of a binding agent (e.g., antibody) from a particular species or belonging to a particular class, and the remaining segments of the chains are homologous to the corresponding sequence of another. Typically, the variable regions of both the light and heavy chains mimic the variable regions of an antibody from one species of mammal, and the constant portions are homologous to the sequences of an antibody from another species. One distinct advantage to such chimeric forms is that the variable regions can be conveniently obtained from currently known sources, for example using readily available B cells or hybridomas from non-human host organisms in combination with constant regions from human cell preparations. The variable regions have the advantage of being easy to prepare and the specificity is not affected by the source, while the constant regions are human and less likely to elicit an immune response from a human subject when the binding agent is injected, compared to constant regions from non-human sources. However, this definition is not limited to this particular example.

[0198] The binding agents or fragments thereof, such as the variable and / or constant regions, can be derived from various species, including, but not limited to, mouse, rat, rabbit, guinea pig, and human.

[0199] Immunoglobulins described herein include IgA (e.g., IgA1 or IgA2), IgG1 (e.g., allotypes having polymorphisms at amino acid positions 356 (D or E) and 358 (L or M) according to EU numbering), IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the immunoglobulin is an IgG1 antibody, more specifically an IgG1, kappa, or IgG1, lambda isotype (i.e., IgG1, κ, λ), an IgG2 antibody (e.g., IgG2a, κ, λ), an IgG2b antibody (e.g., IgG2b, κ, λ), an IgG3 antibody (e.g., IgG3, κ, λ), or an IgG4 antibody (e.g., IgG4, κ, λ). The amino acid sequences described herein for the IgG1 allotype 356D / 358M also include the allotype 356E / 358L.

[0200] The term "IgG subclass modification" or "isotype modification" refers to an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid of a different aligned IgG isotype. For example, since IgG1 contains tyrosine and IgG2 contains phenylalanine, the F296Y substitution in IgG2, which is a phenylalanine at amino acid position 296 according to EU numbering, is considered an IgG subclass modification.

[0201] As used herein, a "heterologous binding agent" includes a "heterologous antibody" and is defined in relation to the transgenic organism producing such a binding agent. The term refers to a binding agent that has an amino acid sequence or encoding nucleic acid sequence that corresponds to one found in an organism other than the transgenic organism, and generally is derived from a species other than the transgenic organism.

[0202] As used herein, "heterohybrid binding agent" includes "heterohybrid antibodies" and refers to binding agents having light and heavy chains of different biological origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.

[0203] Binding agents such as antibodies described herein are preferably isolated. "Isolated" as used herein is intended to refer to a binding agent that is substantially free of other agents with different antigen specificity (e.g., an isolated binding agent that specifically binds to CLDN18.2 and CD3 is substantially free of binding agents that specifically bind to antigens other than CLDN18.2 and CD3). However, an isolated binding agent that specifically binds to an epitope, isoform, or variant of human CLDN18.2 may have cross-reactivity to other related antigens, such as related antigens from other species (e.g., homologs of the CLDN18.2 species). Additionally, an isolated binding agent may be substantially free of other cellular material and / or chemicals.

[0204] The term "antigen-binding portion" (or simply "binding portion") of a binding agent such as an antibody, or "antigen-binding fragment" (or simply "binding fragment") of a binding agent such as an antibody, or similar terms, refer to one or more fragments of the binding agent that retain the ability to specifically bind to an antigen. It has been recognized that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of a binding agent such as an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH domains; (ii) an F(ab') fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. 2 Fragment;(iii)F(ab') 2 (iv) an Fd fragment consisting of the VH and CH domains; (v) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment consisting of the VH domain (Ward et al., (1989)). 9) Nature 341:544-546; (vii) an isolated complementarity determining region (CDR), and (viii) a combination of two or more isolated CDRs, which may be optionally linked by a synthetic linker. Furthermore, the two domains VL and VH of the Fv fragment are encoded by separate genes, but they can be linked by a synthetic linker using recombinant techniques that allows the VL and VH domains to be produced as a single protein chain that pairs to form a monovalent molecule (also known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of a binding agent such as an antibody. A further example is a binding domain immunoglobulin fusion protein comprising: (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are further disclosed in US Patent Publication Nos. 2003 / 0118592 and 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0205] Single chain variable fragments (scFv) are fusion proteins of the variable regions of immunoglobulin heavy (VH) and light (VL) chains linked by a short linker peptide, usually 10 to about 30 amino acids, such as the scFv linkers represented by SEQ ID NOs: 2 to 20. This linker is usually enriched in glycine for flexibility and serine or threonine for solubility, and may link the N-terminus of VH to the C-terminus of VL, or vice versa. Divalent (or bivalent) single chain variable fragments (di-scFvs, bi-scFvs) can be engineered by linking two scFvs. This can be done by creating a single peptide chain with two VH and two VL domains, resulting in tandem scFvs. The invention also includes multispecific molecules comprising multiple scFv binding domains. A common flexible linking peptide is (G4S) x where x can be 2, 3, 4, 5, or 6. Preferably, the linker connecting the VH and VL domains of the scFv has the amino acid sequence (GKPGS): x or a functional variant thereof, wherein x can be 2, 3, 4, 5, or 6. Optionally, the association of VH and VL can be stabilized by one or more intermolecular disulfide bonds.

[0206] Another possibility is the creation of scFvs with a linker peptide that is too short (about 5 amino acids) for the two variable regions to fold together, and the scFvs are dimerized. This type is known as a diabody. Even shorter linkers (1 or 2 amino acids) form trimers (so-called triabodies or tribodies). Tetrabodies have also been generated. These show even higher affinity for the target compared to diabodies.

[0207] The term "specificity", as used herein, is intended to have the following meaning, unless the context contradicts: Two binding agents have the "same specificity" if they bind to the same antigen and the same epitope.

[0208] As used herein, the term "binding domain" or "antigen-binding domain" refers to the site of a binding agent, e.g., an antibody, that binds to an antigen, and includes the antigen-binding portion of a binding agent. A binding domain may be composed of heavy and light chain variable domains (VH and VL), each of which contains four conserved framework regions (FR) and three CDRs. The CDRs differ in sequence and determine the specificity for a particular antigen. The VH and VL domains together can form a site that specifically binds to a particular antigen. A "binding domain with specificity" for an antigen means that a binding agent comprising said binding domain binds to the antigen at a specific binding site, e.g., at about 10 -7 K below M D A binding agent is specific for an antigen if it binds to the antigen through said binding domain in a standard assay, but does not significantly (particularly not detectably) bind to an antigen different from the indicated antigen for which the binding domain is specific in a standard assay through said binding domain. Of course, if a binding agent contains multiple binding domains with different specificities, it will bind to multiple antigens with specificity, as described herein. The binding of the binding agent to the antigen can be determined, for example, using Bio-Layer Interferometry (BLI) or can be determined, for example, using the antigen as the ligand and the binding agent as the analyte, using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument.

[0209] A Fab (fragment antigen-binding) antibody fragment is an immunoreactive polypeptide comprising a monovalent antigen-binding domain of an antibody made up of a polypeptide consisting of a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1) portion, and a polypeptide consisting of a light chain variable region (VL) and a light chain constant region (CL and CH1 portions are linked together, e.g., by disulfide bonds between Cys residues). Preferably, in the Fab fragments described herein, CH1 and CL are of human origin. In one embodiment, CL is a kappa-type CL. In one embodiment, CH1 is derived from IgG1, preferably from human IgG1.

[0210] For the purposes of the present invention, all antibodies and antibody derivatives (e.g., antibody fragments) described herein are encompassed by the term "antibody". The term "antibody derivative" refers to any modified form of an antibody, such as a conjugate of an antibody with another agent or antibody, or an antibody fragment. Furthermore, the antibodies and antibody derivatives described herein are useful in the manufacture of binding agents of the present invention.

[0211] Naturally occurring antibodies are generally monospecific, i.e., bind to a single antigen. The present invention provides binding agents that bind to cytotoxic cells such as T cells (by binding to the CD3 receptor) and to target cells such as cancer cells (by binding to CLDN18.2). The binding agents of the present invention bind to at least two different types of antigens and are at least bispecific or multispecific, such as trispecific, tetraspecific, etc.

[0212] The binding agents of the present invention may be at least bivalent. In one embodiment, the binding agents of the present invention are at least trivalent. As used herein, "valence", "valences", "valencies", or other grammatical variations thereof, refer to the number of antigen binding sites or binding domains in a binding agent. Antigen binding sites that bind to the same antigen may recognize different epitopes, or preferably the same epitope. Trivalent and tetravalent bispecific antibodies are known in the art. The binding agents of the present invention may also have a valency of more than four.

[0213] The binding agents described herein may preferably be composed of fragments of at least two different antibodies (fragments of said at least two different antibodies forming at least two different binding domains), and are therefore artificial proteins (including protein complexes) that bind to at least two different types of antigens. The binding agents according to the invention are engineered to simultaneously bind to immune cells (e.g. immune effector cells, in particular T cells, such as cytotoxic cells) (e.g. by binding to CD3) and to target cells, such as cancer cells, to be destroyed (e.g. by binding to the tumor-associated antigen CLDN18.2).

[0214] Several types of bivalent and trivalent antibodies have been developed, and all types are within the scope of the present invention. Bispecific full-length antibodies can be obtained by covalently linking two monoclonal antibodies or by conventional hybrid hybridoma technology. Covalent linking of two monoclonal antibodies is described in Anderson, Blood 80 (1992), 2826-34. Another example of a bispecific antibody fragment is a diabody (Kipriyanov, Int. J. Cancer 77 (1998), 763-772), which is a small bivalent bispecific antibody fragment. Diabodies contain a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain, connected by a peptide linker that is too short to allow pairing between the two domains on the same chain. This forces pairing with the complementary domain of another chain, promoting the assembly of a dimeric molecule with two functional antigen binding sites.

[0215] In bispecific molecules comprising two Fab fragments, each of the individual Fab fragments may be arranged in a single chain (preferably VL-CL-CH-VH) and the individual variable and constant domains may be linked by a peptide linker, such as the peptide linkers discussed herein. In general, the individual single chains and the Fab fragments may be linked via disulfide bonds, attachment domains, chemical linkages, and / or peptide linkers. Bispecific molecules may also comprise more than two Fab fragments, in particular the molecules may be Fab3, Fab4 or multimeric Fab complexes with specificity for two, three, four or more different antigens. The present invention also includes chemically linked Fabs.

[0216] Triabodies or single chain triabodies (sctbs) are composed of three different scFv regions linked by a linker sequence. Similarly, the natural in vivo heterodimerization of heavy (CH1) and light (CL) chains can be used to form a scaffold onto which multiple scFvs can be added. For example, an scFv specific for one antigen can be linked to CH1, which is also linked to an scFv specific for another antigen, and this chain can interact with another chain containing an scFv specific for either antigen linked to CL (scFv3-CH1 / CL). Another example of a trivalent structure includes the use of a Fab fragment specific for one epitope C-terminally linked to two scFvs specific for different epitopes, one on each chain (Fab-scFv2). Yet another example of a trivalent construct includes the use of two Fab fragments specific for one antigen or epitope, where one of these Fab fragments is C-terminally linked to one scFv specific for another antigen or epitope (Fab2-scFv). Yet another example of a trivalent (or tetravalent) molecule includes various formats that include additional conjugates attached to the N-terminus or C-terminus of the antibody. For example, one format consists of an intact antibody molecule specific for one antigen, to which a single-chain Fab (scFab) is linked at the C-terminus of the molecule (IgG-scFab). The dock-and-lock (DNL) approach has also been used to generate trivalent antibodies (DNL-F(ab)3) (Chang, C.-H. et al. In:Bispecific Antibodies. Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 199-216 (2011)). Each of the above antibodies is within the scope of the present invention.

[0217] Tetravalent antibodies have also been constructed, and all types are within the scope of the present invention. Examples of tetravalent antibodies include, but are not limited to, scFv2-Fc molecules, F(ab')2-scFv2 molecules, scFv2-H / L molecules, and scFv-dhlx-scFv molecules. A bispecific scFv2-Fc construct has an Fc domain with two scFvs specific for one molecule linked to the N-terminus of the Fc chain and two other scFvs specific for another molecule linked to the C-terminus of the Fc chain. A bispecific F(ab')2-scFv2 construct contains an scFv fragment linked to the C-terminus of the F(ab')2 fragment. A scFv2-H / L construct has an Fc domain with two scFvs specific for one molecule linked to the N-terminus of the Fc chain and two other scFvs specific for another molecule linked to the C-terminus of the Fc chain. , has an scFv specific for one molecule linked to the heavy chain and an scFv specific for another molecule linked to the light chain. Finally, the scFv-dhlx-scFv construct contains one type of scFv linked to a helical dimerization domain followed by another type of scFv. Two chains of this type can dimerize to generate a tetravalent antibody.

[0218] The binding agent of the present invention may be in the format of an antibody molecule, or an antibody-like molecule, or a protein scaffold with antibody-like properties, or a cyclic peptide with at least two binding specificities. Thus, the binding agent may comprise one or more of the antibodies described herein, or functional fragments thereof.

[0219] In one embodiment, the binding agent of the invention comprises a heavy chain (Fd fragment) and a light chain (L) of a Fab fragment, capable of interacting and which may incorporate further binding functions or binding domains. Such further binding domains may be independently selected from the group consisting of a binding domain comprising two antibody variable regions (i.e. VH-VL or VL-VH), such as an scFv binding domain, and a binding domain comprising one antibody variable region, such as a VH binding domain and a VHH binding domain.

[0220] In one embodiment, the binding agent of the invention is in the format of a Fab-scFv construct, i.e. in the form of a construct comprising a Fab fragment (comprising the VH and CH1 domains of one polypeptide chain and the corresponding VL and CL domains of another polypeptide chain, wherein an antigen-binding domain is formed by interaction of said polypeptide chains) and an scFv portion (comprising the VH and VL domains of the same polypeptide chain linked together by a polypeptide linker, wherein the VH and VL interact to form the antigen-binding domain). In one embodiment, the binding agent of the invention is a trimer made up of three polypeptide chains, a first polypeptide chain comprising a VH from an immunoglobulin (e.g. an immunoglobulin having a first specificity), a second polypeptide comprising an scFv portion comprising a VH from an immunoglobulin (e.g. an immunoglobulin having a second specificity) and a VL from an immunoglobulin (e.g. an immunoglobulin having a second specificity), and a third polypeptide chain comprising a VL from an immunoglobulin (e.g. an immunoglobulin having a first specificity). In one embodiment, the first polypeptide chain further comprises a CH1 from an immunoglobulin and the third polypeptide chain further comprises a CL from an immunoglobulin. In one embodiment, the first and second polypeptide chains further comprise CH2 and CH3 (CH2-CH3) domains from an immunoglobulin (e.g., C-terminal to the Fab fragment and scFv portion, respectively). Thus, in one embodiment, a binding agent of the invention comprises a first polypeptide chain comprising a VH-CH1 linked to CH2-CH3, a second polypeptide chain comprising an scFv portion (VH-VL or VL-VH) linked to CH2-CH3, and a third polypeptide chain comprising a VL-CL. In some embodiments, the first polypeptide chain interacts with the second polypeptide chain. In some embodiments, the first polypeptide chain interacts with the third polypeptide chain. In some embodiments, the first and second polypeptide chains interact and the first polypeptide chain further interacts with the third polypeptide chain.In one embodiment, the CH2 of the first polypeptide chain interacts with the CH2 of the second polypeptide chain, and / or the CH3 of the first polypeptide chain interacts with the CH3 of the second polypeptide chain. In some embodiments, the VH of the first polypeptide chain interacts with the VL of the third polypeptide chain to form a binding domain, and / or the CH1 of the first polypeptide chain interacts with the CL of the third polypeptide chain. In some embodiments, a disulfide bridge is formed between a cysteine ​​residue in the CL and a cysteine ​​residue in the CH1. One or both polypeptide chains comprising CH2-CH3 may include one or more amino acid modifications described herein (e.g., Fc modifications, e.g., pI modifications, asymmetric modifications, additional Fc modifications, and ablation modifications), for example, to facilitate interactions of the polypeptide chains. According to the invention, the VH and VL of the scFv moiety are preferably linked by a peptide linker ("scFv linker"). In some embodiments, the CH1 of a first polypeptide chain is linked to the CH2 of the same polypeptide chain by a peptide linker. In some embodiments, the scFv is linked to the CH2 by a peptide linker.

[0221] In one embodiment, the binding agent of the invention is in the format of a Fab2-scFv construct, i.e. in the form of a construct comprising two Fab fragments (each comprising the VH and CH1 domains of one polypeptide chain and the corresponding VL and CL domains of another polypeptide chain, whereby an antigen-binding domain is formed by the interaction of each of said polypeptide chains) and an scFv portion (comprising the VH and VL domains of the same polypeptide chain linked together by a polypeptide linker, whereby VH and VL interact to form the antigen-binding domain). In one embodiment, the binding agent of the invention is a tetramer composed of four polypeptide chains, a first polypeptide chain comprises a VH from an immunoglobulin (e.g., an immunoglobulin having a first specificity), a second polypeptide comprises a VH from an immunoglobulin (e.g., an immunoglobulin having a first specificity) and an scFv portion comprising a VH from an immunoglobulin (e.g., an immunoglobulin having a second specificity) and a VL from an immunoglobulin (e.g., an immunoglobulin having a second specificity), a third polypeptide chain comprises a VL from an immunoglobulin (e.g., an immunoglobulin having a first specificity), and a fourth polypeptide chain is identical to the third polypeptide chain. In one embodiment, the first and second polypeptide chains further comprise a CH1 from an immunoglobulin (e.g., C-terminal to the VH from the immunoglobulin having the first specificity), and the third and fourth polypeptide chains further comprise a CL from an immunoglobulin. In one embodiment, the first and second polypeptide chains further comprise CH2 and CH3 (CH2-CH3) domains from an immunoglobulin (e.g., C-terminal to the Fab fragment and scFv portion, respectively). Thus, in one embodiment, a binding agent of the invention comprises a first polypeptide chain comprising VH-CH1 linked to CH2-CH3, a second polypeptide chain comprising VH-CH1 linked to an scFv portion (VH-VL or VL-VH) linked to CH2-CH3, and third and fourth polypeptide chains each comprising VL-CL. In some embodiments, the first polypeptide chain interacts with the second polypeptide chain.In some embodiments, the first polypeptide chain interacts with a third polypeptide chain. In some embodiments, the second polypeptide chain interacts with a fourth polypeptide chain. In one embodiment, the first and second polypeptide chains interact, the first polypeptide chain further interacts with a third polypeptide chain, and the second polypeptide chain further interacts with a fourth polypeptide chain. In one embodiment, the CH2 of the first polypeptide chain interacts with the CH2 of the second polypeptide chain, and / or the CH3 of the first polypeptide chain interacts with the CH3 of the second polypeptide chain. In some embodiments, the VH of the first polypeptide chain interacts with the VL of the third polypeptide chain to form a binding domain, and / or the CH1 of the first polypeptide chain interacts with the CL of the third polypeptide chain. In some embodiments, the VH of the second polypeptide chain (not part of the scFv moiety) interacts with the VL of the fourth polypeptide chain to form a binding domain and / or the CH1 of the second polypeptide chain interacts with the CL of the fourth polypeptide chain. In some embodiments, a disulfide bridge is formed between a cysteine ​​residue in the CL and a cysteine ​​residue in the CH1. One or both polypeptide chains comprising CH2-CH3 may include one or more amino acid modifications described herein (e.g., Fc modifications, e.g., pI modifications, asymmetric modifications, additional Fc modifications, and ablation modifications), for example, to facilitate interaction of the polypeptide chains. According to the present invention, the VH and VL of the scFv moiety are preferably linked by a peptide linker ("scFv linker"). In some embodiments, the CH1 of the first and / or second polypeptide chain is a peptide. In some embodiments, the scFv is linked to CH2 of the same polypeptide chain by a peptide linker.

[0222] In a preferred embodiment, in a bivalent binding agent of the invention (in Fab-scFv format), the VH of the first polypeptide chain interacts with the VL of the third polypeptide chain to form a binding domain with specificity for CLDN18.2, and the VH and VL of the scFv of the second polypeptide chain interact to form a binding domain with specificity for CD3. However, in some embodiments, the binding domain formed by the VH of the first polypeptide chain and the VL of the third polypeptide chain has specificity for CD3, and the binding domain formed by the VH and VL of the scFv of the second polypeptide chain has specificity for CLDN18.2. In another preferred embodiment, in a trivalent binding agent of the invention (in Fab2-scFv format), an additional VH of the second polypeptide chain that is not part of the scFv preferably interacts with the VL of the fourth polypeptide chain to form a binding domain with specificity for CLDN18.2. However, also within the scope of the application is a binding agent in which the binding domain formed by the scFv of the second polypeptide chain and the binding domain formed by the VH and VL of the first and third polypeptide chains have specificity for CLDN18.2, and the binding domain formed by the VH of the second polypeptide chain that is not part of an scFv, and the binding domain formed by the VL of the fourth polypeptide chain have specificity for CD3.

[0223] The term "linker" refers to any means that serves to connect two different functional units (e.g., domains or regions of a polypeptide chain). Types of linkers include, but are not limited to, chemical linkers, peptide linkers, and polypeptide linkers. The sequences of peptide linkers and polypeptide linkers are not limited. Peptide linkers are preferably non-immunogenic and flexible, for example, including serine and glycine sequences. Depending on the particular construct, linkers can be long or short.

[0224] In a preferred embodiment, the scFv linker (i.e., the linker connecting the VH and VL that form the scFv portion) is a flexible peptide linker as described herein, preferably having the amino acid sequence (GKPGS): x or a functional variant thereof, where x is 2, 3, 4, 5, or 6. In an even more preferred embodiment, the scFv linker preferably comprises, and preferably consists of, the amino acid sequence (GKPGS) 4 (SEQ ID NO: 11) or a functional variant thereof. Preferably, the scFv portion comprises, and preferably consists of, EPKSCDKTHTCPPCP (SEQ ID NO: 27), EPKSSDKTHTCPPCP (SEQ ID NO: 22), and (G 4 S) 2 KTHTCPPC (SEQ ID NO: 23), or a functional variant thereof, is linked to CH2 of the second polypeptide chain by a peptide linker comprising an amino acid sequence selected from the group consisting of: KTHTCPPC (SEQ ID NO: 23), or a functional variant thereof. Other useful linkers include those having amino acid sequences according to SEQ ID NOs: 24 and 25.

[0225] According to the present invention, the linker connecting the scFv and CH1, preferably at the C-terminus of CH1, has the amino acid sequence (G 4 S) x or a functional variant thereof, where x is 2, 3, 4, 5, or 6, preferably (G 4 S) 2 (SEQ ID NO: 26) or a functional variant thereof. According to the present invention, the linker linking CH2 and scFv, preferably at the N-terminus of CH2, is selected from the group consisting of EPKSCDKTHTCPPCP (SEQ ID NO: 27), EPKSSDKTHTCPPCP (SEQ ID NO: 22), and (G 4 S) 2 According to the present invention, the linker connecting CH1 and CH2 preferably comprises, and preferably consists of, an amino acid sequence selected from the group consisting of KTHTCPPC (SEQ ID NO: 23), or a functional variant thereof. According to the invention, for example, the CH1 and scFv of the second polypeptide chain preferably comprise, and preferably consist of, the amino acid sequence (G 4 S) 2 (SEQ ID NO: 26) or a functional variant thereof, are linked by a peptide linker that preferably comprises, and preferably consists of, SEQ ID NO: 26, although other linkers may be used as known in the art.

[0226] In one embodiment, a binding agent of the invention comprises a first, second, and third polypeptide chain; i) the first polypeptide chain comprises, from N-terminus to C-terminus, the following domains: VH(CLDN18.2)-CH1-CH2-CH3 Including, ii) the second polypeptide chain comprises, from N-terminus to C-terminus, the following domains: VH(CD3)-VL(CD3)-CH2-CH3, or VL(CD3)-VH(CD3)-CH2-CH3 Including, iii) the third polypeptide chain comprises, from N-terminus to C-terminus, the following domains: VL(CLDN18.2)-CL Including, Preferably, VH(CLDN18.2) and VL(CLDN18.2) interact to form a binding domain with specificity for CLDN18.2, and VH(CD3) and VL(CD3) interact to form a binding domain with specificity for CD3, and the domains of the polypeptide chains are preferably linked to each other by a peptide linker as described herein.

[0227] In another embodiment, a binding agent of the invention comprises a first, second, third, and fourth polypeptide chain; i) the first polypeptide chain comprises, from N-terminus to C-terminus, the following domains: VH(CLDN18.2)-CH1-CH2-CH3 Including, ii) the second polypeptide chain comprises, from N-terminus to C-terminus, the following domains: VH(CLDN18.2)-CH1-VH(CD3)-VL(CD3)-CH2-CH3, or VH(CLDN18.2)-CH1-VL(CD3)-VH(CD3)-CH2-CH3 Including, iii) the third polypeptide chain comprises, from N-terminus to C-terminus, the following domains: VL(CLDN18.2)-CL Including, iv) the fourth polypeptide chain is identical to the third polypeptide chain; Preferably, the VH(CLDN18.2) of the first polypeptide chain and the VL(CLDN18.2) of the third polypeptide chain interact to form a binding domain having specificity for CLDN18.2, the VH(CLDN18.2) of the second polypeptide chain and the VL(CLDN18.2) of the fourth polypeptide chain interact to form a binding domain having specificity for CLDN18.2, and the VH(CD3) and VL(CD3) interact to form a binding domain having specificity for CD3, and the domains of the polypeptide chains are preferably linked to each other by a peptide linker as described herein.

[0228] In one embodiment, a binding agent of the invention comprises: a) a VH (CLDN18.2) containing an aspartic acid residue at position 208, a proline residue at position 233, a valine residue at position 234, an alanine residue at position 235, a deletion at position 236, a lysine residue at position 267, a glutamic acid residue at position 295, an aspartic acid residue at position 368, and a nucleotide sequence similar to that of the VH (CLDN18.2) according to EU numbering. a) a first polypeptide chain comprising a CH1, a CH2 and a CH3 comprising a carboxyl group, a serine residue at position 370, an aspartic acid residue at position 384, a glutamic acid residue at position 418, and an aspartic acid residue at position 421; 4(SEQ ID NO: 11), and a second polypeptide chain comprising a CH2 and a CH3 that comprise a proline residue at position 233, a valine residue at position 234, an alanine residue at position 235, a deletion at position 236, a lysine residue at position 267, a glutamine residue at position 357, and a lysine residue at position 364 according to EU numbering; and c) a third polypeptide chain comprising a VL(CLDN18.2) and a CL. In one embodiment, the second polypeptide chain further comprises a VH(CLDN18.2) and a CH1 as described herein, and the binding agent further comprises a fourth polypeptide chain identical to the third polypeptide chain.

[0229] Other embodiments include a Fab2-scFv format comprising: a) a first polypeptide chain comprising a VH(CLDN18.2) and a CH1 and a CH2 and a CH3 comprising the asymmetric modifications L368D / K370S, pI modifications N208D / Q295E / N384D / Q418E / N421D, ablation modifications E233P / L234V / L235A / G236del / S267K, additional Fc modifications M428L / N434S; b) a VH(CD 3) a second polypeptide chain comprising CH2 and CH3 with the asymmetric modifications S364K / E357Q, the ablation modifications E233P / L234V / L235A / G236del / S267K, the further Fc modifications M428L / N434S, and VH(CLDN18.2); and c) a third polypeptide chain comprising VL(CLDN18.2) and CL; and d) a fourth polypeptide chain identical to the third polypeptide chain.

[0230] In one embodiment, a binding agent of the invention comprises a first, second, and third polypeptide chain; i) the first polypeptide chain comprises, from the N-terminus to the C-terminus: VH(CLDN18.2)-CH1-linker-CH2-CH3 Including, ii) the second polypeptide chain is arranged from the N-terminus to the C-terminus as follows: VH(CD3)-linker-VL(CD3)-linker-CH2-CH3, or VL(CD3)-linker-VH(CD3)-linker-CH2-CH3 Including, iii) the third polypeptide chain comprises, from the N-terminus to the C-terminus: VL(CLDN18.2)-CL Including, Preferably, VH(CLDN18.2) and VL(CLDN18.2) interact to form a binding domain having specificity for CLDN18.2, and VH(CD3) and VL(CD3) interact to form a binding domain having specificity for CD3.

[0231] In one embodiment, a binding agent of the invention comprises a first, second, and third polypeptide chain; i) the first polypeptide chain comprises, from the N-terminus to the C-terminus: VH(CLDN18.2)-CH1-Linker1-CH2-CH3 Including, ii) the second polypeptide chain is arranged, from the N-terminus to the C-terminus, VH(CD3)-linker3-VL(CD3)-linker4-CH2-CH3, or VL(CD3)-linker3-VH(CD3)-linker4-CH2-CH3 Including, iii) the third polypeptide chain comprises, from the N-terminus to the C-terminus: VL(CLDN18.2)-CL Including, Linker 1 comprises the amino acid sequence EPKSCDKTHTCPPCP or a functional variant thereof, and Linker 3 comprises the amino acid sequence (GKPGS) x or a functional variant thereof, wherein x is 2, 3, 4, 5, or 6, preferably x is 4; and Linker 4 is selected from the group consisting of EPKSCDKTHTCPPCP, EPKSSDKTHTCPPCP, and (G 4 S) 2 KTHTCPPCP or a functional variant thereof, Preferably, VH(CLDN18.2) and VL(CLDN18.2) interact to form a binding domain having specificity for CLDN18.2, and VH(CD3) and VL(CD3) interact to form a binding domain having specificity for CD3.

[0232] In one embodiment, a binding agent of the invention comprises a first, second, third, and fourth polypeptide chain, i) the first polypeptide chain comprises, from the N-terminus to the C-terminus: VH(CLDN18.2)-CH1-linker-CH2-CH3, Including, ii) the second polypeptide chain is arranged, from the N-terminus to the C-terminus, VH(CLDN18.2)-CH1-linker-VH(CD3)-linker-VL(CD3)-linker-CH2-CH3, or VH(CLDN18.2)-CH1-linker-VL(CD3)-linker-VH(CD3)-linker-CH2-CH3 Including, iii) the third polypeptide chain comprises, from N-terminus to C-terminus, the following domains: VL(CLDN18.2)-CL Including, iv) the fourth polypeptide chain is identical to the third polypeptide chain; Preferably, the VH(CLDN18.2) of the first polypeptide chain and the VL(CLDN18.2) of the third polypeptide chain interact to form a binding domain having specificity for CLDN18.2, the VH(CLDN18.2) of the second polypeptide chain and the VL(CLDN18.2) of the fourth polypeptide chain interact to form a binding domain having specificity for CLDN18.2, and the VH(CD3) and VL(CD3) interact to form a binding domain having specificity for CD3.

[0233] In one embodiment, a binding agent of the invention comprises a first, second, third, and fourth polypeptide chain; i) the first polypeptide chain is, from the N-terminus to the C-terminus VH(CLDN18.2)-CH1-Linker1-CH2-CH3 Including, ii) the second polypeptide chain is arranged, from the N-terminus to the C-terminus, VH(CLDN18.2)-CH1-linker2-VH(CD3)-linker3-VL(CD3)-linker4-CH2-CH3, or VH(CLDN18.2)-CH1-linker2-VL(CD3)-linker3-VH(CD3)-linker4-CH2-CH3, iii) the third polypeptide chain comprises, from the N-terminus to the C-terminus: VL(CLDN18.2)-CL Including, iv) the fourth polypeptide chain is identical to the third polypeptide chain; Linker 1 comprises the amino acid sequence EPKSCDKTHTPPCP or a functional variant thereof, and linker 2 comprises the amino acid sequence (G 4 S) x or a functional variant thereof (wherein is 2, 3, 4, 5, or 6, preferably, x is 2), and linker 3 comprises the amino acid sequence (GKPGS) x or a functional variant thereof, wherein x is 2, 3, 4, 5, or 6, preferably x is 4; and Linker 4 is selected from the group consisting of EPKSCDKTHTCPPCP, EPKSSDKTHTCPPCP, and (G 4 S) 2 KTHTCPPCP or a functional variant thereof, Preferably, the VH(CLDN18.2) of the first polypeptide chain and the VL(CLDN18.2) of the third polypeptide chain interact to form a binding domain having specificity for CLDN18.2, the VH(CLDN18.2) of the second polypeptide chain and the VL(CLDN18.2) of the fourth polypeptide chain interact to form a binding domain having specificity for CLDN18.2, and the VH(CD3) and VL(CD3) interact to form a binding domain having specificity for CD3.

[0234] The binding agents described herein, and / or the first, second and third (and optional fourth) polypeptide chains of said binding agents described herein may also comprise an amino acid sequence to facilitate secretion of the binding agent or polypeptide chain (e.g. an N-terminal secretion signal), and / or one or more epitope tags to facilitate binding, purification or detection of the molecule. Preferably, the secretion signal is a signal sequence (e.g. the amino acid sequence MGWSCIILFLVATATGVHS) that allows sufficient passage through the secretory pathway and / or secretion of the binding agent or its polypeptide chain into the extracellular environment. Preferably, the secretion signal sequence is cleavable and removed from the mature binding agent or polypeptide chain. The secretion signal sequence is preferably selected for the cell or organism in which the binding agent or polypeptide chain is produced.

[0235] The amino acid sequence of the epitope tag may be introduced at any position within the amino acid sequence of the binding agent or polypeptide chain, may take the form of a loop within the encoded protein structure, or may be fused N- or C-terminally to the binding agent or polypeptide chain. Preferably, the epitope tag is fused C-terminally to the binding agent or polypeptide chain. The epitope tag may comprise a cleavage site that allows removal of the tag from the binding agent or polypeptide chain. The epitope tag may be any kind of epitope tag that functions under native and / or denaturing conditions, preferably a histidine tag, most preferably a tag that contains six histidines.

[0236] The binding agents of the invention may, in addition to said first, second and optional third binding domains, comprise one or more further binding domains which serve, for example, to enhance selectivity for tumour cells. This may be achieved, for example, by providing binding domains which bind to other antigens expressed on tumour cells.

[0237] The term "post-translational modification" or similar terms refers to changes in proteins (e.g., covalent and enzymatic modifications) that occur after the biosynthesis of the protein. As is generally known in the art, binding agents such as antibodies expressed in cells are often post-translationally modified. For example, post-translational modifications of binding agents such as antibodies can occur at the amino acid side chains or the N- or C-terminus of the heavy or light chain (e.g., the first and / or second polypeptide chains described herein). Examples of post-translational modifications that may occur in the binding agents described herein include, but are not limited to, cleavage of lysine, e.g., at the C-terminus of the heavy chain of the first and / or second polypeptide chain, e.g., by carboxypeptidase; modification of glutamine or glutamic acid, e.g., at the N-terminus of the heavy chain of the first and / or second polypeptide chain, to pyroglutamic acid, e.g., by pyroglutamylation; modification of glutamine or glutamic acid, e.g., at the N-terminus of the light chain of the third and fourth polypeptide chains, to pyroglutamic acid, e.g., by pyroglutamylation; glycosylation; oxidation; deamidation; and glycation. Such post-translational modifications are known to occur in various binding agents (Liu et al., 2008, JP harmacol. Sci. 97(7):2426-2447). Post-translational modifications due to pyroglutamylation at the N-terminus and deletion of a lysine at the C-terminus generally do not have any effect on the activity of the binders (Lyubarskaya et al., 2006, Analyt. Biochem. 348(1):24-39).

[0238] Thus, in one embodiment, the binding agent described herein may comprise one or more post-translational modifications. In one embodiment, the one or more post-translational modifications comprise pyroglutamylation at the N-terminus of one or more polypeptide chains of the binding agent. In one embodiment, the one or more post-translational modifications comprise pyroglutamylation at the N-terminus of one or more VH(CLDN18.2). In one embodiment, the one or more post-translational modifications comprise pyroglutamylation at the N-terminus of VH(CD3). In one embodiment, the one or more post-translational modifications comprise deletion of a lysine at the C-terminus of the first polypeptide chain. In one embodiment, the one or more post-translational modifications comprise deletion of a lysine at the C-terminus of the second polypeptide chain.

[0239] In the context of the present invention, the binding agents produced are preferably capable of eliciting one or more immune effector functions as described herein, preferably directed against cells bearing the cancer associated antigen CLDN18.2 on their surface.

[0240] The term "immune effector function" in the context of the present invention includes any function mediated by components of the immune system that results in, for example, inhibition of cancer growth and / or inhibition of cancer development (including inhibition of cancer dissemination and metastasis). Preferably, the immune effector function results in the killing of cancer cells. Immune effector 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 cancer-associated antigen, cytolysis of cells bearing a cancer-associated antigen, and / or inhibition of proliferation of cells bearing a cancer-associated antigen. The binding agents described herein preferably target CD4 and / or CD8 T cells (particularly CD107a+

[0241] The binding agent of the present invention may recruit and redirect T cells, such as CD4+ T cells, to disease-associated cells, such as cancer cells, thereby acting via redirected T cell cytotoxicity (RTCC), i.e., the redirected T cells preferably kill the disease-associated cells (e.g., cancer cells). It is known that CD107a expression is associated with the cytolytic ability of CD4 and CD8 T cells. Preferably, the CD107a+ T cells may degranulate, i.e., release cytotoxic molecules, such as perforin, granzymes, and may also release cytokines, such as one or more of tumor necrosis factor alpha (TNFα), interleukin-2 (IL2), interferon gamma (IFNγ), and the like, thereby killing the target cells (e.g., cancer cells) to which the T cells are redirected by the binding agent of the present invention. The binding agent may also exert an effect by simply binding to a cancer-associated antigen on the surface of a cancer cell. For example, the binding agent may block the function of the cancer-associated antigen or induce apoptosis by simply binding to the cancer-associated antigen on the surface of a cancer cell.

[0242] The term "immune effector cell" or "effector cell" in the context of the present invention relates to a cell that exerts an effector function during an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTL, CD8+ T cells), including cytolytic T cells. The term "MHC-dependent T cell", or similar terms, relates to a T cell that recognizes an antigen when presented in the context of MHC and preferably exerts an effector function of the T cell (e.g., killing a target cell expressing the antigen).

[0243] T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of a specialized receptor called the T cell receptor (TCR) on the cell surface. The thymus is the main organ responsible for the maturation of T cells. Several different subsets of T cells have been found, each with a distinct function.

[0244] Among other functions, T helper cells assist other white blood cells in immunological processes such as maturation of B cells into plasma cells, activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when presented with peptide antigens, usually by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, the cells divide rapidly and secrete small proteins called cytokines to control or assist in active immune responses.

[0245] Cytotoxic T cells destroy virus-infected and cancer cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells normally recognize targets by binding to antigens associated with MHC class I, which is present on the surface of almost all cells in the body.

[0246] All T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The TCR of a T cell binds to major histocompatibility complex (MHC) molecules and can also interact with immunogenic peptides (epitopes) displayed on the surface of target cells. Specific binding of the TCR triggers a signaling cascade in the T cell, leading to proliferation and differentiation into mature effector T cells. In the majority of T cells, the actual T cell receptor is produced by independent T cell receptor alpha and beta (TCRα and TCRβ) genes and consists of two separate peptide chains called the α-TCR chain and the β-TCR chain. A less common group of T cells (2% of all T cells), γδ T cells (gamma delta T cells), has a unique T cell receptor (TCR) on its surface, consisting of one γ chain and one δ chain.

[0247] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells reside in the thymus and proliferate by cell division to generate large numbers of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8 and are therefore classified as double negative (CD4-CD8-) cells. As development progresses, they become double positive thymocytes (CD4+CD8+) and finally mature into single positive (CD4+CD8- or CD4-CD8+) thymocytes, which are then released from the thymus into peripheral tissues.

[0248] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of T cell receptors.

[0249] Human MHC molecules are usually referred to as HLA (human leukocyte antigen) molecules. There are two major classes of MHC molecules: class I and class II. MHC class I antigens are found on almost all nucleated cells in the body. The main function of this class of MHC molecules is to display (or present) peptide fragments of intracellular proteins to CTLs. Based on this display, CTLs attack those that present MHC-bound peptides, including disease-related peptides (antigens) such as cancer antigens. CD8+ T cells are usually cytotoxic (hence the name cytotoxic T cells = CTLs) and act on all intracellular proteins processed intracellularly and presented on the cell surface by MHC class I molecules9-10. They recognize peptides of 10 amino acids. Therefore, surface expression of MHC class I molecules plays an important role in determining the susceptibility of target cells to CTLs.

[0250] The binding agents described herein may be conjugated to a therapeutic moiety or agent (e.g., a cytotoxin, a drug (e.g., an immunosuppressant), or a radioisotope). A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells, and in particular kills cells. Examples include: taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Suitable therapeutic agents for forming conjugates include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepaclorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU)), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly In a preferred embodiment, the therapeutic agent is a cytotoxic or radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another embodiment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide, or ricin A.

[0251] The binding agents may also be conjugated to radioisotopes (eg, iodine-131, yttrium-90, or indium-111) to generate cytotoxic radiopharmaceuticals.

[0252] Techniques for conjugating such therapeutic moieties to binding agents are known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And See, "Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).

[0253] The term "binding" according to the present invention preferably relates to specific binding.

[0254] According to the present invention, an agent, such as an antibody, is capable of binding to a given target if it has a significant affinity for the given target and binds to the given target in a standard assay. "Affinity" or "binding affinity" is defined as the equilibrium dissociation constant (K D ) Preferably, the term "significant affinity" refers to a -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 The dissociation constant (K D ) to a given target.

[0255] An agent is not (substantially) capable of binding to a target if the agent has no significant affinity for the target and does not bind significantly (particularly does not detectably bind) to the target in a standard assay. Preferably, the agent does not detectably bind to the target when present at high concentrations of up to 2, preferably 10, more preferably 20, especially 50 or 100 μg / ml or more. Preferably, an agent has a K D At least 10 times, 100 times, or 10 times 3 Double, 10 4 Double, 10 5 Double or 10 6 Twice as high as K D If the agent binds to the target at a K, then the agent does not have significant affinity for the target. For example, D But, 10-7 M, the K for binding to a target for which the drug does not have significant affinity D is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 It turns out to be M. A binding agent, such as an antibody, is specific for a given target if it can bind to a given target but not to other targets, i.e. if it has no significant affinity for other targets and does not bind significantly to other targets in standard assays. According to the present invention, a binding agent is specific for CLDN18.2 if it can bind to CLDN18.2 but cannot (substantially) bind to other targets. Preferably, a binding agent is specific for CLDN18.2 if its affinity and binding to such other targets does not significantly exceed its affinity or binding to CLDN18.2-unrelated proteins (bovine serum albumin (BSA), casein, human serum albumin (HSA)), or non-claudin transmembrane proteins (e.g. MHC molecules or transferrin receptors), or any other specific polypeptide. Preferably, a binding agent has a K for binding to a non-specific target. D At least 10 times, 100 times, or 10 times 3 Double, 10 4 Double, 10 5 Double or 10 6 Twice lower K D For example, a binding agent is specific for a given target if it binds to that target at a K D 10 -7 If M, then the K for binding to a non-specific target D is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 It turns out to be M.

[0256] "kd" (seconds -1 The term k ) as used herein refers to the dissociation rate constant of a particular antibody-antigen interaction. off Also called the value.

[0257] "K D The term "(M)" as used herein refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0258] Binding of a binding agent to a target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984), Ku See, for example, Janis Immunology, W.H. Freeman and Company New York, NY (1992), and the methods described therein. Affinity can be readily determined using conventional techniques such as equilibrium dialysis; by using a BIAcore 2000 instrument using the general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by other methods known to those skilled in the art. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann NYAcad. ScL, 51:660 (1949). The measured affinity of a particular interaction between a binding agent and an antigen can vary when measured under various conditions (e.g., salt concentration, pH). Thus, affinity and other antigen binding parameters (e.g., K D ,I C 50 ) is preferably performed with standardized solutions of binding agent and antigen and with a standardized buffer.

[0259] The term "compete" refers to the competition between two binding agents, such as antibodies, for binding to a target antigen. If two binding agents do not block each other from binding to a target antigen, such binding agents are non-competitive, which indicates that the binding agents do not bind to the same part (i.e. epitope) of the target antigen. Methods for testing the competition of binding agents for binding to a target antigen are known to those skilled in the art. One example of such a method is the so-called cross-competition assay, which can be carried out, for example, as an ELISA or by flow cytometry.

[0260] For example, an ELISA-based assay may be performed by coating ELISA plate wells with each binder; adding and incubating a competing binder and a His-tagged extracellular domain of the antigen / target, and detecting whether the added binder inhibits the binding of the His-tagged protein to the coated binder by adding a biotinylated anti-His antibody, followed by streptavidin-poly-HRP, further developing the reaction with ABTS, and measuring absorbance at 405 nm. For example, a flow cytometry assay may be performed by incubating cells expressing the antigen / target with an excess of unlabeled binder, incubating the cells with a suboptimal concentration of biotin-labeled antibody, followed by incubation with fluorescently labeled streptavidin, and analyzing by flow cytometry.

[0261] Two binding agents, such as antibodies, have "the same specificity" if they bind to the same antigen and the same epitope. Such binding agents will compete for binding in a competitive binding assay. In one embodiment, binding agents that bind to the same epitope will be considered to bind to the same amino acid on the target molecule. The binding of antibodies to the same epitope on a target antigen can be determined by standard alanine scanning experiments or antibody-antigen crystallization experiments known to those skilled in the art.

[0262] The ability of a binder to compete for binding to an antigen indicates that the binder may bind to the same epitope region of the antigen or, if it binds to another epitope, may sterically inhibit the binding of the binder to that particular epitope region. Competitive binders can be easily identified based on their ability to compete with one or more binders in standard binding assays such as surface plasmon resonance analysis, ELISA assays, or flow cytometry (see WO 2013 / 173223). For example, competition between binders can be detected by a cross-blocking assay. For example, a competitive ELISA assay can be performed by coating the target antigen on a well of a microtiter plate and adding the antigen binder and a candidate competitive test binder. The amount of antigen binder bound to the antigen in the well indirectly correlates to the binding ability of a candidate competitive test binder that competes with it for binding to the same epitope, for example. Specifically, the greater the affinity of a candidate competitive test binder for the same epitope, the greater the affinity of the antigen. The amount of antigen binding agent bound to the well coated with the original is reduced. The amount of antigen binding agent bound to the well can be measured by labeling the binding agent with a detectable or measurable labeling substance. As described in WO2013 / 173223, surface plasmon resonance analysis (e.g., using a Biacore instrument) can be used to identify overlapping and different epitope regions recognized by binding agents, as is known in the art. Alternatively, competition can be determined using biolayer interferometry.

[0263] A binding agent that competes for binding to an antigen with another binding agent (e.g., a binding agent comprising heavy and light chain variable regions as described herein) or has specificity for an antigen of another binding agent (e.g., a binding agent comprising heavy and light chain variable regions as described herein, e.g., an antibody) may be a binding agent that comprises a variant of the heavy and / or light chain variable regions as described herein (e.g., modifications and / or a degree of identity of the CDRs as described herein).

[0264] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0265] As used herein, "isotype switching" refers to the phenomenon in which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.

[0266] The term "naturally occurring," as used herein as applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and has not been artificially modified in a laboratory is naturally occurring.

[0267] The term "rearranged," as used herein, refers to the configuration of a heavy or light chain immunoglobulin locus in which a V segment is positioned immediately adjacent to a DJ or J segment in a conformation that encodes essentially a complete VH or VL domain, respectively. Rearranged immunoglobulin (antibody) loci can be identified by comparison to germline DNA, and rearranged loci have at least one recombined heptamer / nonamer homology element.

[0268] The term "unrearranged" or "germline configuration," as used herein with respect to a V segment, refers to an arrangement in which the V segment has not been immediately recombined with a D segment or a J segment.

[0269] In one embodiment, the binding agent of the invention has the ability to bind to CLDN18.2, i.e. has the ability to bind to, and preferably binds to, an epitope present in CLDN18.2 (preferably an epitope located within the extracellular domain of CLDN18.2 (particularly the first extracellular loop, preferably amino acids 29-78 of CLDN18.2). In a particular embodiment, an agent capable of binding to CLDN18.2 binds to an epitope in CLDN18.2 that is not present in CLDN18.1.

[0270] The agent capable of binding to CLDN18.2 preferably binds to CLDN18.2 (preferably in human, mouse and / or cynomolgus monkey) but not to CLDN18.1 (preferably in human, mouse and / or cynomolgus monkey). Preferably, the agent that binds to CLDN18.2 does not bind to CLDN9 (preferably in human, mouse and / or cynomolgus monkey). Preferably, the agent that has the ability to bind to CLDN18.2 is specific for CLDN18.2. Preferably, the agent that has the ability to bind to CLDN18.2 binds to CLDN18.2 expressed on the cell surface. Particularly preferred In a preferred embodiment, the agent capable of binding to CLDN18.2 binds to a natural epitope of CLDN18.2 present on the surface of a living cell.

[0271] The term "fragment" refers in particular to one or more of the complementarity determining regions (CDRs), preferably at least the CDR3 variable region, of the heavy chain variable region (VH) and / or the light chain variable region (VL). In one embodiment, said one or more of the complementarity determining regions (CDRs) are selected from the set of complementarity determining regions CDR1, CDR2 and CDR3. In a particularly preferred embodiment, the term "fragment" refers to the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and / or the light chain variable region (VL).

[0272] In one embodiment, a binding domain comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs described herein comprises said CDRs and their intervening framework regions. Preferably, this portion also comprises at least about 50% of either or both of the first and fourth framework regions, the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. The construction of binding agents made by recombinant DNA techniques may result in the introduction of residues at the N- or C-terminus of the variable region that are introduced by linkers to facilitate cloning or other engineering steps, including the introduction of linkers for linking the variable region of the invention to further protein sequences, including immunoglobulin heavy chains, other variable regions, or protein tags.

[0273] In one embodiment, a binding domain comprising one or more CDRs, a set of CDRs or a combination of a set of CDRs described herein comprises said CDRs in a human antibody framework.

[0274] The exact identification of CDR regions depends on the calculation method used to determine the amino acid residues involved. For example, according to Kabat et al. (see above), the variable region generally includes amino acid residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in VL and about 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) in VH; the variable region may also include residues forming the hypervariable loops (e.g., residues 26-32 (CDR1), 50-52 (CDR2), and 91-96 (CDR3) in VL and 26-32 (CDR1), 53-55 (CDR2), and 96-101 (CDR3) in VH (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917)).

[0275] Those of skill in the art will appreciate that the exact identification of the CDR positions within the sequences disclosed herein may vary slightly depending on the numbering system used, as shown in Table 1 below (see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)):

[0276] [Table 1]

[0277] Thus, the CDR sequences disclosed herein include variants thereof derived according to the various numbering systems, and thus, disclosure of each VH is a disclosure of the CDRs (e.g., CDR1, CDR2, and CDR3) derivable therefrom, and disclosure of each VL is a disclosure of the CDRs (e.g., CDR1, CDR2, and CDR3) derivable therefrom.

[0278] Throughout the specification, when referring to residues in the variable regions of the binding domains with specificity for CD3 or CLDN18.2 as described herein (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is used, and for the CH1 and CH2-CH3 (optionally including the hinge) regions, the EU numbering system is used.

[0279] In a preferred embodiment, the binding domain of the binding agent of the present invention having specificity for CLDN18.2 comprises a VH comprising the complementarity determining regions CDR1, CDR2, and / or CDR3 specified in the amino acid sequence selected from the group consisting of SEQ ID NO: 38, 39, and 40. More preferably, the binding domain of the binding agent of the present invention having specificity for CLDN18.2 comprises a VH comprising the complementarity determining regions CDR1, CDR2, and / or CDR3 specified in the amino acid sequence represented by SEQ ID NO: 39.

[0280] In a preferred embodiment, the binding domain of the binding agent of the present invention having specificity for CLDN18.2 comprises a VL comprising the complementarity determining regions CDR1, CDR2, and / or CDR3 specified in the amino acid sequence selected from the group consisting of SEQ ID NO: 41 and 42. More preferably, the binding domain of the binding agent of the present invention having specificity for CLDN18.2 comprises a VL comprising the complementarity determining regions CDR1, CDR2, and / or CDR3 specified in the amino acid sequence represented by SEQ ID NO: 42.

[0281] In a preferred embodiment, the binding domain with specificity for CLDN18.2 of the binding agent of the invention comprises the following set of CDRs: VH comprises a CDR3 comprising the sequence set forth in SEQ ID NO: 34 or a functional variant thereof; and The VL comprises a CDR3 comprising the sequence set forth in SEQ ID NO: 37 or a functional variant thereof.

[0282] In one embodiment, the VH comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 32 or a functional variant thereof, and / or a CDR2 comprising the sequence set forth in SEQ ID NO: 33 or a functional variant thereof. and / or the VL further comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 35 or a functional variant thereof, and / or a CDR2 comprising the sequence set forth in SEQ ID NO: 36 or a functional variant thereof.

[0283] In a preferred embodiment, the binding domain with specificity for CLDN18.2 of the binding agent of the invention comprises the following set of CDRs: The VH comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 32 or a functional variant thereof, a CDR2 comprising the sequence set forth in SEQ ID NO: 33 or a functional variant thereof, and a CDR3 comprising the sequence set forth in SEQ ID NO: 34 or a functional variant thereof, and the VL comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 35 or a functional variant thereof, a CDR2 comprising the sequence set forth in SEQ ID NO: 36 or a functional variant thereof, and a CDR3 comprising the sequence set forth in SEQ ID NO: 37 or a functional variant thereof. Preferably, the VH comprises CDR1, 2, and 3 of SEQ ID NOs: 32, 33, and 34, and the VL comprises CDR1, 2, and 3 of SEQ ID NOs: 35, 36, and 37.

[0284] In one embodiment, the heavy and light chain variable regions comprise the complementarity determining regions interspersed within framework regions. In one embodiment, each variable region comprises three complementarity determining regions (CDR1, 2, and 3) and four framework regions (FR1, 2, 3, and 4). In one embodiment, the complementarity determining regions and the framework regions are arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0285] In a preferred embodiment, the binding domain of the binding agent of the present invention having specificity for CLDN18.2 comprises a VH(CLDN18.2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 38, 39, 40 or a functional variant thereof, and preferably comprises an amino acid sequence represented by SEQ ID NO: 39 or a functional variant thereof.

[0286] In a preferred embodiment, the binding domain of the binding agent of the present invention having specificity for CLDN18.2 comprises a VL(CLDN18.2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41, 42 or a functional variant thereof, preferably comprising the amino acid sequence represented by SEQ ID NO: 42 or a functional variant thereof.

[0287] In one embodiment, the binding domain with specificity for CLDN18.2 of the binding agent of the invention comprises the following combination of VH(CLDN18.2) and VL(CLDN18.2): VH(CLDN18.2) comprises the amino acid sequence represented by SEQ ID NO: 38 or a functional variant thereof, and VL(CLDN18.2) comprises the amino acid sequence represented by SEQ ID NO: 41 or a functional variant thereof. In one embodiment, the binding domain with specificity for CLDN18.2 of the binding agent of the invention comprises the following combination of VH(CLDN18.2) and VL(CLDN18.2): VH(CLDN18.2) comprises the amino acid sequence represented by SEQ ID NO: 40 or a functional variant thereof, and VL(CLDN18.2) comprises the amino acid sequence represented by SEQ ID NO: 42 or a functional variant thereof.

[0288] In a particularly preferred embodiment, the binding domain with specificity for CLDN18.2 of the binding agent of the invention comprises the following combination of VH(CLDN18.2) and VL(CLDN18.2): VH(CLDN18.2) comprises the amino acid sequence represented by SEQ ID NO: 39 or a functional variant thereof, and VL(CLDN18.2) comprises the amino acid sequence represented by SEQ ID NO: 42 or a functional variant thereof. includes functional variants thereof.

[0289] In preferred embodiments, the framework regions of the VH and VL domains present in the binding agents of the invention may contain amino acid changes but retain at least 80%, 85%, or 90% identity to human germline sequences.

[0290] In a further embodiment, the binding domain of the binding agent of the present invention having specificity for CLDN18.2 comprises the heavy and light chain variable regions of an antibody that (i) competes for CLDN18.2 binding with an antibody comprising the heavy and light chain variable regions described above, and / or (ii) has the specificity for CLDN18.2 of an antibody comprising the heavy and light chain variable regions described above.

[0291] In one embodiment, the binding domain with specificity for CLDN18.2 of the binding agent of the invention has the format of a Fab molecule as described herein. In this embodiment, VH(CLDN18.2) is part of one polypeptide chain, such as a first polypeptide chain, and VL(CLDN18.2) is part of another polypeptide chain, such as a third polypeptide chain, of the binding agent of the invention. In one embodiment, VH(CLDN18.2) is part of the first and second polypeptide chains described herein, and VL(CLDN18.2) is part of the third polypeptide chain and a fourth polypeptide chain that is identical to the third polypeptide chain of the binding agent of the invention.

[0292] It is to be understood that the binding domains that bind CLDN18.2 of the binding agents of the invention in Fab2-scFv format may be identical, essentially identical, or different, and thus may bind to the same or essentially identical epitope of CLDN18.2, or different epitopes. Thus, both binding domains that bind CLDN18.2 of the binding agents of the invention in Fab2-scFv format may correspond to or essentially correspond to one of the binding domains that bind CLDN18.2 described herein, or they may be independently selected from the binding domains that bind CLDN18.2 described herein.

[0293] Generally, any CD3 binding domain or fragment thereof known in the art may be used in the binding agents of the invention (e.g., the CD3 binding domain of an anti-CD3 antibody). Anti-CD3 antibodies useful for providing binding agents of the invention include, but are not limited to, UCHT1-HS (humanized mAb), UCHT1-MM (mouse mAb), CLB-T3, TR66, 145-2C11.

[0294] UCHT1 is a monoclonal IgG1 anti-CD3 antibody that detects CD3 in human and primate sample types. CLB-T3 is a mouse monoclonal anti-CD3 antibody against the CD3 antigen, reacting with 80-90% of human peripheral T lymphocytes and medullary thymocytes. TR66 is a mouse IgG1 monoclonal anti-CD3 antibody that recognizes the epsilon chain of human CD3. 145-2C11 is an Armenian hamster monoclonal anti-mouse CD3 antibody.

[0295] Preferably, the VH and VL domains of the binding domain with specificity for CD3 are derived from antibodies / antibody molecules and antibody-like molecules capable of specifically recognizing human CD3 in the context of other TCR subunits present on activated primary human T cells expressing the TCR in the native configuration. Most preferred are VH and VL domains derived from antibodies specific for the CD3-epsilon chain, said (parent) antibody should be able to specifically bind to an epitope that reflects an epitope of the native or near-native structure or conformation of human CD3 presented in the context of the TCR complex. In one embodiment of the invention, the C The VH and VL domains of the binding domain with specificity for D3 are derived from a CD3-specific antibody selected from the group consisting of UCHT1-HS, UCHT1-MM, CLB-T3, and TR66.

[0296] In a preferred embodiment, the binding domain with specificity for CD3 of the binding agent of the invention comprises a VH comprising CDR1, CDR2, and / or CDR3 identified within an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 58, 61, 64, 67, and 70.

[0297] In a preferred embodiment, the binding domain with specificity for CD3 of the binding agent of the invention comprises a VH comprising a set of CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (vi): (i) CDR1: SEQ ID NO: 43 or a functional variant thereof, CDR2: SEQ ID NO: 44 or a functional variant thereof, CDR3: SEQ ID NO: 45 or a functional variant thereof, (ii) CDR1: SEQ ID NO: 43 or a functional variant thereof, CDR2: SEQ ID NO: 50 or a functional variant thereof, CDR3: SEQ ID NO: 45 or a functional variant thereof, (iii) CDR1: SEQ ID NO: 43 or a functional variant thereof, CDR2: SEQ ID NO: 44 or a functional variant thereof, CDR3: SEQ ID NO: 51 or a functional variant thereof, (iv) CDR1: SEQ ID NO: 43 or a functional variant thereof, CDR2: SEQ ID NO: 44 or a functional variant thereof, CDR3: SEQ ID NO: 52 or a functional variant thereof, (v) CDR1: SEQ ID NO: 43 or a functional variant thereof, CDR2: SEQ ID NO: 44 or a functional variant thereof, CDR3: SEQ ID NO: 53 or a functional variant thereof, and (vi) CDR1: SEQ ID NO: 49 or a functional variant thereof, CDR2: SEQ ID NO: 44 or a functional variant thereof, CDR3: SEQ ID NO: 45 or a functional variant thereof.

[0298] In a preferred embodiment, a binding domain with specificity for CD3 of a binding agent of the invention comprises a VL comprising CDR1, CDR2 and / or CDR3 as specified within the amino acid sequence according to SEQ ID NO:55.

[0299] In a preferred embodiment, the binding domain with specificity for CD3 of a binding agent of the invention comprises a VL comprising the following set of CDR1, CDR2 and CDR3: CDR1: SEQ ID NO: 46 or a functional variant thereof, CDR2: SEQ ID NO: 47 or a functional variant thereof, CDR3: SEQ ID NO: 48 or a functional variant thereof.

[0300] In a preferred embodiment, the binding domain with specificity for CD3 of the binding agent of the invention comprises a VH and VL combination, each comprising a set of CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (vi): (i) VH: CDR1: SEQ ID NO: 43 or a functional variant thereof, CDR2: SEQ ID NO: 44 or a functional variant thereof, CDR3: SEQ ID NO: 45 or a functional variant thereof, VL: CDR1: SEQ ID NO: 46 or a functional variant thereof, CDR2: SEQ ID NO: 47 or a functional variant thereof, CDR3: SEQ ID NO: 48 or a functional variant thereof, (ii) VH: CDR1: SEQ ID NO: 43 or a functional variant thereof, CDR2: SEQ ID NO: 50 or a functional variant thereof, CDR3: SEQ ID NO: 45 or a functional variant thereof, VL: CDR1: SEQ ID NO: 46 or a functional variant thereof, CDR2: SEQ ID NO: 47 or a functional variant thereof, CDR3: SEQ ID NO: 48 or a functional variant thereof, (iii) VH: CDR1: SEQ ID NO: 43 or a functional variant thereof, CDR2: SEQ ID NO: 44 or a functional variant thereof, CDR3: SEQ ID NO: 51 or a functional variant thereof, VL: CDR1: SEQ ID NO: 46 or a functional variant thereof, CDR2: SEQ ID NO: 47 or a functional variant thereof, CDR3: SEQ ID NO: 48 or a functional variant thereof, (iv) VH: CDR1: SEQ ID NO: 43 or a functional variant thereof, CDR2: SEQ ID NO: 44 or a functional variant thereof, CDR3: SEQ ID NO: 52 or a functional variant thereof, VL: CDR1: SEQ ID NO: 46 or a functional variant thereof, CDR2: SEQ ID NO: 47 or a functional variant thereof, CDR3: SEQ ID NO: 48 or a functional variant thereof, (v) VH: CDR1: SEQ ID NO: 43 or a functional variant thereof, CDR2: SEQ ID NO: 44 or a functional variant thereof, CDR3: SEQ ID NO: 53 or a functional variant thereof, VL: CDR1: SEQ ID NO: 46 or a functional variant thereof, CDR2: SEQ ID NO: 47 or a functional variant thereof, CDR3: SEQ ID NO: 48 or a functional variant thereof, and (vi) VH: CDR1: SEQ ID NO: 49 or a functional variant thereof, CDR2: SEQ ID NO: 44 or a functional variant thereof, CDR3: SEQ ID NO: 45 or a functional variant thereof, VL: CDR1: SEQ ID NO: 46 or a functional variant thereof, CDR2: SEQ ID NO: 47 or a functional variant thereof, CDR3: SEQ ID NO: 48 or a functional variant thereof.

[0301] In a preferred embodiment, the binding domain with specificity for CD3 comprises a VH and a VL selected from the following embodiments (i) to (vi): (i) the VH comprises or consists of an amino acid sequence according to SEQ ID NO: 54 or a functional variant thereof, and the VL comprises or consists of an amino acid sequence according to SEQ ID NO: 55 or a functional variant thereof; (ii) the VH comprises or consists of an amino acid sequence according to SEQ ID NO: 58 or a functional variant thereof, and the VL comprises or consists of an amino acid sequence according to SEQ ID NO: 55 or a functional variant thereof; (iii) the VH comprises or consists of an amino acid sequence according to SEQ ID NO: 61 or a functional variant thereof, and the VL comprises or consists of an amino acid sequence according to SEQ ID NO: 55 or a functional variant thereof; (iv) VH comprises or consists of an amino acid sequence according to SEQ ID NO: 64 or a functional variant thereof, and VL comprises or consists of an amino acid sequence according to SEQ ID NO: 55 or a functional variant thereof; (v) VH comprises or consists of an amino acid sequence according to SEQ ID NO: 67 or a functional variant thereof, and VL comprises or consists of an amino acid sequence according to SEQ ID NO: 55 or a functional variant thereof; and (vi) the VH comprises or consists of an amino acid sequence according to SEQ ID NO: 70 or a functional variant thereof, and the VL comprises or consists of an amino acid sequence according to SEQ ID NO: 55 or a functional variant thereof.

[0302] In one embodiment, the binding domain having specificity for CD3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, and 72, or a functional variant thereof. Of course, the scFv linker contained in the sequence may be replaced with any other scFv linker, for example, with an scFv linker selected from the group consisting of SEQ ID NOs: 2 to 20, or a functional variant thereof.

[0303] In a preferred embodiment, the binding domain with specificity for CD3 of the binding agent of the invention comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 58, and 61, or a functional variant thereof.

[0304] In a preferred embodiment, a binding domain with specificity for CD3 of a binding agent of the invention comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 55 or a functional variant thereof.

[0305] In a preferred embodiment, the binding domain with specificity for CD3 of the binding agent of the invention comprises a VH and a VL selected from the following embodiments (i) to (iii): (i) VH comprises an amino acid sequence according to SEQ ID NO: 54 or a functional variant thereof; or consisting of, and VL comprises or consists of an amino acid sequence according to SEQ ID NO: 55 or a functional variant thereof; (ii) VH comprises or consists of an amino acid sequence according to SEQ ID NO: 58 or a functional variant thereof, and VL comprises or consists of an amino acid sequence according to SEQ ID NO: 55 or a functional variant thereof; and (iii) the VH comprises or consists of an amino acid sequence according to SEQ ID NO: 61 or a functional variant thereof, and the VL comprises or consists of an amino acid sequence according to SEQ ID NO: 55 or a functional variant thereof.

[0306] In some embodiments, a binding agent comprising a binding domain having specificity for CD3 binds to CD3 with high affinity, i.e., is a strong CD3 binder (e.g., a binding agent comprising SEQ ID NO: 56 or 57; or SEQ ID NO: 54, or CDR1, 2, and 3 as specified therein, and 55, or CDR1, 2, and 3 as specified therein). In some embodiments, a binding agent comprising a binding domain having specificity for CD3 binds to CD3 with moderate affinity, i.e., is a moderate CD3 binder. In some embodiments, a binding agent comprising a binding domain having specificity for CD3 binds to CD3 with low affinity, i.e., is a low CD3 binder. In some embodiments, a higher binding affinity for CD3 enhances RTCC. In one embodiment, bivalent binding to CLDN18.2 enhances RTCC compared to monovalent binding.

[0307] In certain embodiments, as described herein, one or more polypeptide chains of the binding agent of the invention comprise a CH1. In a preferred embodiment, the first polypeptide chain of the binding agent of the invention comprises a CH1. In another preferred embodiment, the first and second polypeptide chains of the binding agent of the invention comprise a CH1. Preferably, the CH1 of the first and / or second polypeptide chain of the binding agent of the invention is derived from IgG, preferably from IgG1, more preferably from human IgG1. In certain embodiments, one or more polypeptide chains of the binding agent of the invention comprise a CH2 domain and a CH3 domain. In a preferred embodiment, the first and / or second polypeptide chain of the binding agent of the invention comprises a CH2 domain and a CH3 domain. Preferably, the CH2 domain and the CH3 domain of the first and / or second polypeptide chain of the binding agent of the invention is derived from IgG, preferably from IgG1, more preferably from human IgG1. In a preferred embodiment, the first polypeptide chain of the binding agent of the invention comprising VH(CLDN18.2) comprises CH1, CH2, and CH3, and the second polypeptide chain of the binding agent of the invention comprising VH(CD3) and VL(CD3) comprises CH2 and CH3, said domains preferably being derived from IgG (e.g., IgG1, e.g., human IgG1). In another preferred embodiment, the first polypeptide chain of the binding agent of the invention comprising VH(CLDN18.2) comprises CH1, CH2, and CH3, and the second polypeptide chain of the binding agent of the invention comprising VH(CLDN18.2), VH(CD3), and VL(CD3) comprises CH1, CH2, and CH3, said domains preferably being derived from IgG (e.g., IgG1, e.g., human IgG1).

[0308] In certain embodiments, the polypeptide chains of the binding agents of the invention, such as the third and optional fourth polypeptide chains comprising VL(CLDN18.2), comprise a CL, such as a CL derived from Igκ or Igλ (preferably Igκ, more preferably human Igκ).

[0309] In a preferred embodiment, the first polypeptide chain of the binding agent of the invention comprises the amino acid sequence represented by SEQ ID NO: 28 or a functional variant thereof. In a preferred embodiment, the second polypeptide chain of the binding agent of the invention comprises the amino acid sequence represented by SEQ ID NO: 29 or 30 or a functional variant thereof. In a preferred embodiment, the third and optional fourth polypeptide chain of the binding agent comprises the amino acid sequence represented by SEQ ID NO: 31 or a functional variant thereof. Includes potential variants.

[0310] In a preferred embodiment, the binding agent of the present invention has the following embodiment: (i) the first polypeptide chain comprises SEQ ID NO: 28 or a functional variant thereof, the second polypeptide chain comprises SEQ ID NO: 29 or a functional variant thereof, and the third polypeptide chain comprises SEQ ID NO: 31 or a functional variant thereof; or (ii) the first polypeptide chain comprises SEQ ID NO: 28 or a functional variant thereof, the second polypeptide chain comprises SEQ ID NO: 30 or a functional variant thereof, and the third polypeptide chain comprises SEQ ID NO: 31 or a functional variant thereof. and a first, second, third, and optionally fourth polypeptide chain comprising an amino acid sequence selected from The fourth polypeptide chain, when present, is identical to the third polypeptide chain.

[0311] In a preferred embodiment, the binding agent of the invention comprises at least one binding domain having specificity for CLDN18.2 and at least one binding domain having specificity for CD3. In a preferred embodiment, the first polypeptide chain of the binding agent of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73 or a functional variant thereof. In a preferred embodiment, the second polypeptide chain of the binding agent of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75 or a functional variant thereof. In a preferred embodiment, the third polypeptide chain of the binding agent of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 78 or a functional variant thereof. In a preferred embodiment, the binding agent of the invention comprising at least one binding domain having specificity for CLDN18.2 and at least one binding domain having specificity for CD3 comprises the set of first, second and third polypeptide chains of SEQ ID NOs: 73, 75 and 78 or functional variants thereof.

[0312] In another preferred embodiment, the binding agent of the invention comprises at least two binding domains with specificity for CLN18.2 and at least one binding domain with specificity for CD3. In a preferred embodiment, the first polypeptide chain of the binding agent of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73 or a functional variant thereof. In a preferred embodiment, the second polypeptide chain of the binding agent of the invention comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 74, 76, and 77 or a functional variant thereof. In a preferred embodiment, the third polypeptide chain of the binding agent of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 78 or a functional variant thereof. In a preferred embodiment, a binding agent of the present invention comprising at least two binding domains that bind to CLDN18.2 and at least one binding domain having specificity for CD3 comprises a set of first, second, third and fourth polypeptide chains selected from the group consisting of SEQ ID NOs: (i) 73, 74 and 78; (ii) 73, 76 and 78; (iii) 73, 77 and 78, or functional variants thereof, wherein the fourth binding domain is identical to the third binding domain.

[0313] It should be understood that the binding agents described herein can be delivered to a patient by administering a nucleic acid, such as an RNA, encoding the agent, and / or by administering a host cell that contains a nucleic acid, such as an RNA, encoding the agent. When the binding agent comprises multiple polypeptide chains, the different polypeptide chains may be encoded by the same nucleic acid or different nucleic acids (e.g., a set of nucleic acids). Thus, the administered nucleic acid may be a mixture of various nucleic acid molecules, such as a set of nucleic acids. The nucleic acid or set of nucleic acids encoding the binding agent, when administered to a subject, such as a patient, may be present in naked form, in a suitable delivery vehicle, such as in the form of a liposome, or nanoparticle, or viral particle, or may be present within a host cell. The nucleic acid or set of nucleic acids provided may be The set may produce the agent over time in a sustained manner that at least partially alleviates the instability observed with therapeutic antibodies. The nucleic acid or set of nucleic acids delivered to the patient may be produced by recombinant means. If the nucleic acid or set of nucleic acids is administered to the patient without being present within a host cell, it is preferably incorporated into the patient's cells for expression of the binding agent encoded by the nucleic acid. If the nucleic acid or set of nucleic acids is administered to the patient while being present within a host cell, it is preferably expressed by the host cell in the patient to produce the binding agent encoded by the nucleic acid.

[0314] The term "recombinant" in the context of the present invention means "produced by genetic engineering." Preferably, "recombinants," such as recombinant nucleic acids, in the context of the present invention are not naturally occurring.

[0315] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), that can be isolated from a natural source, and that has not been artificially modified in a laboratory, is naturally occurring.

[0316] The term "nucleic acid" as used herein is intended to include DNA and RNA, including, for example, genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0317] A nucleic acid or a set of nucleic acids may be included in a vector. A set of nucleic acids may also be included in a set of vectors, such that each nucleic acid of the set of nucleic acids is included in a vector. The term "vector" as used herein includes any vector known to those skilled in the art, including, for example, a plasmid vector, a cosmid vector, a phage vector, such as lambda phage, a viral vector, such as an adenovirus vector or a baculovirus vector, or an artificial chromosome vector, such as a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a P1 artificial chromosome (PAC). The vector includes an expression vector and a cloning vector. Expression vectors include plasmids and viral vectors, and generally include a desired coding sequence and appropriate DNA sequences required for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a particular desired DNA fragment, and may lack functional sequences required for the expression of the desired DNA fragment.

[0318] In the context of the present invention, the term "RNA" refers to a molecule that comprises ribonucleotide residues, preferably composed entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may include, for example, the addition of non-nucleotide material at the end or within the RNA, for example at one or more nucleotides of the RNA. The nucleotides of an RNA molecule may also include non-standard nucleotides, for example non-naturally occurring nucleotides, or chemically synthesized nucleotides or deoxynucleotides. This modified RNA may be referred to as an analog of naturally occurring RNA.

[0319] According to the present invention, the term "RNA" is used in the context of "messenger RNA". The term "mRNA" includes "RNA" and preferably in this regard relates to "transcripts" which may be produced using DNA as a template and code for a peptide or protein. An mRNA typically comprises a 5' untranslated region (5'-UTR), a region coding for a protein or peptide, and a 3' untranslated region (3'-UTR). An mRNA has a limited half-life in cells and in vitro. Preferably, the mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, the RNA is obtained by in vitro transcription or chemical synthesis. Methodologies for in vitro transcription are known to those skilled in the art. For example, various in vitro transcription kits are commercially available.

[0320] In one embodiment of the invention, the RNA is a self-replicating RNA, such as a single-stranded self-replicating RNA. In one embodiment, the self-replicating RNA is a positive-sense single-stranded RNA. In one embodiment, the self-replicating RNA is a viral RNA or RNA derived from a viral RNA. In one embodiment, the self-replicating RNA is an alphavirus genomic RNA or is derived from an alphavirus genomic RNA. Alphavirus RNA can act as an mRNA, as known in the art. In one embodiment, the self-replicating RNA is a viral gene expression vector. In one embodiment, the virus is Semliki forest virus. In one embodiment, the self-replicating RNA comprises one or more transgenes, at least one of which encodes a binding agent as described herein. In one embodiment, when the RNA is a viral RNA or is derived from a viral RNA, the transgene can partially or completely replace viral sequences, such as viral sequences encoding structural proteins. In one embodiment, the self-replicating RNA is an in vitro transcribed RNA.

[0321] In order to increase the expression and / or stability of the RNA used according to the invention, it may be modified, preferably without changing the sequence of the expressed peptide or protein.

[0322] The term "modification" in relation to RNA as used according to the present invention includes any modification of RNA that does not naturally occur in said RNA.

[0323] In one embodiment of the invention, the RNA used according to the invention does not have uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates may be achieved by treating the RNA with a phosphatase.

[0324] The RNA of the present invention may have naturally occurring or synthetic modified ribonucleotides to increase its stability and / or reduce its cytotoxicity and / or immunogenicity.For example, in one embodiment, in the RNA used according to the present invention, 5-methylcytidine is partially or completely replaced by cytidine, preferably completely replaced by cytidine.Alternatively or in addition, in one embodiment, in the RNA used according to the present invention, pseudouridine is partially or completely replaced by uridine, preferably completely replaced by uridine.

[0325] In one embodiment, the term "modification" relates to providing an RNA with a 5'-cap or 5'-cap analog. The term "5'-cap" refers to the cap structure found at the 5' end of an mRNA molecule, generally consisting of a guanosine nucleotide attached to the mRNA via a unique 5'-5'-triphosphate linkage. In one embodiment, the guanosine is methylated at the 7 position. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably a 7-methylguanosine cap (m7G). In the context of the present invention, the term "5'-cap" refers to a cap structure that resembles an RNA cap structure and is preferably associated in vivo and / or in a cell. The present invention includes 5'-cap analogs that have been modified to have the ability to stabilize RNA when

[0326] Providing a 5'-cap or 5'-cap analog to RNA can be achieved by in vitro transcription of a DNA template in the presence of the 5'-cap or 5'-cap analog, and the 5'-cap is co-transcriptionally incorporated into the generated RNA strand, or RNA can be generated, for example, by in vitro transcription, and a 5'-cap can be post-transcriptionally attached to the RNA using a capping enzyme (e.g., vaccinia virus capping enzyme).

[0327] The RNA may contain further modifications. For example, further modifications of the RNA used in the present invention may be an extension or shortening of the naturally occurring poly(A) tail, or a modification of the 5'- or 3'-untranslated region (UTR), such as the introduction of a UTR that is not associated with the coding region of said RNA, for example the insertion of one or more (preferably two) copies of the 3'-UTR from a globin gene (e.g. alpha2-globin, alpha1-globin, beta-globin, preferably beta-globulin, more preferably human beta-globin).

[0328] Thus, to increase the stability and / or expression of the RNA used according to the invention, this RNA may be modified to be present with a polyA sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, especially 100 to 150 adenosine residues. In a particularly preferred embodiment, the polyA sequence has a length of about 120 adenosine residues. In addition, the translation efficiency may be improved by incorporating two or more 3'-untranslated regions (UTRs) in the 3'-untranslated region of the RNA molecule. In a particular embodiment, the 3'-UTR is derived from the human β-globin gene.

[0329] Preferably, when the RNA is delivered to (ie, transfected into) a cell, particularly a cell existing in vivo, the protein, peptide, or antigen that it encodes is expressed.

[0330] The term "transfection" relates to the introduction of a nucleic acid, particularly RNA, into a cell. For the purposes of the present invention, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such a cell, which may be present in a subject (e.g. a patient). Thus, according to the present invention, the cells for transfection of a nucleic acid as described herein may be present in vitro or in vivo, for example, the cells may form part of an organ, tissue and / or organ of a patient. According to the present invention, transfection may be transient or stable. Depending on the application of the transfection, it may be sufficient that the transfected genetic material is only expressed transiently. Since the nucleic acid introduced in the transfection process is not usually integrated into the nuclear genome, this foreign nucleic acid is diluted or degraded by mitosis. Cells that allow episomal amplification of the nucleic acid greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must occur. The RNA can be transfected into cells to transiently express the encoded protein.

[0331] The term "stability" of an RNA relates to the "half-life" of the RNA. "Half-life" relates to the period required to remove half of the activity, amount, or number of a molecule. In the context of the present invention, the half-life of an RNA indicates the stability of said RNA. The half-life of an RNA may affect the "expression period" of the RNA. An RNA with a long half-life can be expected to be expressed for a long period of time.

[0332] In the context of the present invention, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription", which refers to the process in which RNA (especially mRNA) is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is applied to generate the transcription product. This cloning vector is generally called a transcription vector and is included in the term "vector" according to the present invention.

[0333] The term "translation" according to the present invention relates to the process in the ribosomes of a cell where a chain of messenger RNA directs the construction of a sequence of amino acids to make a peptide or protein.

[0334] The term "expression" is used according to the present invention in its most general sense and includes, for example, the production of RNA and / or peptides or proteins by transcription and / or translation. With respect to RNA, the terms "expression" or "translation" particularly relate to the production of peptides or proteins. This term also includes partial expression of a nucleic acid. Furthermore, expression may be transient or stable. According to the present invention, the term expression also includes "aberrant expression" or "abnormal expression".

[0335] "Aberrant expression" or "abnormal expression" means, in accordance with the present invention, that expression is altered, preferably increased, compared to a reference (e.g., a state in a subject not having a disease associated with aberrant or abnormal expression of a particular protein (e.g., a tumor antigen). Increased expression refers to an increase of at least 10%, in particular at least 20%, at least 50%, or at least 100%, or more. In one embodiment, expression is found only in diseased tissue and expression in healthy tissue is suppressed. The term "specifically expressed" means that a protein is essentially expressed only in a particular tissue or organ. For example, a tumor antigen specifically expressed in the gastric mucosa means that the protein is mainly expressed in the gastric mucosa and not in other tissues or not expressed to a significant extent in other tissues or other organ types. Thus, a protein that is expressed only in cells of the gastric mucosa and to a significantly lower extent in any other tissue, such as the testis, is specifically expressed in cells of the gastric mucosa. In some embodiments, a tumor antigen may also be specifically expressed in multiple tissue types or organs (e.g., two or three tissue types or organs) under normal conditions, but preferably not more than three different tissue types or organ types. In this case, the tumor antigen is then specifically expressed in these organs. For example, if a tumor antigen is expressed to approximately the same extent, preferably in the lung and stomach under normal conditions, the tumor antigen is specifically expressed in the lung and stomach.

[0336] According to the present invention, the term "encoding RNA" means that the RNA, when present in an appropriate environment (preferably within a cell), can be expressed to produce the protein or peptide that it encodes.

[0337] Some aspects of the invention rely on the adoptive transfer of host cells that are transfected in vitro with a nucleic acid, such as an RNA, encoding a binding agent as described herein, and preferably transferred into a recipient, such as a patient, after ex vivo expansion from low precursor frequencies to clinically relevant cell numbers. Host cells used in treatments according to the invention may be autologous, allogeneic, or syngeneic to the treated recipient.

[0338] The term "autologous" is used to describe something that is derived from the same subject. For example, "autologous transplant" refers to the transplantation of a tissue or organ derived from the same subject. Such procedures are advantageous because they overcome immunological barriers that would otherwise cause rejection.

[0339] The term "allogenic" is used to describe something that is derived from different individuals of the same species. Two or more individuals are said to be allogenic to one another if the genes at one or more loci are not identical.

[0340] The term "syngeneic" is used to describe those derived from individuals or tissues having the same genotype (i.e., identical twins or inbred strains of animals, or tissues thereof).

[0341] The term "xenogeneic" is used to describe something that is made up of multiple dissimilar elements. As an example, the transplantation of bone marrow from one individual into another individual constitutes a xenogeneic transplant. Xenogeneic genes are genes that are derived from a source other than the subject.

[0342] The term "peptide" according to the present invention includes oligopeptides and polypeptides and refers to a substance comprising 2 or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 9 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 21 or more, and preferably up to 8, 10, 20, 30, 40 or 50, especially 100 amino acids covalently linked by peptide bonds. The term "protein" refers to large peptides, preferably peptides having more than 100 amino acid residues, although in general the terms "peptide" and "protein" are synonymous and are used interchangeably herein.

[0343] The teachings given herein with respect to specific amino acid sequences (e.g., those shown in the sequence listing) should also be interpreted as relating to variants of said specific sequences, resulting in sequences that are functionally equivalent to said specific sequences (e.g., those that exhibit the same or similar properties as said specific amino acid sequences). One important property is to retain binding to a target or to retain an effector function. Preferably, a sequence that is variant with respect to a specific sequence, when replacing a specific sequence in an antibody, retains the binding of said antibody to CLDN18.2 and / or CD3, and preferably retains the function of said antibody as described herein. Furthermore, preferably, a sequence that is variant with respect to a specific sequence, when replacing a specific sequence in a binding agent, retains the binding of said binding agent to CLDN18.2 and / or CD3, and preferably retains the function of said binding agent as described herein (e.g., cytotoxic T cell-mediated lysis).

[0344] For example, the sequences shown in the sequence listing may be modified to remove one or more, preferably all, free cysteine ​​residues, in particular by replacing the cysteine ​​residue with an amino acid other than cysteine, preferably serine, alanine, threonine, glycine, tyrosine, tryptophan, leucine, or methionine.

[0345] In particular, it will be appreciated by those skilled in the art that the sequences of the CDRs, hypervariable regions and variable regions may be altered without losing the ability to bind to CLDN18.2 and / or CD3. For example, the CDR regions may be identical or highly homologous to the regions defined herein. By "highly homologous", it is contemplated that 1 to 5, preferably 1 to 4, such as 1 to 3, or 1 or 2 substitutions may be made in the CDRs. In addition, the hypervariable and variable regions may be altered to show substantial homology with the regions specifically disclosed herein. In one embodiment, the variable region sequences are identical to the variable regions specifically disclosed herein. The only deviations from the domain sequences are in the framework sequences.

[0346] The binding agents of the invention may be produced intracellularly (e.g., in the cytosol, periplasm, or in inclusion bodies) and then isolated from the host cell and optionally further purified; or may be produced extracellularly (e.g., in the medium in which the host cell is cultured) and then isolated from the culture medium and optionally further purified. The methods and reagents used for recombinant production of polypeptides (e.g., specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc.) are known in the art. Similarly, protein isolation and purification techniques are known to those of skill in the art.

[0347] The term "cell" or "host cell" preferably relates to an intact cell, i.e. a cell with an intact membrane that has not released normal intracellular components such as enzymes, organelles or genetic material. An intact cell preferably refers to a living cell, i.e. a living cell capable of carrying out normal metabolic functions. Preferably, the term relates according to the invention to any cell that can be transfected with an exogenous nucleic acid. Preferably, this cell, when transfected with an exogenous nucleic acid and transplanted into a recipient, is capable of expressing the nucleic acid in the recipient. The term "cell" includes bacterial cells; other useful cells are yeast cells, fungal cells or mammalian cells. Suitable bacterial cells include cells from gram-negative bacterial strains such as Escherichia coli, Proteus, and Pseudomonas, and cells from gram-positive bacterial strains such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cells include cells from species of the genera Trichoderma, Neurospora, and Aspergillus. Suitable yeast cells include cells from species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, 293HEK cells, and the like. However, amphibian cells, insect cells, plant cells, and other cells used in the art for expression of heterologous proteins may be used as well.Mammalian cells (e.g., human, mouse, hamster, pig, goat, and primate cells) are particularly preferred for adoptive transfer. Cells can be derived from many tissue types and include primary cells and cell lines, such as cells of the immune system (particularly antigen-presenting cells such as dendritic cells and T cells, stem cells such as hematopoietic stem cells and mesenchymal stem cells, as well as other cell types). Antigen-presenting cells are cells that present antigens in association with the major histocompatibility complex on their surface. T cells can recognize this complex using the T cell receptor (TCR).

[0348] "Reduce," "reduce," or "inhibit," as used herein, refers to an overall decrease or the ability to cause an overall decrease in a cellular level (e.g., expression level or proliferation level), preferably by 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more.

[0349] Terms such as "increase" or "enhancement" preferably mean an increase or decrease in a concentration of at least about 10%, preferably at least about 20%, preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 80%, and most preferably at least about 10%. Preferably, the increase or enhancement is at least about 100%, at least about 200%, at least about 500%, at least about 1000%, at least about 10000%, or even greater.

[0350] Antibody-dependent cell-mediated cytotoxicity ADCC describes the cell killing capacity of effector cells (particularly lymphocytes) as described herein, which preferably requires that a target cell be labeled with an antibody.

[0351] ADCC preferably occurs when an antibody binds to an antigen on a tumor cell and the antibody Fc domain binds to an Fc receptor (FcR) on the surface of an immune effector cell. Several families of Fc receptors have been identified, and certain cell populations characteristically express defined Fc receptors. ADCC can be considered as a mechanism for directly inducing various degrees of immediate tumor destruction leading to antigen presentation and induction of tumor-directed T cell responses. Preferably, induction of ADCC in vivo results in tumor-directed T cell responses and host-derived antibody responses.

[0352] Antibody-dependent cell-mediated phagocytosis ADCP is one mechanism of action of many antibody therapeutics. It is defined as a highly regulated process in which an antibody removes bound targets by attaching its Fc domain to specific receptors on phagocytes and inducing phagocytosis. ADCP can be mediated by monocytes, macrophages, neutrophils, and dendritic cells via FcγRIIa, FcγRI, and FcγRIIIa, with FcγRIIa (CD32a) on macrophages representing the major pathway.

[0353] ADCP preferably occurs when non-specific phagocytes expressing FcγR recognize antibodies bound to target cells, such as diseased cells, including tumor cells, and then trigger phagocytosis of the target cells, such as diseased cells, including tumor cells. ADCP also stimulates downstream adaptive immune responses by promoting antigen presentation or stimulating secretion of inflammatory mediators. ADCP can be improved in vivo by co-treatment with immunomodulatory agents. The Fc receptor-dependent function of ADCP provides a mechanism for the clearance of viruses and virus-infected cells, as well as a mechanism for stimulating downstream adaptive immune responses by promoting antigen presentation or stimulating secretion of inflammatory mediators.

[0354] Complement-dependent cytotoxicity CDC is yet another method of cell killing that can be induced by antibodies. IgM is the most effective isotype in complement activation. IgG1 and IgG3 are also very effective in inducing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex leads to the CDC of participating antibody molecules, such as IgG molecules. H Multiple closely spaced C1q binding sites on the 2 domain are exposed (C1q is one of three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert the previously low affinity C1q-IgG interaction to high affinity, which triggers a series of events involving a series of other complement proteins, leading to the proteolytic release of effector cell chemotactic / activating factors C3a and C5a. Preferably, the complement cascade culminates in the formation of a membrane attack complex, which forms a pore in the cell membrane, facilitating the free passage of water and solutes in and out of the cell.

[0355] The binding agents and antibodies described herein may be produced by a variety of techniques, including conventional monoclonal antibody methodology (e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975)). Although somatic cell hybridization procedures are preferred, in principle any method can be used to produce monoclonal antibodies. Other techniques for producing antibodies may be used, such as viral or oncogenic transformation of B lymphocytes, or phage display techniques using libraries of antibody genes.

[0356] The preferred animal system for preparing hybridomas secreting monoclonal binding agents, such as monoclonal antibodies, is the mouse system. Hybridoma production in mice is a very well-established procedure. Immunization protocols and techniques for isolation of 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 preparing hybridomas secreting monoclonal binding agents, such as monoclonal antibodies, are the rat and rabbit systems (e.g., as described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92:9348 (1995); see also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

[0357] In yet another preferred embodiment, human binding agents, such as human antibodies, can be generated using transgenic or transchromosomal mice that have parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice." Production of human binding agents, such as human antibodies, in such transgenic mice can be performed as detailed for CD20 in WO2004035607.

[0358] Yet another strategy for generating monoclonal binding agents, such as monoclonal antibodies, is to directly isolate antibody-encoding genes from lymphocytes that produce binding agents of defined specificity (e.g., Babcock et al., 1996; A novel ... agents such as monoclonal antibodies from single, isolated lymphocytes producing (See, for details on recombinant binding agent engineering, see also Welschof and Kraus, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny KC Lo Antibody Engineering ISBN 1-58829-092-1.

[0359] To generate antibodies, mice may be immunized with carrier-bound peptides derived from the antigen sequence (i.e., sequences for antibodies), enriched preparations of recombinantly expressed antigen or fragments thereof, and / or cells expressing the antigen, as described. Alternatively, mice may be immunized with DNA encoding the antigen or fragments thereof. In cases where immunization with purified or enriched preparations of antigen does not yield antibodies, mice may also be immunized with cells expressing the antigen (e.g., cell lines) to stimulate an immune response. Immune responses may be monitored over the course of the immunization protocol with plasma and serum samples obtained by tail vein or retroorbital bleeds. Mice with sufficient titers of immunoglobulin may be used for fusions. Mice may be boosted intraperitoneally or intravenously with antigen-expressing cells 3 days before sacrifice and removal of the spleen to increase the proportion of specific antibody-secreting hybridomas.

[0360] To generate monoclonal antibody-producing hybridomas, spleen and lymph node cells from immunized mice can be isolated and fused with a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. Individual wells are then screened by ELISA for antibody-secreting hybridomas. Cells may be screened for specificity for the antigen. Antibodies with specificity for the antigen may be identified by immunofluorescence and FACS analysis using antigen-expressing cells. Hybridomas secreting antibodies can be replated, screened again, and if still positive for monoclonal antibodies, can be subcloned by limiting dilution. Stable subclones can be cultured in vivo to generate antibody in tissue culture medium for characterization.

[0361] Binding agents such as antibodies can also be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection methods known in the art (Morrison, S. (1985) Science 229:1202).

[0362] For example, in one embodiment, a gene of interest (e.g., an antibody gene) can be ligated into an expression vector, such as a eukaryotic expression plasmid used by the GS gene expression system disclosed in, for example, WO 87 / 04462, WO 89 / 01036, EP 338841, or other expression systems known in the art. The purified plasmid carrying the cloned antibody gene can be introduced into eukaryotic host cells, such as CHO cells, NS / 0 cells, HEK293T cells, or HEK293 cells, or other eukaryotic cells, such as plant-derived cells, fungal cells, or yeast cells. The method used to introduce the gene can be a method described in the art, such as electroporation, lipofectin, lipofectamine, etc. After introduction of the antibody gene into the host cells, cells expressing the antibody can be identified and selected. The cells represent transfectomas that can amplify expression levels and be scaled up to produce the antibody. The recombinant antibody can be isolated and purified from the culture supernatant and / or cells.

[0363] Alternatively, cloned binding agent (e.g., antibody) genes can be expressed in other expression systems, including prokaryotic cells such as microorganisms (e.g., E. coli). Additionally, binding agents (e.g., antibodies) can be produced in transgenic non-human animals (e.g., in 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).

[0364] Chimerization Unlabeled mouse antibodies are highly immunogenic in humans when applied repeatedly, leading to reduced therapeutic efficacy. The main immunogenicity is mediated by the heavy chain constant region. The immunogenicity of binding agents derived from mouse antibodies in humans can be reduced or completely avoided if the respective binding agents are chimerized or humanized. Chimeric binding agents are binding agents in which different portions are derived from different animal species (e.g., binding agents having a variable region derived from a mouse antibody and a human immunoglobulin constant region). Chimerization of binding agents is achieved by combining the variable regions of mouse antibody heavy and light chains with the constant regions of human heavy and light chains (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 binding agents are generated by combining a human kappa-light chain constant region with a mouse light chain variable region. In an equally preferred embodiment, chimeric binding agents may be generated by combining a human lambda-light chain constant region with a mouse light chain variable region. Preferred heavy chain constant regions for generating chimeric binding agents are IgG1, IgG3, and IgG4. Other preferred heavy chain constant regions for generating chimeric binding agents are IgG2, IgA, IgD, and IgM.

[0365] Humanization Binders such as antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Because the CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant binders (e.g., antibodies) that mimic the properties of a particular naturally occurring antibody by constructing an expression vector that contains the CDR sequences of a particular naturally occurring antibody grafted onto the framework sequences of 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. USA 86:10029-10033). Such framework sequences can be obtained from public DNA databases that contain the sequences of germline antibody genes. This germline sequence differs from the mature antibody gene sequences because it does not contain the fully assembled variable genes formed by V(D)J joining during the B cell maturation process. The germline gene sequences also differ from the sequences of high affinity secondary repertoire antibodies, individually and uniformly across the entire variable region.

[0366] The ability of antibodies and other binding agents to bind to an antigen can be determined using standard binding assays (eg, ELISA, Western blot, immunofluorescence, and flow cytometric analysis).

[0367] To purify the binding agent, such as an antibody, the selected production cell line may be grown in a 2 liter spinner flask to purify the recombinant antibody. Alternatively, the binding agent, such as an antibody, may be produced in a dialysis-based bioreactor. The supernatant may be filtered and, if necessary, concentrated prior to affinity chromatography using protein L-sepharose. The eluted binding agent may be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer may be exchanged into PBS and the concentration may be determined by OD280 using the respective extinction coefficients. The recombinant binding agent may be aliquoted and stored at -65 to -85°C.

[0368] Flow cytometry may be used to demonstrate the binding of monoclonal binders, such as monoclonal antibodies, to live cells expressing the antigen. Cell lines expressing the antigen naturally or after transfection, and negative controls lacking antigen expression (grown under standard growth conditions), may be mixed with various concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1% FBS and incubated for 30 minutes at 4°C. After washing, fluorescently labeled detection reagents (e.g., fluorescently conjugated anti-IgG, anti-Fab, or Protein-L) may bind to the antigen-bound monoclonal binders under the same conditions as the staining of the primary binders. Samples may be analyzed by flow cytometry with a FACS machine using light and side scatter properties to gate on single live cells. Co-transfection methods may be employed to distinguish between antigen-specific monoclonal binders and non-specific binders in a single measurement. Cells transiently transfected with plasmids encoding antigen and fluorescent markers may be stained as described above. Transfected cells can be detected in a different fluorescent channel than cells stained with the binder. Because the majority of transfected cells express both transgenes, antigen-specific monoclonal binders will preferentially bind to fluorescent marker expressing cells, while non-specific binders will bind to untransfected cells in equal proportions. In addition to or instead of the flow cytometry assay, an alternative assay using a fluorescent microscope can be used. Cells can be stained exactly as described above and examined under a fluorescent microscope.

[0369] Immunofluorescence microscopy can be used to demonstrate the binding of monoclonal binding agents, such as monoclonal antibodies, to live cells expressing the antigen. For example, cell lines expressing the antigen naturally or after transfection and negative controls lacking antigen expression are grown in chamber slides under standard growth conditions in DMEM / F12 medium (supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin). The cells can then be fixed with methanol or paraformaldehyde or left untreated. The cells can then be reacted with monoclonal binding agents against the antigen for 30 minutes at 25° C. After washing, the cells can be reacted with Alexa555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. The cells can then be examined under a fluorescent microscope.

[0370] Cell extracts from cells expressing the antigen, and appropriate negative controls, can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked, and probed with the monoclonal binders to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed with ECL substrate.

[0371] Binding agents such as antibodies can be further tested for reactivity with antigens by immunohistochemistry using paraformaldehyde or acetone fixed frozen sections or paraformaldehyde fixed paraffin embedded tissue sections from non-cancerous or cancerous tissue samples taken from patients during routine surgery or from mice with xenograft tumors inoculated with cell lines expressing the antigen, either spontaneously or after transfection.For immunostaining, binding agents that react with antigens can be incubated followed by horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibody (DAKO) according to the supplier's instructions.

[0372] Preclinical trials The binding agents described herein may also be tested in in vivo models (e.g., immunodeficient mice bearing xenograft tumors inoculated with cell lines expressing CLDN18.2) to determine their effectiveness in controlling the growth of tumor cells expressing CLDN18.2. In vivo studies after xenografting of CLDN18.2-expressing tumor cells into immunodeficient mice or other animals may be performed using the binding agents described herein. The binding agents and optional effector cells, such as PBMCs, may be administered to tumor-free mice followed by injection of tumor cells to measure the effectiveness of the binding agents in preventing tumor formation or tumor-associated symptoms. The binding agents and optional effector cells, such as PBMCs, may be administered to tumor-bearing mice to determine the therapeutic efficacy of each binding agent to reduce tumor growth, metastasis, or tumor-associated symptoms. Application of the binding agents and optional effector cells may be combined with application of other agents, such as cystostatic drugs, growth factor inhibitors, cell cycle inhibitors, angiogenesis inhibitors, or antibodies, to determine increased efficacy and potential toxicity of the combination. To analyze toxic side effects mediated by the binding agents, animals may be inoculated with the binding agents or control agents described herein and thoroughly examined for symptoms believed to be related to CLDN18.2-binding agent treatment.

[0373] Mapping of epitopes recognized by binding agents is described in “Epitope Mapping Protocols (Methods in Molecular Biology) by Glenn E. Morris ISBN-089603-375-9 and “Epitope Mapping: A Practical Approach” Practical Approach Series, 248 by ISBN-0896 03-375-9, and in “Epitope Mapping: A Practical Approach” Practical Approach Series, 248 by Olwyn MRWestwood, Frank C. Hay.

[0374] The compounds and agents described herein may be administered in the form of any suitable pharmaceutical composition.

[0375] The pharmaceutical compositions of the present invention are preferably sterile and contain an effective amount of the binding agent described herein and optionally additional agents discussed herein to obtain the desired reaction or desired effect.

[0376] The pharmaceutical compositions are usually provided in a uniform dosage form and may be prepared in a manner known per se The pharmaceutical composition may, for example, be in the form of a solution or a suspension.

[0377] Pharmaceutical compositions may contain salts, buffer substances, preservatives, carriers, diluents, and / or excipients, all of which are preferably pharma- ceutically acceptable. The term "pharmaceutical acceptable" refers to the non-toxicity of substances that do not interact with the action of the active ingredients of the pharmaceutical composition.

[0378] Pharmaceutically unacceptable salts may be used to prepare pharma-ceutically acceptable salts and are included in the present invention. Such pharma-ceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts may also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, or calcium salts.

[0379] Buffering substances suitable for use in the pharmaceutical compositions include acetate, citrate, borate, and phosphate.

[0380] Preservatives suitable for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0381] Injectable formulations may contain pharma- ceutically acceptable excipients such as lactated Ringer's.

[0382] The term "carrier" refers to an organic or inorganic component of natural or synthetic nature, in which the active ingredient is formulated to facilitate, enhance or enable application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a patient.

[0383] Possible carrier materials for parenteral administration are, for example, sterile water, Ringer's, lactated Ringer's, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes, and especially biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0384] The term "excipient" as used herein is intended to indicate any substance (e.g., carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring or coloring agents, etc.) that may be present in a pharmaceutical composition and which is not an active ingredient.

[0385] The agents and compositions described herein may be administered by any conventional route, such as parenteral administration, for example, by injection or infusion. Administration is preferably parenteral, for example, intravenous. , intra-arterial, subcutaneous, intradermal, or intramuscular.

[0386] Compositions suitable for parenteral administration usually comprise a sterile aqueous or non-aqueous preparation of the active compound, which is preferably isotonic with the blood of the recipient. Examples of suitable carriers and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are usually used as a medium for the solution or suspension.

[0387] The agents and compositions described herein are administered in an effective amount. "Effective amount" refers to an amount that alone or together with further doses achieves a desired response or a desired effect. In the case of the treatment of a particular disease or a particular condition, the desired response preferably relates to the inhibition of the course of the disease. This includes delaying the progression of the disease, and in particular includes preventing or reversing the progression of the diseas...

Claims

1. A bispecific binding agent that binds to CLDN18.2 and CD3, wherein the bispecific binding agent is A first heavy chain variable region (VH) and a first light chain variable region (VL), wherein the first VH and the first VL bind to CLDN18.2; A second VH and a second VL, wherein the second VH and the second VL bind to CLDN18.2; and a third VH and a third VL, wherein said third VH and said third VL bind to CD3; Including, wherein each of the first VH and the second VH comprises a CDR1 comprising the amino acid sequence SYWIN (SEQ ID NO: 32), a CDR2 comprising the amino acid sequence NIYPSDSYTNYNQKFQG (SEQ ID NO: 33), and a CDR3 comprising the amino acid sequence SWRGNSFDY (SEQ ID NO: 34); Each of the first VL and the second VL comprises a CDR1 comprising the amino acid sequence KSSQSLLNSGNQKNYLT (SEQ ID NO: 35), a CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO: 36), and a CDR3 comprising the amino acid sequence QNDYSYPFT (SEQ ID NO: 37); the third VH comprises a CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 43), a CDR2 comprising the amino acid sequence RIRSKANNYATYYADSVKG (SEQ ID NO: 50), and a CDR3 comprising the amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO: 45); and The third VL comprises a CDR1 comprising the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 46), a CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 47), and a CDR3 comprising the amino acid sequence ALWYSNHWV (SEQ ID NO: 48); The bispecific binding agent.

2. The bispecific binding agent of claim 1, wherein the first VH comprises an amino acid sequence represented by SEQ ID NO:

39.

3. The bispecific binding agent of claim 1, wherein the first VL comprises an amino acid sequence represented by SEQ ID NO:

42.

4. The bispecific binding agent of claim 1, wherein the second VH comprises an amino acid sequence represented by SEQ ID NO:

39.

5. The bispecific binding agent of claim 1, wherein the second VL comprises an amino acid sequence represented by SEQ ID NO:

42.

6. The bispecific binding agent of claim 1, wherein the third VH comprises an amino acid sequence represented by SEQ ID NO:

58.

7. The bispecific binding agent of claim 1, wherein the third VL comprises an amino acid sequence represented by SEQ ID NO:

55.

8. The method of claim 1, wherein each of the first and second VHs comprises an amino acid sequence represented by SEQ ID NO:39; Each of the first and second VLs comprises an amino acid sequence represented by SEQ ID NO: 42; The third VH comprises an amino acid sequence represented by SEQ ID NO:58; and The third VL comprises an amino acid sequence represented by SEQ ID NO:

55. The bispecific binding agent of claim 1 .

9. One or more nucleic acid molecules encoding the bispecific binding agent of claim 1.

10. A host cell comprising one or more nucleic acid molecules described in claim 9.

11. A pharmaceutical composition comprising the bispecific binding agent of claim 1.

12. The bispecific binding agent of claim 1 for use in a method for treating a cancer involving cancer cells expressing CLDN18.2, the method comprising administering the bispecific binding agent of claim 1 to a subject.

13. A bispecific binding agent that binds to CLDN18.2 and CD3, comprising: The bispecific binding agent is (a) from the N-terminus to the C-terminus (i) a first heavy chain variable region (VH1); (ii) constant region 1 (CH1) of the heavy chain; (iii) a constant region 2 (CH2) of the heavy chain, and (iv) constant region 3 (CH3) of the heavy chain; a first polypeptide chain comprising: (b) from the N-terminus to the C-terminus: (i) a first light chain variable region (VL1), and (ii) a light chain constant region (CL); a second polypeptide chain comprising: (c) from the N-terminus to the C-terminus: (i) a second light chain variable region (VL2), and (ii) a light chain constant region (CL); and a third polypeptide chain comprising: (d) from the N-terminus to the C-terminus: (i) a second heavy chain variable region (VH2); (ii) constant region 1 (CH1) of the heavy chain; (iii) a third heavy chain variable region (VH3) and a third light chain variable region (VL3), or a third light chain variable region (VL3) and a third heavy chain variable region (VH3); (iv) a constant region 2 (CH2) of the heavy chain, and (v) constant region 3 (CH3) of the heavy chain; Including, Where: The VH1 and the VL1 bind to CLDN18.2, the VH2 and the VL2 bind to CLDN18.2, and the VH3 and the VL3 bind to CD3; each of the VH1 and the VH2 comprises a CDR1 comprising the amino acid sequence SYWIN (SEQ ID NO: 32), a CDR2 comprising the amino acid sequence NIYPSDSYTNYNQKFQG (SEQ ID NO: 33), and a CDR3 comprising the amino acid sequence SWRGNSFDY (SEQ ID NO: 34); Each of the VL1 and the VL2 comprises a CDR1 comprising the amino acid sequence KSSQSLLNSGNQKNYLT (SEQ ID NO: 35), a CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO: 36), and a CDR3 comprising the amino acid sequence QNDYSYPFT (SEQ ID NO: 37); the VH3 comprises a CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 43), a CDR2 comprising the amino acid sequence RIRSKANNYATYYADSVKG (SEQ ID NO: 50), and a CDR3 comprising the amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO: 45); and The VL3 comprises a CDR1 comprising the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 46), a CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 47), and a CDR3 comprising the amino acid sequence ALWYSNHWV (SEQ ID NO: 48); The bispecific binding agent.

14. The bispecific binding agent of claim 13, wherein the VH1 comprises an amino acid sequence represented by SEQ ID NO:

39.

15. The bispecific binding agent of claim 13, wherein the VL1 comprises an amino acid sequence represented by SEQ ID NO:

42.

16. The bispecific binding agent of claim 13, wherein the VH2 comprises an amino acid sequence represented by SEQ ID NO:

39.

17. The bispecific binding agent of claim 13, wherein the VL2 comprises an amino acid sequence represented by SEQ ID NO:

42.

18. The bispecific binding agent of claim 13, wherein the VH3 comprises an amino acid sequence represented by SEQ ID NO:

58.

19. The bispecific binding agent of claim 13, wherein the VL3 comprises an amino acid sequence represented by SEQ ID NO:

55.

20. The bispecific binding agent of claim 13, wherein the VH3 or VL3 is linked to CH1 by a peptide linker.

21. The bispecific binding agent described in claim 20, wherein the peptide linker comprises an amino acid sequence represented by SEQ ID NO: 2, 13, 26, 96, or 97.

22. The bispecific binding agent of claim 13, wherein the VH3 or VL3 is linked to CH2 by a peptide linker.

23. The bispecific binding agent described in claim 22, wherein the peptide linker comprises an amino acid sequence represented by SEQ ID NO: 2, 13, 26, 96, or 97.

24. The bispecific binding agent of claim 13, wherein the first polypeptide chain comprises an amino acid sequence represented by SEQ ID NO:73 or a variant thereof, wherein the variant of SEQ ID NO:73 has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:73 over the entire length of SEQ ID NO:

73.

25. The bispecific binding agent of claim 13, wherein the second polypeptide chain comprises an amino acid sequence represented by SEQ ID NO:78 or a variant thereof, wherein the variant of SEQ ID NO:78 has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:78 over the entire length of SEQ ID NO:

78.

26. The bispecific binding agent of claim 13, wherein the third polypeptide chain comprises an amino acid sequence represented by SEQ ID NO:78 or a variant thereof, wherein the variant of SEQ ID NO:78 has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:78 over the entire length of SEQ ID NO:

78.

27. The bispecific binding agent of claim 13, wherein the fourth polypeptide chain comprises an amino acid sequence represented by SEQ ID NO:74 or a variant thereof, wherein the variant of SEQ ID NO:74 has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74 over the entire length of SEQ ID NO:

74.

28. One or more nucleic acid molecules encoding the bispecific binding agent of claim 13.

29. A host cell comprising one or more nucleic acid molecules according to claim 28.

30. A pharmaceutical composition comprising the bispecific binding agent of claim 13.

31. The bispecific binding agent of claim 13 for use in a method for treating a cancer involving cancer cells expressing CLDN18.2, the method comprising administering to a subject the bispecific binding agent of claim 13.

32. The bispecific binding agent for use according to claim 31, wherein the cancer involving cancer cells expressing CLDN18.2 is gastric cancer, cancer of the gastroesophageal junction, esophageal cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), or colorectal cancer.

33. The bispecific binding agent for use according to claim 12, wherein the cancer involving cancer cells expressing CLDN18.2 is gastric cancer, cancer of the gastroesophageal junction, esophageal cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), or colorectal cancer.

Images