Combination therapy involving bispecific binding agents binding to cldn18.2 and cd3 and agents stabilizing or increasing expression of cldn18.2

Abstract

The present invention provides a combination therapy involving bispecific binding agents comprising two binding domains binding to CLDN18.2 and a binding domain binding to CD3 and agents stabilizing or increasing the expression of CLDN 18.2. The combination therapy is effective in treating cancer involving cancer cells expressing CLDN18.2.

Description

[0001] DESCRIPTION

[0002] Title of Invention: COMBINATION THERAPY INVOLVING BISPECIFIC BINDING

[0003] AGENTS BINDING TO CLDN18.2 AND CD3 AND AGENTS STABILIZING OR

[0004] INCREASING EXPRESSION OF CLDN18.2

[0005] Cancer is the second leading cause of death globally and is expected to be responsible for an estimated 9.6 million deaths in 2018 (Bray, F. et al., CA: A Cancer Journal for Clinicians, 68: 394- 424, 2018). In general, once a solid tumor has metastasized, with a few exceptions such as germ cell and some carcinoid tumors, 5-year survival rarely exceeds 25%. The poor prognosis of certain cancers highlights the need for additional treatment approaches.

[0006] The tight junction molecule claudin 18 (CLDN18) is an integral transmembrane protein (tetraspanin) having four membrane spanning hydrophobic regions and two extracellular loops (loopl embraced by hydrophobic region 1 and hydrophobic region 2; loop2 embraced by hydrophobic regions 3 and 4). CLDN18 exists in two different splice variants, which are described in mouse and in human (Niimi, Mol. Cell. Biol. 21:7380-90, 2001). The splice variants (Genbank accession number: splice variant 1 (CLDN18.1): NP 057453, NM 016369, and splice variant 2 (CLDN18.2): NM 001002026, NP 001002026) have a molecular weight of approximately 27.9 / 27.72 kD. The splice variants CLDN18.1 and CLDN18.2 differ in the N-terminal portion which comprises the first transmembrane (TM) region and loopl, whereas the primary protein sequence of the C-terminus is identical.

[0007] In normal tissues, there is no detectable expression of CLDN 18.2 with exception of stomach where CLDN18.2 is expressed exclusively on short-lived differentiated gastric epithelial cells. CLDN 18.2 is maintained in the course of malignant transformation and thus frequently displayed on the surface of human gastric cancer cells. Moreover, this pan-tumoral antigen is ectopically activated at significant levels in esophageal, pancreatic and lung adenocarcinomas. The CLDN18.2 protein is also localized in lymph node metastases of gastric cancer adenocarcinomas and in distant metastases especially into the ovary (so-called Krukenberg tumors).

[0008] The differential expression of claudins such as CLDN18.2 between cancer and normal cells, their membrane localization and their absence from the vast majority of toxicity relevant normal tissues makes these molecules attractive targets for cancer immunotherapy and the use of antibody-based therapeutics for targeting CLDN 18.2 in cancer therapy promises a high level of therapeutic specificity.

[0009] The chimeric IgGl antibody IMAB362 (Zolbetuximab (previously named Claudiximab)) which is directed against CLDN18.2 has been 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 claudin family member including the closely related splice variant 1 of Claudin 18 (CLDN18.1). IMAB362 shows precise tumor cell specificity and bundles two independent highly potent mechanisms of action. Upon target binding IMAB362 mediates cell killing mainly by ADCC and CDC. Thus, IMAB362 lyses efficiently CLDN 18.2-positive cells, including human gastric cancer cell lines in vitro and in vivo. Anti-tumor efficacy of IM AB 362 was demonstrated in mice carrying xenografted tumors inoculated with CLDN 182-positive cancer cell lines.

[0010] IgGl antibodies typically engage the cellular immune system via interaction of the Fc domain with Fcγ receptors ( Fcγ Rs) expressed on various immune cells including natural killer cells which are the main agents of ADCC. However, IgGl monoclonal antibodies (mAbs) triggering ADCC face several limitations including broad distribution of low affinity Fc receptor variants in the population (up to 80%) as well as in vivo IgGl modifications reducing the mAb efficacy (Chames et al., (2009) Br J Pharmacol, 157(2):220-233). Therapeutic antibodies also have to compete with the patients IgGs resulting in high doses of mAbs necessary in vivo. Furthermore, therapeutic antibodies may interact with FcγRIIb (an inhibitory FcγR expressed by B cells, macrophages, dendritic cells and neutrophils) resulting in negative signaling that decreases their efficacy.

[0011] It has been an object of the invention to provide novel therapies for cancer diseases.

[0012] The present invention generally embraces the treatment of a subject comprising the administration of a bispecific binding agent described herein that comprises two CLDN 18.2 binding domains in the Fab format that are specific for cancer cells, and a CD3 binding domain in the scFv format that is specific for the T cell-specific antigen CD3 allowing to bind to T cells and to pull the T cells into the complex, thus making it possible to target the cytotoxic effect of the T cells to the cancer cells. Formation of this complex can induce signalling in cytotoxic T cells, either on its own or in combination with accessory cells, which leads to the release of cytotoxic mediators. The treatment using a bispecific binding agent described herein is combined with an additional treatment comprising administration of one or more agents stabilizing or increasing expression of CLDN 18.2.

[0013] It is demonstrated herein that a treatment involving a bispecific binding agent described herein and an agent stabilizing or increasing expression of CLDN18.2 can induce a potent antitumor effect.

[0014] SUMMARY OF THE INVENTION

[0015] The invention generally provides a combination therapy involving a bispecific binding agent that binds to CLDN18.2 and CD3 and an agent stabilizing or increasing expression of CLDN18.2. In some embodiments, the bispecific binding agent is a bispecific tetrameric binding agent. In some embodiments, the agent stabilizing or increasing expression of CLDN18.2 comprises gemcitabine, paclitaxel, or a combination thereof.

[0016] The invention provides a composition or medical preparation comprising:

[0017] (a) a bispecific binding agent comprising a first binding domain that binds to human CLDN18.2, a second binding domain that binds to human CLDN18.2, and a third binding domain that binds to human CD3, wherein the binding agent comprises four polypeptide chains, wherein

[0018] (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 27;

[0019] (ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 28;

[0020] (iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and

[0021] (iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and

[0022] (b) an agent stabilizing or increasing expression of CLDN18.2.

[0023] In some embodiments, the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 27.

[0024] In some embodiments, the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29.

[0025] In some embodiments, the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29.

[0026] In some embodiments,

[0027] (i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 27;

[0028] (ii) the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 28;

[0029] (iii) the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and

[0030] (iv) the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29.

[0031] The invention further provides a composition or medical preparation comprising:

[0032] (a) a bispecific binding agent comprising a first binding domain that binds to human CLDN18.2, a second binding domain that binds to human CLDN18.2, and a third binding domain that binds to human CD3, wherein the binding agent comprises four polypeptide chains, wherein

[0033] (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27;

[0034] (ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28;

[0035] (iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and

[0036] (iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and

[0037] (b) an agent stabilizing or increasing expression of CLDN18.2. In some embodiments, the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27.

[0038] In some embodiments, the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28.

[0039] In some embodiments, the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29.

[0040] In some embodiments, the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29.

[0041] In some embodiments,

[0042] (i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27;

[0043] (ii) the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28;

[0044] (iii) the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29; and

[0045] (iv) the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29.

[0046] The invention further provides a composition or medical preparation comprising:

[0047] (a) a bispecific binding agent comprising a first binding domain that binds to human CLDN18.2, a second binding domain that binds to human CLDN18.2, and a third binding domain that binds to human CD3, wherein the binding agent is encoded by one or more nucleic acid molecules comprising:

[0048] (i) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 27;

[0049] (ii) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 28; and

[0050] (iii) a third nucleic acid sequence encoding a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 29; and

[0051] (b) an agent stabilizing or increasing expression of CLDN18.2.

[0052] In some embodiments, the one or more nucleic acid molecules is a set of nucleic acids.

[0053] In some embodiments, the bispecific binding agent comprises four polypeptide chains, wherein (i) the first polypeptide chain is encoded by the first nucleic acid sequence;

[0054] (ii) the second polypeptide chain is encoded by the second nucleic acid sequence;

[0055] (iii) the third polypeptide chain is encoded by the third nucleic acid sequence; and

[0056] (iv) the fourth polypeptide chain is encoded by the third nucleic acid sequence.

[0057] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 27 comprises a deletion of lysine at position 447 of SEQ ID NO: 27.

[0058] In some embodiments, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 28 comprises a deletion of lysine at position 720 of SEQ ID NO: 28.

[0059] In some embodiments, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29.

[0060] In some embodiments, the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29.

[0061] In some embodiments,

[0062] (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or a C- terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 27 comprises a deletion of lysine at position 447 of SEQ ID NO: 27;

[0063] (ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or a C- terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 28 comprises a deletion of lysine at position 720 of SEQ ID NO: 28;

[0064] (iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and

[0065] (iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29.

[0066] In some embodiments,

[0067] (i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27 or a C- terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 27 comprises a deletion of lysine at position 447 of SEQ ID NO: 27; (ii) the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28 or a C- terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 28 comprises a deletion of lysine at position 720 of SEQ ID NO: 28;

[0068] (iii) the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29; and

[0069] (iv) the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29.

[0070] In some embodiments, the first polypeptide chain interacts with the second polypeptide chain and with the third polypeptide chain.

[0071] In some embodiments, the second polypeptide chain interacts with the fourth polypeptide chain.

[0072] In some embodiments, the first polypeptide chain interacts with the second polypeptide chain and with the third polypeptide chain and the second polypeptide chain interacts with the fourth polypeptide chain.

[0073] In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus,

[0074] (i) a variable region of a heavy chain (VH) derived from an immunoglobulin that binds to human CLDN18.2 (VH(CLDN18.2)),

[0075] (ii) a constant region 1 of a heavy chain (CHI) derived from an immunoglobulin or a functional variant thereof,

[0076] (iii) a constant region 2 of a heavy chain (CH2) derived from an immunoglobulin or a functional variant thereof, and

[0077] (iv) a constant region 3 of a heavy chain (CH3) derived from an immunoglobulin or a functional variant thereof.

[0078] In some embodiments, the second polypeptide chain comprises, from N-terminus to C-terminus,

[0079] (i) a variable region of a heavy chain (VH) derived from an immunoglobulin that binds to human CLDN18.2 (VH(CLDN18.2)),

[0080] (ii) a constant region 1 of a heavy chain (CHI) derived from an immunoglobulin or a functional variant thereof,

[0081] (iii) a variable region of a light chain (VL) derived from an immunoglobulin that binds to human CD3 (VL(CD3)),

[0082] (iv) a variable region of a heavy chain (VH) derived from an immunoglobulin that binds to human CD3 (VH(CD3)), (v) a constant region 2 of a heavy chain (CH2) derived from an immunoglobulin or a functional variant thereof, and

[0083] (vi) a constant region 3 of a heavy chain (CH3) derived from an immunoglobulin or a functional variant thereof.

[0084] In some embodiments, the third polypeptide chain comprises, from N-terminus to C-terminus,

[0085] (i) a variable region of a light chain (VL) derived from an immunoglobulin that binds to human CLDN18.2 (VL(CLDN18.2)), and

[0086] (ii) a constant region of a light chain (CL) derived from an immunoglobulin or a functional variant thereof.

[0087] In some embodiments, the fourth polypeptide chain comprises, from N-terminus to C-terminus,

[0088] (i) a variable region of a light chain (VL) derived from an immunoglobulin that binds to human CLDN18.2 (VL(CLDN18.2)), and

[0089] (ii) a constant region of a light chain (CL) derived from an immimoglobulin or a functional variant thereof.

[0090] In some embodiments, the VH(CLDN18.2) on the first polypeptide chain and the VL(CLDN18.2) on the third polypeptide chain interact to form a binding domain that binds to human CLDN 18.2.

[0091] In some embodiments, the VH(CLDN18.2) on the second polypeptide chain and the VL(CLDN18.2) on the fourth polypeptide chain interact to form a binding domain that binds to human CLDN 18.2.

[0092] In some embodiments, the VH(CD3) and the VL(CD3) interact to form a binding domain that binds to human CD3.

[0093] In some embodiments, the VH(CLDN18.2) comprises a CDR1 comprising the amino acid sequence SYWIN (SEQ ID NO: 10), a CDR2 comprising the amino acid sequence NIYPSDSYTNYNQKFQG (SEQ ID NO: 11), and a CDR3 comprising the amino acid sequence SWRGNSFDY (SEQ ID NO: 12).

[0094] In some embodiments, the VL(CLDN18.2) comprises a CDR1 comprising the amino acid sequence KSSQSLLNSGNQKNYLT (SEQ ID NO: 13), a CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO: 14), and a CDR3 comprising the amino acid sequence QNDYSYPFT (SEQ ID NO: 15).

[0095] In some embodiments, the VH(CD3) comprises a CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 18), a CDR2 comprising the amino acid sequence RIRSKANNYATYYADSVKG (SEQ ID NO: 23), and a CDR3 comprising the amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO: 19).

[0096] In some embodiments, the VL(CD3) comprises a CDR1 comprising the ammo acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 20), a CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 21), and a CDR3 comprising the amino acid sequence ALWYSNHWV (SEQ ID NO: 22).

[0097] In some embodiments, the CH2 on the first polypeptide chain interacts with the CH2 on the second polypeptide chain and / or the CH3 on the first polypeptide chain interacts with the CH3 on the second polypeptide chain.

[0098] In some embodiments, the CHI on the first polypeptide chain interacts with the CL on the third polypeptide chain.

[0099] In some embodiments, the CHI on the second polypeptide chain interacts with the CL on the fourth polypeptide chain.

[0100] In some embodiments, the immunoglobulin is IgGl. In some embodiments, the IgGl is human IgGl.

[0101] In some embodiments, the VH(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 16.

[0102] In some embodiments, the VL(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 17.

[0103] In some embodiments, the VL(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 24. In some embodiments, the VH(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 25.

[0104] In some embodiments, the VH(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO:

[0105] 16, the VL(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO:

[0106] 17, the VH(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 25, and the VL(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 24.

[0107] In some embodiments, the VH(CLDN18.2), VL(CLDN18.2), VH(CD3) and / or VL(CD3) are humanized.

[0108] In some embodiments, on the first polypeptide chain the CHI is connected to the CH2 by a peptide linker LI. In some embodiments, the peptide linker LI comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 6) or a functional variant thereof.

[0109] In some embodiments, the VL(CD3) is connected to the CHI by a peptide linker L2. In some embodiments, the peptide linker L2 comprises the amino acid sequence (G4S)xor a functional variant thereof, wherein x is 2, 3, 4, 5 or 6. In some embodiments, the peptide linker L2 comprises the amino acid sequence (G4S)2(SEQ ID NO: 5) or a functional variant thereof.

[0110] In some embodiments, the VL(CD3) and the VH(CD3) are connected to one another by a peptide linker L3. In some embodiments, the peptide linker L3 comprises the amino acid sequence (GKPGS)xor a functional variant thereof, wherein x is 2, 3, 4, 5 or 6. In some embodiments, the peptide linker L3 comprises the amino acid sequence (6KP6S)4(SEQ ID NO: 2) or a functional variant thereof.

[0111] In some embodiments, the VH(CD3) is connected to the CH2 by a peptide linker L4. In some embodiments, the peptide linker L4 comprises the amino acid sequence (G4S)xor a functional variant thereof, wherein x is 2, 3, 4, 5 or 6. In some embodiments, the peptide linker L4 comprises the amino acid sequence (G4S)2(SEQ ID NO: 5) or a functional variant thereof.

[0112] In certain embodiments, the CHI, CH2 and / or CH3 domains of the bispecific binding agent described herein comprise one or more amino acid modifications, in particular substitutions and / or deletions, in positions corresponding to positions of human IgGl according to EU numbering. In some embodiments, the CHI on the first and / or the second polypeptide chain comprises an amino acid sequence comprising an aspartic acid residue at position 208 according to EU numbering, In some embodiments, the CHI on the first polypeptide chain comprises an amino acid sequence comprising an aspartic acid residue at position 208 according to EU numbering and the CHI on the second polypeptide chain comprises an amino acid sequence comprising an asparagine residue at position 208 according to EU numbering.

[0113] Furthermore, in certain embodiments, the binding agent described herein does not substantially, e.g., not detectably, bind to human FcγRI, Ila, lib, and / or Illa. In some embodiments, the CH2 on the first and / or the second polypeptide chain comprises an amino acid sequence comprising 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 some embodiments, the CH2s on the first 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 wherein the CH2 on the first polypeptide chain further comprises a glutamic acid residue at position 295 according to EU numbering and the CH2 on the second polypeptide chain further comprises a glutamine residue at position 295 according to EU numbering.

[0114] In some embodiments, the CH3 on the first 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 some embodiments, the CH3 on the first polypeptide chain comprises an amino acid sequence comprising a glutamic acid 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 the CH3 on 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.

[0115] In some embodiments, the first polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 7.

[0116] In some embodiments, the second polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 8.

[0117] In some embodiments, the third and / or fourth polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 9.

[0118] In some embodiments, CD3 is expressed on the surface of T cells. In some embodiments, the bispecific binding agent described herein binds to the epsilon chain of CD3. In some embodiments, binding of the binding agent to CD3 on T cells results in proliferation and / or activation of the T cells. In some embodiments, proliferation and / or activation of T cells includes proliferation and / or activation of CD4 and / or CDS T cells, preferably CD107a+ T cells. In some embodiments, said proliferated and / or activated T cells are capable of degranulation. In some embodiments, said activated T cells release cytotoxic factors, e.g., perforins and granzymes, and initiate cytolysis and / or apoptosis of cancer cells.

[0119] In some embodiments, CLDN18.2 is expressed in cancer cells. In some embodiments, CLDN18.2 is expressed on the surface of cancer cells. In some embodiments, the bispecific binding agent binds to an extracellular portion of CLDN18.2. In some embodiments, the binding agent induces T cell-mediated cytotoxicity against cancer cells expressing CLDN18.2.

[0120] In some embodiments, the cancer cells are from a cancer selected from the group consisting of gastric cancer, particularly gastric adenocarcinoma, esophageal cancer, cancer of the gastroesophageal junction (GEJ), particularly GEJ adenocarcinoma, pancreatic cancer, particularly pancreatic adenocarcinoma, lung cancer such as non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, rectal cancer, colorectal cancer, hepatic cancer, head- neck cancer, bile duct cancer, cancer of the gallbladder and the metastasis thereof, a Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis.

[0121] In some embodiments, the bispecific binding agent comprises one or more posttranslational modifications. In some embodiments, the bispecific binding agent is derived from one or more posttranslational modifications of a binding agent described herein. In some embodiments, the one or more posttranslational modifications are selected from pyroglutamylation at the N-terminus of one or more VH(CLDN18.2), deletion of lysine at the C-terminus of the first polypeptide chain and deletion of lysine at the C-terminus of the second polypeptide chain.

[0122] In some embodiments, a bispecific binding agent is provided as nucleic acid encoding the binding agent. In some embodiments, a bispecific binding agent is provided as a set of nucleic acids together encoding the binding agent In some embodiments, the nucleic acid or the set of nucleic acids is capable of expressing the bispecific binding agent.

[0123] In some embodiments, a vector comprises the nucleic acid or the set of nucleic acids. In some embodiments, a set of vectors comprises the set of nucleic acids. In some embodiments, each nucleic acid of the set of nucleic acids is contained in a vector of the set of vectors. In some embodiments, the vector or the set of vectors is capable of expressing the bispecific binding agent.

[0124] Expression of the nucleic acid or set of nucleic acids, or of the vector or set of vectors, e.g., in a subject may provide the bispecific binding agent.

[0125] In some embodiments, a nucleic acid is operably linked to any number of regulatory elements (promoters, origin of replication, selectable markers, ribosomal binding sites, inducers, etc.). The vector can be an expression vector and can be extra-chromosomal or integrating vector. In some embodiments, nucleic acids encoding a polypeptide chain of the binding agent described herein are each contained within a single expression vector. The nucleic acids can be under control of different or the same promoters. In such embodiments, different vector ratios can be used to drive formation of the binding agent described herein.

[0126] In some embodiments, the agent stabilizing or increasing expression of CLDN18.2 comprises a chemotherapeutic agent. In some embodiments, the agent stabilizing or increasing expression of CLDN18.2 comprises a cytotoxic and / or cytostatic agent.

[0127] In some embodiments, the agent stabilizing or increasing expression of CLDN18.2 comprises an agent selected from the group consisting of anthracyclines, nucleoside analogs, platinum compounds, camptothecin analogs and taxanes, prodrugs thereof, salts thereof, and combinations thereof.

[0128] In some embodiments, the agent stabilizing or increasing expression of CLDN18.2 comprises a nucleoside analog, a prodrug thereof, or a salt thereof.

[0129] In some embodiments, the agent stabilizing or increasing expression of CLDN18.2 comprises a taxane, a prodrug thereof, or a salt thereof.

[0130] In some embodiments, the agent stabilizing or increasing expression of CLDN18.2 comprises (i) gemcitabine, a prodrug thereof, or a salt thereof, (ii) paclitaxel, a prodrug thereof, or a salt thereof, or (iii) a combination of gemcitabine, a prodrug thereof, or a salt thereof, and paclitaxel, a prodrug thereof, or a salt thereof.

[0131] In some embodiments, the composition or medical preparation comprises:

[0132] (i) the bispecific binding agent;

[0133] (ii) oxaliplatin;

[0134] (iii) fluorouracil or a precursor thereof; and optionally

[0135] (iv) folinic acid.

[0136] In some embodiments, the composition or medical preparation further comprises pembrolizumab or irinothecan.

[0137] In some embodiments, the composition or medical preparation comprises:

[0138] (i) the bispecific binding agent;

[0139] (ii) paclitaxel; and

[0140] (iii) ramucirumab. In some embodiments, the composition or medical preparation is a pharmaceutical composition.

[0141] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0142] In some embodiments, the composition or medical preparation is a kit. In some embodiments, the bispecific binding agent and the agent stabilizing or increasing expression of CLDN 18.2 are in separate vials.

[0143] In some embodiments, the composition or medical preparation further comprises instractions for use of the bispecific binding agent and the agent stabilizing or increasing expression of CLDN 18.2 for treating or preventing cancer.

[0144] The invention further provides the composition or medical preparation described herein for pharmaceutical use.

[0145] In some embodiments, the pharmaceutical use comprises a therapeutic or prophylactic treatment of a disease or disorder.

[0146] In some embodiments, the therapeutic or prophylactic treatment of a disease or disorder comprises treating or preventing cancer.

[0147] In some embodiments, the cancer involves cancer cells expressing CLDN18.2.

[0148] In some embodiments, the cancer is selected from the group consisting of gastric cancer, particularly gastric adenocarcinoma, esophageal cancer, cancer of the gastroesophageal junction (GEJ), particularly GEJ adenocarcinoma, pancreatic cancer, particularly pancreatic adenocarcinoma lung cancer such as non small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, rectal cancer, colorectal cancer, hepatic cancer, head-neck cancer, bile duct cancer, cancer of the gallbladder and the metastasis thereof, a Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis.

[0149] In some embodiments, the composition or medical preparation described herein is for administration to a human. The invention further provides a method of treating or preventing cancer in a subject comprising administering to the subject:

[0150] (a) a bispecific binding agent as described herein; and

[0151] (b) an agent stabilizing or increasing expression of CLDN18.2.

[0152] In some embodiments, the bispecific binding agent is a bispecific binding agent as described in the context of the composition or medical preparation described herein. In some embodiments, the agent stabilizing or increasing expression of CLDN18.2 is an agent stabilizing or increasing expression of CLDN18.2 as described herein, e.g., as described in the context of the composition or medical preparation described herein.

[0153] In some embodiments, the subject is a human.

[0154] The invention further provides the composition or medical preparation described herein for use in the method described herein.

[0155] According to the invention, CLDN18.2 preferably has the amino acid sequence according to SEQ ID NO: 1.

[0156] In some embodiments, the first polypeptide chain does not comprise a VL(CLDN18.2). In some embodiments, the second polypeptide chain does not comprise a VL(CLDN18.2). In some embodiments, the third polypeptide chain does not comprise a VH(CLDN18.2). In some embodiments, the fourth polypeptide chain does not comprise a VH(CLDN18.2).

[0157] In some embodiments, the CLDN18.2 binding domains of the bispecific binding agent are in the format of a Fab fragment. In some embodiments, the CD3 binding domain of the bispecific binding agent is in the format of an scFv moiety.

[0158] In some embodiments, the bispecific binding agent does not bind to CLDN18.1. In some embodiments, the bispecific binding agent does not bind to CLDN18.1 of human, mouse or cynomolgus. In some embodiments, the bispecific binding agent does not bind to CLDN9, such as human CLDN9. In some embodiments, the bispecific binding agent binds to CLDN18.2 of more than one species such as CLDN18.2 of human, mouse and cynomolgus.

[0159] In some embodiments, treating a subject such as a patient with the bispecific binding agent and an agent stabilizing or increasing expression of CLDN18.2 as described herein results in prolonged survival of said subject. In some embodiments, treating a patient with the bispecific binding agent and an agent stabilizing or increasing expression of CLDN18.2 as described herein reduces, preferably significantly reduces, growth and / or volume of a tumor of a subject such as a patient.

[0160] In some embodiments, the bispecific binding agent described herein is able to redirect T cells to attack cancer cells and, thus, acts through redirected T cell cytotoxicity (RTCC). In some embodiments, the bispecific binding agent is not capable, or not substantially capable, of inducing ADCC. In some embodiments, the binding agent is not capable, or not substantially capable, of inducing CDC.

[0161] In some embodiments, the bispecific binding agent is produced by a method comprising transfecting host cells with a nucleic acid, a set of nucleic acids, a vector or a set of vectors encoding the polypeptide chains of the bispecific binding agent. In some embodiments, the host cells express the nucleic acid, the set of nucleic acids, the vector or the set of vectors. In some embodiments, a host cell co-expresses a nucleic acid encoding the first polypeptide chain of the bispecific binding agent, a nucleic acid encoding the second polypeptide chain of the bispecific binding agent, a nucleic acid encoding the third polypeptide chain of the bispecific binding agent and a nucleic acid encoding the fourth polypeptide chain of the bispecific binding agent. In some embodiments, said nucleic acids are contained in a vector or in a set of vectors. In some embodiments, a host cell expresses all polypeptide chains of the bispecific binding agent. In some embodiments, the host cell after transfection produces the bispecific binding agent, preferably when grown under appropriate conditions for binding agent production such as those described herein or known in the art. In some embodiments, the bispecific binding agent can be obtained from the host cell.

[0162] Thus, in some embodiments, a method of producing the bispecific binding agent comprises the steps of transfecting a host cell with a nucleic acid encoding the first polypeptide chain of the bispecific binding agent, a nucleic acid encoding the second polypeptide chain of the bispecific binding agent, a nucleic acid encoding the third polypeptide chain of the bispecific binding agent, and a nucleic acid encoding the fourth polypeptide chain of the bispecific binding agent, expressing said nucleic acids in the host cell and obtaining the bispecific binding agent. In some embodiments, the host cell is a mammalian cell, preferably selected from the group consisting of CHO cells, BHK cells, HeLa cells, COS cells, HEK293 cells, HEK293 T cells and the like. In some embodiments, the host cell is a bacterial cell, a yeast cell, a fungal cell, a plant cell or an insect cell. In some embodiments, the binding agent is produced in vitro. In some embodiments, the binding agent is produced in vivo, e.g., in a subject to be treated such as a subject having a disease, in particular a disease associated with cells expressing CLDN18.2, e.g. cancer. In some embodiments, the different polypeptide chains of the bispecific binding agent are produced in two or more different host cells. In some embodiments, all polypeptide chains of the bispecific binding agent are produced in the same host cell.

[0163] In some embodiments, the polypeptide chains of the bispecific binding agent are linked to one another, e.g., covalently linked. In some embodiments, the polypeptide chains of the bispecific binding agent are produced as one polypeptide comprising all polypeptide chains of the bispecific binding agent. In some embodiments, at least two polypeptide chains of the bispecific binding agent are linked together and are produced as one polypeptide. In some embodiments, the polypeptide chains of the bispecific binding agent described herein are produced separately, i.e., as separate polypeptides, e.g., in the same or in different cells and interact upon or after production to form the bispecific binding agent, e.g., within a cell or outside of a cell. In some embodiments, 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 the fourth polypeptide chain is produced as one polypeptide, and the polypeptide chains interact to form the bispecific binding agent.

[0164] Other features and advantages of the instant invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0165] Figure 1 depicts the "Fab2-scFv" format of the bispecific binding agent described herein which comprises a VH recombinantly fused to one side of a heterodimeric Fc (the first polypeptide chain described herein), a VH recombinantly fused to an scFv fused to the other side of the heterodimeric Fc (the second polypeptide chain described herein), and a LC (third and fourth polypeptide chains described herein) forming Fab domains with the VH of the first polypeptide chain and the additional VH of the second polypeptide chain.

[0166] Figure 2: Claudinl8.2 Expression on Pancreatic Carcinoma Cells with or without Gemcitabine Hydrochloride and Paclitaxel Treatment (First Experiment)

[0167] Histograms show claudinl8.2 expression on the surface of MIA PaCa-2_GFP_CLDN18.2_27.

[0168] Opened: treated with 0.1 μg / mL of gemcitabine and 0.003 μg / mL of paclitaxel for 2 days, Filled: untreated.

[0169] Figure 3: Cytotoxicity of ASP2138 in Co-culture of PBMC and Pancreatic Carcinoma Cells with or without Gemcitabine Hydrochloride and Paclitaxel Treatment (First Experiment) MIA PaCa-2 cells treated with or without gemcitabine hydrochloride and paclitaxel were co- cultured with human peripheral blood mononuclear cell (PBMC) at effector cell to target cell ratio of 10 : 1 and ASP2138 (0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 ng / mL) for 1 day. The number of living target cells was measured by flow cytometry and cytotoxicity (%) was calculated. Each datapoint represents the mean of duplicate in single experiment. Datapoints treated with gemcitabine hydrochloride, paclitaxel and 1 ng / mL or 30 ng / mL of ASP2138 were conducted in singlet. Each line represents the fitted non-linear regression curve.

[0170] MIA PaCa-2: MIA PaCa-2_GFP_CLDN18.2_27, Chemo Treatment: treatment with 0.1 μg / mL of gemcitabine and 0.003 μg / mL of paclitaxel for 2 days.

[0171] Figure 4: Claudinl8.2 Expression on Pancreatic Carcinoma Cells with or without Gemcitabine Hydrochloride and Paclitaxel Treatment (Second Experiment)

[0172] Histograms show claudin18.2 expression on the surface of MIA PaCa-2_GFP_CLDN18.2_27.

[0173] Opened: treated with 0.1 μg / mL of gemcitabine and 0.003 μg / mL of paclitaxel for 2 days, Filled: untreated. Figure 5: Cytotoxicity of ASP2138 in Co-culture of PBMC and Pancreatic Carcinoma Cells with or without Gemcitabine Hydrochloride and Paclitaxel Treatment (Second Experiment) MIA PaCa-2 cells treated with or without gemcitabine hydrochloride and paclitaxel were co- cultured with human peripheral blood mononuclear cell (PBMC) at effector cell to target cell ratio of 10 : 1 and ASP2138 (0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300 and 1000 ng / mL) for 1 day. The number of living target cells was measured by flow cytometry and cytotoxicity (%) was calculated. Each datapoint represents the mean of triplicate in single experiment. Each line represents the fitted non-linear regression curve.

[0174] MIA PaCa-2: MIA PaCa-2_GFP_CLDN18.2_27, Chemo Treatment: treatment with 0.1 μg / mL of gemcitabine and 0.003 μg / mL of paclitaxel for 2 days.

[0175] Figure 6: Anti-tumor Effect of ASP2138 in Combination with Gemcitabine Hydrochloride and Paclitaxel in a Mouse CLDN18.2-Expressing B16-F10 Tumor-Bearing Human CD3ε Knock-in Mouse Model

[0176] B16F10_mCLDN18.2 cells were subcutaneously inoculated into the flank of mice at 2.0 x 105cells on day -4. hCD3ε KI mice received intraperitoneal administration of ASP2138 (0.01 mg / kg) and paclitaxel (10 mg / kg) on day 0 and 7, and gemcitabine hydrochloride (40 mg / kg) on day 0, 3, 7 and 10. Upper: the tumor volumes in each group were plotted at each time point as the mean ± SEM (n=15). Lower: scatter plots indicate the individual tumor volume on day 14 and short horizontal lines and error bars represent the mean ± SEM (n=15). Statistical analysis was performed on the values on day 14.

[0177] **: P<0.01 compared with the value of the combination group on day 14 (unpaired Student’s t test).

[0178] B16F10_mCLDN18.2: mouse claudin 18.2-expressing B16-F10; hCD3ε KI: human CD3ε knock- in; Chemotherapy: gemcitabine hydrochloride and paclitaxel

[0179] Figure 7: Body Weight Changes in a Mouse CLDN18.2-Expressing B16-F10 Tumor-Bearing Human CD3ε Knock-in Mouse Treated with ASP2138 and / or Chemotherapy

[0180] Mice received intraperitoneal administration of ASP2138 (0.01 mg / kg) and paclitaxel (10 mg / kg) on day 0 and 7, and gemcitabine hydrochloride (40 mg / kg) on day 0, 3, 7 and 10. Each point represents the mean ± SEM (n=15).

[0181] CLDN18.2: claudin 18.2; Chemotherapy: gemcitabine hydrochloride and paclitaxel Figure 8: Anti-tumor Effect of ASP2138 in Combination with FOLFOX in a Human CLDN18.2-expressing MC38 Tumor-bearing Human CD3ε Knock-in Mouse Model

[0182] MC38_hCLDN18.2 cells were subcutaneously inoculated into the flank of mice at 2.0 x 105cells on day -6. hCD3ε KI mice received intraperitoneal administration of ASP2138 (0.1 mg / kg) on days 0 and 7, and 5-fluorouracil (25 mg / kg) and oxaliplatin (2.5 mg / kg) on days 0, 4, 7 and 11. Upper: the tumor volumes in each group are plotted at each time point as the mean ± SEM (n=15). Lower: scatter plots indicate the individual tumor volume on day 14 and short horizontal lines and error bars represent the mean ± SEM (n=15). Statistical analysis was performed on the values on day 14.

[0183] **: P<0.01 compared with the value of the combination group on day 14 (Student’s t-test). MC38_hCLDN18.2: human claudin 18.2-expressing MC38; hCD3ε KI: human CD3ε knock-in; FOLFOX: 5-fluorouracil and oxaliplatin

[0184] Figure 9: Body Weight Changes in a Human CLDN18.2-expressing MC38 Tumor-bearing Human CD3ε Knock-in Mouse Treated with ASP2138 and / or FOLFOX

[0185] Mice received intraperitoneal administration of ASP2138 (0.1 mg / kg) on days 0 and 7, and 5- fluorouracil (25 mg / kg) and oxaliplatin (2.5 mg / kg) on days 0, 4, 7 and 11. Each point represents the mean ± SEM (n=15).

[0186] CLDN18.2: claudin 18.2; FOLFOX: 5-fluorouracil and oxaliplatin

[0187] Figure 10: Anti-tumor Effect of ASP2138 in Combination with FOLFIRINOX in a Human CLDN18.2-expressing MC38 Tumor-bearing Human CD3ε Knock-in Mouse Model

[0188] MC38_hCLDN18.2 cells were subcutaneously inoculated into the flank of mice at 2.0 x 105cells on day -6. hCD3ε KI mice received intraperitoneal administration of ASP2138 (0.1 mg / kg) on days 0 and 7, and 5-fluorouracil (25 mg / kg), oxaliplatin (2.5 mg / kg) and irinotecan hydrochloride (10 mg / kg) on days 0, 4 and 7.

[0189] Upper: the tumor volumes in each group are plotted at each time point as the mean ± SEM (n=15). Lower: scatter plots indicate the individual tumor volume on day 11 and short horizontal lines and error bars represent the means ± SEM (n=l 5). Statistical analysis was performed on the values on day 11.

[0190] **: P<0.01 compared with the value of the combination group on day 11 (Student’s t-test). MC38_hCLDN18.2: human claudin 18.2-expressing MC38; hCD3ε KI: human CD3ε knock-in; FOLFIRINOX: 5-fluorouracil, oxaliplatin and irinotecan hydrochloride Figure 11: Body Weight Changes in a Human CLDN18.2-expressing MC38 Tumor-bearing Human CD3ε Knock-in Mouse Treated with ASP2138 and / or FOLFIRINOX

[0191] Mice received intraperitoneal administration of ASP2138 (0.1 mg / kg) on days 0 and 7, and 5- fluorouracil (25 mg / kg), oxaliplatin (2.5 mg / kg) and irinotecan hydrochloride (10 mg / kg) on days 0, 4 and 7. Each point represents the mean ± SEM (n=15).

[0192] CLDN18.2: claudin 18.2; FOLFIRINOX: 5-fluorouracil, oxaliplatin and irinotecan hydrochloride

[0193] DETAILED DESCRIPTION OF THE INVENTION

[0194] Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0195] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0196] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0197] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2ndEdition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0198] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps although in some embodiments, such other member, integer or step or group of members, integers or steps may be excluded, i.e. the subject-matter consists in the inclusion of a stated member, integer or step or group of members, integers or steps. It is contemplated as a specific embodiment of the present disclosure that the term "comprising" encompasses the possibility of no further members being present, i.e., for the purpose of this embodiment, "comprising" is to be understood as having the meaning of "consisting of or "consisting essentially of*.

[0199] The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0200] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0201] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0202] The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0203] The term "about" means approximately or nearly, and in the context of a numerical value or range set forth herein in some embodiments, means ± 20%, ± 10%, ± 5%, or ± 3% of the numerical value or range recited or claimed.

[0204] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0205] Bispecific binding agent The first target molecule of the bispecific binding agents described herein is CLDN18.2.

[0206] Claudius are a family of proteins that are the most important components of tight junctions, where they establish the paracellular barrier that controls the flow of molecules in the intercellular space between cells of an epithelium. Claudins are transmembrane proteins spanning the membrane 4 times with the N-terminal and the C-terminal end both located in the cytoplasm. The first extracellular loop, termed ECI or ECL1, consists on average of 53 amino acids, and the second extracellular loop, termed EC2 or ECL2, consists of around 24 amino acids. Cell surface proteins of the claudin family, such as CLDN 18.2, are expressed in tumors of various origins, and are particularly suited as target structures in connection with antibody-mediated cancer immunotherapy due to their selective expression (no expression in a toxicity relevant normal tissue) and localization to the plasma membrane.

[0207] CLDN18.2 has been identified as differentially expressed in tumor tissues, with the only normal tissues expressing CLDN 18.2 being stomach. CLDN 18.2 is selectively expressed in normal tissues in differentiated epithelial cells of the gastric mucosa. CLDN 18.2 is expressed in cancers of various origins such as pancreatic carcinoma, esophageal carcinoma, gastric carcinoma, bronchial carcinoma, breast carcinoma, and ENT tumors. CLDN 18.2 is a valuable target for the prevention and / or treatment of primary tumors, such as gastric cancer, particularly gastric adenocarcinoma, esophageal cancer, cancer of the gastroesophageal junction (GEJ), particularly GEJ adenocarcinoma, pancreatic cancer, particularly pancreatic adenocarcinoma, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, rectal cancer, colorectal cancer, hepatic cancer, head-neck cancer, bile duct cancer, cancers of the gallbladder, and metastases thereof, in particular gastric cancer metastasis such as Krukenberg tumors, peritoneal metastasis, and lymph node metastasis. The term "claudin 18" or "CLDNI8" relates to claudin 18 and includes any variants, including claudin 18 splice variant 1 (claudin 18.1 (CLDN18.1)) and claudin 18 splice variant 2 (claudin 18.2 (CLDN18.2)).

[0208] The term "claudin 18.2" or "CLDN 18.2" preferably relates to human CLDN 18.2, and, in particular, to a protein comprising, preferably consisting of the amino acid sequence according to SEQ ID NO: 1 of the sequence listing or a variant of said amino acid sequence. The first extracellular loop of CLDN 18.2 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. Said first and second extracellular loops preferably form the extracellular portion of CLDN18.2. In general, the antibodies described herein bind to the extracellular portion of human CLDN 18.2.

[0209] The second target molecule of the bispecific binding agents described herein is CD3 (cluster of differentiation 3), in particular CD3ε. The CD3 complex denotes an antigen that is expressed on mature human T-cells, thymocytes and a subset of natural killer cells as part of the multimolecular T-cell receptor (TCR) complex. The T-cell co-receptor is a protein complex and is composed of four distinct chains. In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains associate with a molecule known as the T-cell receptor (TCR) and the ζ-chain to generate an activation signal in T lymphocytes. The TCR, ζ-chain, and CD3 molecules together comprise the TCR complex.

[0210] The human CD3ε is indicated in GenBank Accession No. NM 000733. The human CD3γ is indicated in GenBank Accession No. NM_000073. The human CD3δ is indicated in GenBank Accession No. NM 000732. CD3 is responsible for the signal transduction of the TCR. As described by Lin and Weiss, Journal of Cell Science 114, 243-244 (2001), activation of the TCR complex by binding of MHC-presented specific antigen epitopes results in the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) by Src family kinases, triggering recruitment of further kinases which results in T-cell activation including Ca2+release. Clustering of CD3 on T cells, e.g. by immobilized anti-CD3-antibodies, leads to T-cell activation similar to the engagement of the T-cell receptor, but independent from its clone typical specificity.

[0211] As used herein, "CD3" includes human CD3 and denotes an antigen that is expressed on human T cells as part of the multimolecular T-cell receptor complex. With respect to CD3, the binding agent described herein preferably recognizes the epsilon-chain of CD3. In some embodiments, it recognizes an epitope that corresponds to the first 27 N-terminal amino acids of CD3 epsilon or functional fragments of this 27 amino acid stretch.

[0212] According to the invention, the term "CLDN18.2 positive cancer" or similar terms mean a cancer involving cancer cells expressing CLDN18.2, preferably on the surface of said cancer cells.

[0213] "Cell surface" is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. CLDN18.2 is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by CLDN18.2-specific antibodies added to the cells.

[0214] CD3 is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by CD3-specific antibodies added to the cells.

[0215] The term "extracellular portion" in the context of the present invention refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by antigen-binding molecules such as antibodies located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or a fragment thereof.

[0216] The terms "part" or "fragment" are used interchangeably herein and refer to any portion of a structure such as an amino acid sequence that is smaller than the complete structure. In some embodiments, a part of a structure refers to a continuous element of said structure. In some embodiments, a part of a structure refers to more than one continuous element of said structure which elements may be connected. A portion, a part or a fragment of a structure preferably comprises one or more functional properties of said structure. A part or fragment of an amino acid sequence preferably comprises a sequence of at least 4, in particular 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 the protein sequence.

[0217] "Fragment", with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable e.g. by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable e.g. by translation of a truncated open reading frame that lacks the 5 '-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises e.g. at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from an amino acid sequence.

[0218] By "variant" of an amino acid sequence or similar expressions herein is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of 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 a 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, e.g., from 1 to about 20 amino acid modifications, and preferably from 1 to about 10 or from 1 to about 5 amino acid modifications compared to the parent.

[0219] By "wild type" or "WT" or "native" used with respect to an amino acid sequence herein is meant an amino acid sequence that is 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.

[0220] For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide, protein or polypeptide) comprise 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, posttranslationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" includes, in particular, fragments of an amino acid sequence.

[0221] Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.

[0222] Amino acid addition variants comprise 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.

[0223] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and / or C-terminal end of the protein are also called N-terminal and / or C-terminal truncation variants.

[0224] Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and / or to replacing amino acids with other ones having similar properties. Preferably, amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0225] In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in the following table:

[0226] Preferably the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be 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%. In this respect the term "at least 80% sequence identity" as used herein with respect to a given amino acid sequence includes amino acid sequences having a level of identitiy to the given amino acid sequence of at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably 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, continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS: :needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0227] "Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0228] The terms "% identical", "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by 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 in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_ LOC=align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for 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.

[0229] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0230] In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or about 200 nucleotides, in some embodiments, continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0231] Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues to the reference sequence.

[0232] The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example. Furthermore, the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.

[0233] In some embodiments, a 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 exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to antigen binding domains comprising functional VH and VL variants, one particular function is to retain binding of said binding domain. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more, or all, of the functions of the parent molecule or sequence, e.g., forming a binding domain for a particular antigen. For example, a binding domain comprising functional VH and VL variants or functional CDR variant sequences has the same or similar binding characteristics compared to the parent molecule. In some embodiments, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, characteristics of a molecule comprising the functional fragment or functional variant, e.g. binding characteristics such as binding strength of a binding domain, may be reduced but still significantly present, e.g., binding characteristics such as binding strength of the binding domain comprising 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, a functional variant may comprise 1, 2, 3, 4, 5, or more amino acid insertions, amino acid additions, amino acid substitutions and / or amino acid deletions compared to the parent molecule. However, in other embodiments, characteristics of the molecule comprising the functional variant or functional fragment, e.g., binding characteristics of a binding domain comprising 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 shortened at the N-terminal and / or C-terminal end compared to the parent molecule, but retains or retains essentially one or more, or all, of the functions of the parent molecule, as described above, and in particular is functional equivalent to the parent molecule.

[0234] The term "functional variant" of an amino acid sequence includes "functional" fragments of said amino acid sequence.

[0235] An amino acid sequence (peptide, protein or polypeptide, e.g., VH, VL, CHI, CH2 or CH3) "derived from" a designated amino acid sequence (peptide, protein or polypeptide, e.g., VH, VL, CHI, CH2 or CH3) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof, preferably functional variants thereof as described herein, including functional fragments. For example, it will be understood by one of ordinary skill in the art that the amino acid sequences suitable for use herein may be altered such that they vary in sequence, including amino acid insertions, amino acid deletions, amino acid additions and / or amino acid substitutions, from the naturally occurring or native sequences from which they were derived, while retaining or essentially retaining the desirable activity of the native sequences. For example, the amino acid sequences of the VH, VL, CHI, CH2 and / or CH3 domains on the peptide chains of the binding agent described herein are derived from amino acid sequences of VH, VL, CHI, CH2 and / or CH3 domains of immunoglobulins 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 can be identical to the amino acid sequence of the respective VH or VL it is derived from, or it can differ in one or more amino acid positions compared to the sequence of the respective parent VH or VL. For example, a VH domain of a binding agent described herein may comprise an amino acid sequence comprising one or more amino acid insertions, amino acid additions, amino acid deletions and / or amino acid substitutions compared to the amino acid sequence of the VH domain it is derived from. For example, a VL domain of a binding agent described herein may comprise an amino acid sequence comprising one or more amino acid insertions, amino acid additions, amino acid deletions and / or amino acid substitutions compared to the amino acid sequence of the VL domain it is derived from. Preferably, a VH or VL having an amino acid sequence that is a functional variant of the amino acid sequence of the parent VH or VL provides the same or essentially the same functions as the amino acid sequence of the parent VH or VL, e.g., in terms of binding specificity, binding strength etc. However, as one of ordinary skill in the art will be aware, in some embodiments, it may also be preferable to provide a functional variant of an amino acid sequence, e.g., of a VH or VL, which has altered characteristics compared to the amino acid sequence of the parent molecule. The same considerations apply to amino acid sequences of, e.g., CDRs, and to other amino acid sequences, e.g., those of CHI, CH2, CH3 and / or CL domains.

[0236] When a bispecific binding agent is described to comprise a VH "derived from" an immunoglobulin and a VL "derived from" the same or a different immunoglobulin, the term "derived from" indicates that the bispecific binding agent was generated by recombining, by any known method, said VH and VL from said immunoglobulin(s) into the resulting bispecific binding agent. In this context, "recombining" is not intended to be limited by any particular method of recombining and thus includes all of the methods for producing bispecific binding agents described herein or known in the art, including for example recombining at nucleic acid level and / or through co-expression of different molecules in the same cells. The term "bispecific" as used in the context of an agent such as an antibody, antibody-derived molecule or any other agent refers to an agent having two different antigen-binding domains defined by different amino acid sequences. In some embodiments, said different antigen-binding domains bind different epitopes on the same antigen, or as discussed below, can bind to the same epitope on the same antigen. However, in preferred embodiments, said different antigen-binding domains bind different target antigens. A binding agent can bind each of the different antigens or different epitopes with one, two, or more binding domains, i.e., bind each of the different antigens or different epitopes monovalently, divalently (or bivalently), trivalently, tetravalently and even with valency of higher order. In the case of a trivalent bispecific antibody, two of the binding domains bind to one target (either to the same epitope or a different one) and the other to a second target, for example.

[0237] CLDN18.2 is not substantially expressed in a cell if the level of expression is lower compared to expression in stomach cells or stomach tissue. Preferably, the level of expression is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% of the expression in stomach cells or stomach tissue or even lower. Preferably, CLDN18.2 is not substantially expressed in a cell if the level of expression exceeds the level of expression in non-cancerous tissue other than stomach by no more than 2-fold, preferably 1.5-fold, and preferably does not exceed the level of expression in said non-cancerous tissue. Preferably, CLDN18.2 is not substantially expressed in a cell if the level of expression is below the detection limit and / or if the level of expression is too low to allow binding by CLDN18.2-specific antibodies added to the cell.

[0238] CLDN18.2 is expressed in a cell if the level of expression exceeds the level of expression in non- cancerous tissue other than stomach preferably by more than 2-fold, preferably 10-fold, 100-fold, 1000-fold, or 10000-fold. Preferably, CLDN18.2 is expressed in a cell if the level of expression is above the detection limit and / or if the level of expression is high enough to allow binding by CLDN18.2-specific antibodies added to the cell. Preferably, CLDN18.2 expressed in a cell is expressed or exposed on the surface of said cell.

[0239] According to the invention, the term "disease" refers to any pathological state, including cancer, in particular those forms of cancer described herein. Any reference herein to cancer or particular forms of cancer also includes cancer metastasis thereof. In a preferred embodiment, a disease to be treated according to the present teaching involves cells expressing CLDN18.2. "Disease involving cells expressing CLDN18.2", "disease associated with cells expressing CLDN18.2" or similar expressions, as used herein, mean that CLDN18.2 is expressed in cells of a diseased tissue or organ. In some embodiments, expression of CLDN18.2 in cells of a diseased tissue or organ is increased compared to the state in a healthy tissue or organ. An increase refers to an increase by 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 some embodiments, expression is only found in a diseased tissue, while expression in a healthy tissue is repressed. According to the invention, diseases associated with cells expressing CLDN18.2 include cancer diseases. According to the invention, cancer diseases preferably are those wherein the cancer cells express CLDN18.2.

[0240] As used herein, a "cancer disease" or "cancer" includes a disease characterized by aberrantly regulated cellular growth, proliferation, differentiation, adhesion, and / or migration. By "cancer cell" is meant an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Preferably, a "cancer disease" is characterized by cells expressing CLDN18.2 and a cancer cell expresses CLDN18.2. A cell expressing CLDN18.2 preferably is a cancer cell, preferably of the cancers described herein.

[0241] The term "cancer" according to the invention comprises leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, stomach cancer, intestine cancer, head and neck cancer, bile duct cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, cancer of the gastroesophageal junction (GEJ), colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, cancer of the uterus, ovarian cancer and lung cancer and the metastases thereof. Examples thereof are lung carcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, cervical carcinomas, or metastases of the cancer types or tumors described above. The term cancer according to the invention also comprises cancer metastases.

[0242] According to the invention, a "carcinoma" is a malignant tumor derived from epithelial cells. This group represents the most common cancers, including the common forms of breast, prostate, lung and colon cancer. "Adenocarcinoma" is a cancer that originates in glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissue includes skin, glands and a variety of other tissue that lines the cavities and organs of the body. Epithelium is derived embryologically from ectoderm, endoderm and mesoderm. To be classified as adenocarcinoma, the cells do not necessarily need to be part of a gland, as long as they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Well differentiated adenocarcinomas tend to resemble the glandular tissue that they are derived from, while poorly differentiated may not. By staining the cells from a biopsy, a pathologist will determine whether the tumor is an adenocarcinoma or some other type of cancer. Adenocarcinomas can arise in many tissues of the body due to the ubiquitous nature of glands within the body. While each gland may not be secreting the same substance, as long as there is an exocrine function to the cell, it is considered glandular and its malignant form is therefore named adenocarcinoma. Malignant adenocarcinomas invade other tissues and often metastasize given enough time to do so. Ovarian adenocarcinoma is the most common type of ovarian carcinoma. It includes the serous and mucinous adenocarcinomas, the clear cell adenocarcinoma and the endometrioid adenocarcinoma.

[0243] By "metastasis" is meant the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement manbranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumor at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop metastatic potential. In some embodiments, the term "metastasis" according to the invention relates to "distant metastasis" which relates to a metastasis which is remote from the primary tumor and the regional lymph node system. In some embodiments, the term "metastasis" according to the invention relates to lymph node metastasis. One particular form of metastasis which is treatable using the therapy or methods of treatment of the invention is metastasis originating from gastric cancer as primary site. In preferred embodiments, such gastric cancer metastasis is Krukenberg tumors, peritoneal metastasis and / or lymph node metastasis.

[0244] Krukenberg tumor is an uncommon metastatic tumor of the ovary accounting for 1% to 2% of all ovarian tumors. Prognosis of Krukenberg tumor is still very poor and there is no established treatment for Krukenberg tumors. Krukenberg tumor is a metastatic signet ring cell adenocarcinoma of the ovary. Stomach is the primary site in most Krukenberg tumor cases (70%). Carcinomas of colon, appendix, and breast (mainly invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg tumor originating from carcinomas of the gallbladder, biliary tract, pancreas, small intestine, ampulla of Vater, cervix, and urinary bladder / urachus have been reported.

[0245] By "treat" is meant to administer a compound or composition or a combination of compounds or compositions to a subject in order to prevent, ameliorate or eliminate a disease, including reducing the size of a tumor or the number of tumors in a subject; arrest or slow a disease in a subject; inhibit or slow the development of a new disease in a subject; decrease the frequency or severity of symptoms and / or recurrences in a subject who currently has or who previously has had a disease; and / or prolong, i.e. increase the lifespan of the subject.

[0246] In particular, the term "treatment of a disease" includes curing, shortening the duration, ameliorating, preventing, slowing down or inhibiting progression or worsening, or preventing or delaying the onset of a disease or the symptoms thereof.

[0247] 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 the propagation of a disease in a subject and, in particular, to minimizing the chance that a subject will develop a disease or to delaying the development of a disease. For example, a person at risk for cancer would be a candidate for therapy to prevent cancer.

[0248] By "being at risk" is meant a subject that is identified as having a higher than normal chance of developing a disease, in particular cancer, compared to the general population. In addition, a subject who has had, or who currently has, a disease, in particular cancer, is a subject who has an increased risk for developing a disease, as such a subject may continue to develop a disease. Subjects who currently have, or who have had, a cancer also have an increased risk for cancer metastases.

[0249] The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal (e.g. mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) that can be afflicted with or is susceptible to a disease or disorder (e.g., cancer) but may or may not have the disease or disorder. In many embodiments, the individual or subject is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In embodiments of the present disclosure, the "individual" or "subject" is a "patient".

[0250] The term "patient" means according to the invention a subject for treatment, in particular a diseased subject, including human beings, nonhuman primates or another animals, in particular mammals such as cows, horses, pigs, sheeps, goats, dogs, cats or rodents such as mice and rats. In a particularly preferred embodiment, a patient is a human being.

[0251] "Target cell" shall mean any undesirable cell such as a cancer cell. In preferred embodiments, the target cell expresses CLDN18.2.

[0252] "Activation" or "stimulation", as used herein, refers to the state of an immune effector cell such as T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with initiation of signaling pathways, induced cytokine production, and detectable effector functions. The term "activated immune effector cells" refers to, among other things, immune effector cells that are undergoing cell division.

[0253] The term "priming" refers to a process wherein an immune effector cell such as a T cell has its first contact with its specific antigen and causes differentiation into effector cells such as effector T cells.

[0254] The term "clonal expansion" or "expansion" refers to a process wherein a specific entity is multiplied. In the context of the present disclosure, the term is preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cell recognizing said antigen is amplified. Preferably, clonal expansion leads to differentiation of the immune effector cells.

[0255] As used herein, the term "interact" means that two molecular species, e.g., two polypeptide chains or portions thereof, physically associate with each other. The association that is characterized as an interaction can involve non-covalent and / or covalent, preferably non-covalent interactions, e.g., charge-charge interactions, charge-dipole interactions, dipole-dipole interactions, van der Waals forces, hydrogen bonding and / or hydrophobic forces. The term "bind" or "binding" relates to the non-covalent interaction with a target. In some embodiments, the term "bind" or "binding" relates to a specific binding. By the term "specific binding" or "specifically binds", as used herein, is meant a molecule such as an antibody which recognizes a specific target molecule, but does not substantially recognize or bind other molecules in a sample or in a subject. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.

[0256] In some instances, the term "specific binding" or "specifically binds", can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.

[0257] Disclosed herein are binding moieties and binding agents binding to CLDN18.2 and / or CD3. Such binding moieties and binding agents may form complexes with CLDN18.2 and / or CD3.

[0258] The term "binding agent" as used herein refers to any agent capable of binding to desired antigens. A binding agent may also comprise synthetic, modified or non-naturally occurring moieties. Such moieties may, for example, link desired antigen-binding functionalities or regions such as antibodies or antibody fragments. In some embodiments, a binding agent is a synthetic construct comprising antigen-binding CDRs or variable regions. In some embodiments, binding agents disclosed herein comprise bispecific or multispecific binding agents such as bispecific antibody- derived binding agents comprising a first and a second binding domain, wherein the first binding domain is capable of binding to CLDN18.2 and the second binding domain is capable of binding to CD3.

[0259] The term "binding domain" or "antigen-binding domain" as used herein refers to any region, moiety, group or domain which interacts with an antigen. In some embodiments, the term "binding domain" or "antigen-binding domain" refers to the site of a binding agent described herein, that binds to an antigen and includes the antigen-binding portion of a binding agent. In certain embodiments, the binding domain is or comprises an antibody, antibody fragment, or any other binding protein, or any combination thereof. The binding domain may be comprised of heavy chain and light chain variable domains (VH and VL), each of which includes four conserved framework regions (FR) and three CDRs. The CDRs vary in sequence and determine the specificity to a particular antigen. The VH and VL domains together may form the site that binds, e.g., specifically binds, a particular antigen. A "binding domain for" an antigen designates a binding domain, e.g., of a binding agent described herein, which binds to said antigen and preferably specifically binds to said antigen.

[0260] According to the present invention, an agent such as an antibody is capable of binding to a predetermined target if it has a significant affinity for said predetermined target and binds to said predetermined target in standard assays. "Affinity" or "binding affinity" is often measured by equilibrium dissociation constant (KD). Preferably, the term "significant affinity" refers to the binding to a predetermined target with a dissociation constant (KD) of 10-5M or lower, 10-6M or lower, 10-7M or lower, 10-8M or lower, 10-9M or lower, 10-10M or lower, 10-11M or lower, or 10-12M or lower.

[0261] An agent is not (substantially) capable of binding to a target if it has no significant affinity for said target and does not bind significantly, in particular does not bind delectably, to said target in standard assays. Preferably, the agent does not delectably bind to said target if present in a concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 μg / ml or higher. Preferably, an agent has no significant affinity for a target if it binds to said target with a KDthat is at least 10-fold, 100-fold, 10-3fold, 10-4fold, 10-5fold, or 10-6fold higher than the KDfor binding to the predetermined target to which the agent is capable of binding. For example, if the KDfor binding of an agent to the target to which the agent is capable of binding is 10-7M, the KDfor binding to a target for which the agent has no significant affinity would be at least 10-6M, 10-5M, 10-4M, 10-3M, 10-2M, or 10-1M.

[0262] A binding agent such as an antibody is specific for a predetermined target if it is capable of binding to said predetermined target while it is not capable of binding to other targets, i.e. has no significant affinity for other targets and does not significantly bind to other targets in standard assays. According to the invention, a binding agent is specific for CLDN18.2 if it is capable of binding to CLDN 18.2 but is not (substantially) capable of binding to other targets. Preferably, a binding agent is specific for CLDN18.2 if the affinity for and the binding to such other targets does not significantly exceed the affinity for or binding to CLDN18.2-unrelated proteins such as bovine serum albumin (BSA), casein, human serum albumin (HSA) or non-claudin transmembrane proteins such as MHC molecules or transferrin receptor or any other specified polypeptide. Preferably, a binding agent is specific for a predetermined target if it binds to said target with a KDthat is at least 10-fold, 100-fold, 10-3fold, 10-4fold, 10-5fold, or 10-6fold lower than the KDfor binding to a target for which it is not specific. For example, if the KDfor binding of a binding agent to the target for which it is specific is 10-7M, the KDfor binding to a target for which it is not specific would be at least 10-6M, 10-5M, 10-4M, 10-3M, 10-2M, or 10-1M.

[0263] The term "kd" (sec1), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koffvalue.

[0264] The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0265] 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, W. E., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y (1992), and methods described herein. Affinities may be readily determined using conventional techniques, such as by equilibrium dialysis; by using the BIAcore 2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by another method known to the skilled artisan. The affinity data may be analyzed, for example, by the method of Scatchard et al., Ann N.Y. Acad. ScL, 51 :660 (1949). The measured affinity of a particular interaction between binding agent and antigen can vary if measured under different conditions, e.g., salt concentration, pH. Thus, measurements of affinity and other antigen-binding parameters, e.g., KD, IC50, are preferably made with standardized solutions of binding agent and antigen, and a standardized buffer.

[0266] The term "competes" 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 for binding to a target antigen, such binding agents are non-competing and this is an indication that said binding agents do not bind to the same part, i.e. epitope of the target antigen. It is well known to a person skilled in the art how to test for competition of binding agents for binding to a target antigen. An example of such a method is a so-called cross-competition assay, which may e.g. be performed as an ELISA or by flow-cytometry.

[0267] Two binding agents such as antibodies have the "same specificity" if they bind to the same antigen and to the same epitope. Such binding agents would compete for binding in a competition binding assay. In some embodiments, binding agents binding to the same epitope are considered to bind to the same amino acids on the target molecule. That antibodies bind to the same epitope on a target antigen may be determined by standard alanine scanning experiments or antibody-antigen crystallization experiments known to a person skilled in the art.

[0268] The ability of binding agents to compete for binding to an antigen indicates that these binding agents may bind to the same epitope region of the antigen or when binding to another epitope sterically hinder the binding of binding agents to that particular epitope region. Competing binding agents can be readily identified based on their ability to compete with one or more binding agents in standard binding assays such as Surface Plasmon Resonance analysis, ELISA assays or flow cytometry (see WO 2013 / 173223). For example, the competition between binding agents can be detected by a cross-blocking assay. For example, a competitive ELISA assay may be performed by coating target antigen on the wells of a microtiter plate and adding antigen-binding agent and candidate competing test binding agent. The amount of the antigen-binding agent bound to the antigen in the well indirectly correlates with the binding ability of the candidate competing test binding agent that competes therewith for binding, e.g., to the same epitope. Specifically, the larger the affinity of the candidate competing test binding agent is for the same epitope, the smaller the amount of the antigen-binding agent bound to the antigen-coated well. The amount of the antigen- binding agent bound to the well can be measured by labelling the binding agent with detectable or measurable labelling substances. As described in WO 2013 / 173223 and as known in the art, Surface Plasmon Resonance analysis, e.g., using a Biacore instrument, can be used to identify overlapping versus different epitope regions recognized by binding agents. Alternatively, competition may be determined using biolayer interferometry.

[0269] A binding agent competing 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 a binding agent having the specificity for an antigen of another binding agent, e.g., a binding agent comprising heavy and light chain variable regions as described herein, Such as an antibody, may be a binding agent comprising variants of said heavy and / or light chain variable regions as described herein, e.g. modifications in the CDRs and / or a certain degree of identity as described herein.

[0270] The term "antigen" relates to a molecule such as a protein or peptide comprising an epitope against which an agent is directed and / or is to be directed, preferably to induce an immune response. An antigen or a procession product thereof such as a T-cell epitope is in some embodiments, bound by a T- or B-cell receptor, or by an immunoglobulin molecule such as an antibody. Accordingly, an antigen or a procession product thereof may react specifically with antibodies or T lymphocytes (T cells). In a preferred embodiment, an antigen is a tumor-associated antigen, such as CLDN18.2, i.e., a constituent of cancer cells which may be derived from the cytoplasm, the cell surface and the cell nucleus, in particular those antigens which are produced, preferably in large quantity, intracellular or as surface antigens on cancer cells.

[0271] In the context of the present invention, the term "tumor-associated antigen" or "cancer-associated antigen" preferably relates to proteins that are under normal conditions specifically expressed in a limited number of tissues and / or organs or in specific developmental stages and are expressed or aberrantly expressed in one or more tumor or cancer tissues. In the context of the present invention, the tumor-associated antigen is preferably associated with the cell surface of a cancer cell and is preferably not or only rarely expressed in normal tissues.

[0272] The term "epitope" refers to an antigenic determinant in a molecule, e.g., to the part in a molecule that is recognized by the immune system, for example, that is recognized by an antibody. For example, epitopes are the discrete, three-dimensional sites on an antigen, which are 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 non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, most preferably between 10 and 25 amino acids in length, for example, the epitope may be preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably to antigen receptors such as antibodies or the B cell receptor (BCR). The immunoglobulins are characterized by a structural domain, i.e., the immunoglobulin domain, having a characteristic immunoglobulin (Ig) fold. The term encompasses membrane bound immunoglobulins as well as soluble immunoglobulins. Soluble immunoglobulins are generally termed antibodies. Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains which are linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as the VL (variable light chain) domain, CL (constant light chain) domain, the VH (variable heavy chain) domain and the CH (constant heavy chain) domains CHI, CH2, CH3, and CH4. There are five types of mammalian immunoglobulin heavy chains, i.e., α, δ, ε, γ, and μ which account for the different classes of immunoglobulins, i.e., IgA, IgD, IgE, IgG, and IgM. Immunoglobulin classes are also referred to as "isotypes" (for instance IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) referring to the immunoglobulin class that is encoded by heavy chain constant region genes. When a particular isotype, e.g. IgGl, is mentioned herein, the term is not limited to a specific isotype sequence, e.g. a particular IgGl sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g. IgGl, than to other isotypes. As opposed to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins comprise a transmembrane domain and a short cytoplasmic domain at their caiboxy-terminus. In mammals there are two types of light chains, i.e., lambda and kappa. The immunoglobulin chains comprise a variable region and a constant region. The constant region is essentially conserved within the different isotypes of the immunoglobulins, wherein the variable part is highly divers and accounts for antigen recognition.

[0273] The term "antibody" includes an immunoglobulin comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies and chimeric antibodies. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (amino acid residues 118-447 of human IgGl according to EU numbering) comprising CHI, CH2 and CH3 domains, wherein CHI is typically connected to CH2-CH3 by a peptide linker (also called "hinge"). Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The term "region" and the term "domain" are used interchangeably herein. The VH and VL domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). 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 reference to amino acid positions in the constant regions in the present invention is according to the EU-numbering (Edelman et al., (1969) Proc. Natl. Acad. Sci. USA 63(l):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 a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0274] The term "amino acid corresponding to position... " as used herein refers to an amino acid position number in a human IgGl chain, in particular IgGl heavy chain. Corresponding amino acid positions in other immunoglobulins may be found by alignment with human IgGl . Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings and has at least 50%, at least 80%, at least 90%, or at least 95% identity to a human IgGl heavy chain. It is considered well-known in the art how to align a sequence or segment in a sequence and thereby determine the corresponding position in a sequence to an amino acid position according to the present invention.

[0275] 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 FcγRIs, FcγRIIs, FcγRIIIs, FcRn, Clq, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that are homologous to the FcγRs (Davis et al., (2002) Immunol. Rev. 190:123-136). Particular IgG Fc ligands are FcRn and Fc gamma receptors. An "Fc ligand" as used herein can be from any organism such as mouse, human and cynomolgus. "Fc gamma receptor", "FcγR" or "FcgammaR" refers to any member of the family of proteins that bind the IgG Fc region and is encoded by an FcγR gene. In humans this family includes but is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb- 1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD 16), including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NAl and FcγRIIb-NA2) (Jefferis et al., (2002) Immunol. Lett, 82:57-65). An FcγR may 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).

[0276] "FcRn" or "neonatal Fc Receptor" as used herein relates to a protein that binds the IgG Fc region and is encoded at least in part by an FcRn gene. The FcRn may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. The functional FcRn protein comprises two polypeptides, often referred to as the heavy chain (encoded by the FcRn gene) and light chain (beta-2-microglobulin). Unless specified otherwise, "FcRn" or "FcRn protein" refers to the complex of FcRn heavy chain with beta-2-microglobulin. An "FcRn variant" as used herein is one that increases binding to the FcRn receptor and may also increase serum half-life.

[0277] "Fc" or "Fc region" or "Fc domain" as used herein refers to a polypeptide comprising the CH2 and CH3 domains of an IgG molecule and optionally a peptide linker, including all or part of the hinge. The CH2-CH3 domains of human IgGl comprise amino acid positions 231-447, and the hinge comprises 216-230, according to EU numbering. Thus, with reference to IgG, the term "Fc domain" as used herein includes amino acid positions 231-447 (CH2-CH3) and 216-447 (hinge- CH2-CH3) according to EU numbering, and functional variants thereof including functional fragments thereof. An "Fc fragment" may contain fewer amino acids, e.g., an N-terminal or C- terminal truncation variant, but still retains the ability to form a dimer with another Fc domain or Fc fragment, as can be detected using standard methods, e.g., based on size (e.g. non-denaturing chromatography, size exclusion chromatography, etc.), and thus is a functional variant. According to the present invention, an IgG Fc domain preferably is a human IgG Fc domain including the Fc domain from human IgGl, IgG2 or IgG4.

[0278] The terms "hinge", "hinge region", "antibody hinge region" or "hinge domain" as used herein refer to the peptide linker comprising the amino acids between CHI and CH2 of an immunoglobulin, e.g., IgG. Structurally, in naturally occurring IgG, e.g., IgGl, molecules, the CHI ends at amino acid position 215 according to EU numbering, and the CH2 begins at amino acid position 231 according to EU numbering. Thus, for IgG, the hinge includes amino acid positions 216 to 230 according to EU numbering.

[0279] A "variant Fc domain" contains amino acid modifications as compared to a parental Fc domain. Thus, a "variant IgGl Fc domain", e.g., variant human IgGl Fc domain, contains amino acid modifications (e.g., amino acid substitutions and / or deletions) in positions corresponding to positions of an IgGl Fc domain, e.g., human IgGl Fc domain, and preferably is a functional variant of the parental Fc domain. Such variant IgG Fc domains retain at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the corresponding parental human IgG Fc domain. Optionally, the variant Fc domains can 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 as compared to the parental Fc domain. Optionally, the variant Fc domains can 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 as compared to the parental Fc domain. Preferably, a variant Fc domain retains the ability to form a dimer with another Fc domain as measured using techniques as described herein or known in the art, such as non-denaturing gel electrophoresis.

[0280] Fc modifications

[0281] The binding agents described herein comprise three different polypeptide chains, wherein two polypeptide chains, e.g., the first polypeptide chain and the second polypeptide chain, comprise CH2-CH3 regions, preferably derived from IgG such as IgGl , in particular human IgGl . Said first and second polypeptide chains comprising CH2-CH3 regions are able to interact, e.g., dimerize thereby forming a heterodimer comprising said first and second polypeptide chain of a binding agent described herein. Preferably, the CH2-CH3 regions of the binding agents are derived from IgGl, more preferably human IgGl, even though CH2 and CH3 derived from other serotypes can be used as described herein. Moreover, as discussed herein, the binding agents will self-assemble, e.g., within a producing host cell. For example, it is envisaged that the CH2 on the first polypeptide chain interacts with the CH2 on the second polypeptide chain and / or the CH3 on the first polypeptide chain interacts with the CH3 on the second polypeptide chain, thereby forming an Fc fragment. These Fc fragments may comprise one or more amino acid modifications or "Fc modifications" as discussed herein with respect to human IgGl to promote interaction between the CH2 on the first and second polypeptide chains and / or CH3 on the first and second polypeptide chains, and / or allow for ease of purification of heteromultimers, such as heterodimers, containing first and second polypeptide chains interacting with one another over homomultimers only containing one type of polypeptide chain, and / or confer further beneficial functionalities as discussed herein. Amino acid modifications discussed herein may also be contained in CHI domains, e.g., within the first and / or second polypeptide chains of a binding agent described herein. In addition, peptide linkers such as peptide linkers within scFv moieties or peptide linkers connecting further domains of a binding agent, e.g., CHI and scFv, or CH2 and scFv, or CHI and CH2, may comprise one or more amino acid modifications as described herein. For example, a peptide linker connecting a CH2-CH3 region to another region or domain, such as VH(CD3), or CHI, can have a serine at the amino acid position which corresponds to position 220 according to EU numbering in a naturally occurring IgGl (where normally a cysteine can be found). Using a peptide linker comprising a serine at said position corresponding to position 220 of human IgGl reduces disulfide formation between the two chains comprising CH2-CH3 regions.

[0282] Thus, formation of the binding agents described herein is based on the use of different monomers (e.g., first and second polypeptide chains described herein) comprising CHI, CH2 and / or CH3 containing amino acid substitutions such as those that "skew" formation of heterodimers formed by said monomers over homodimers described herein, preferably coupled with "pl modifications" that allow for simple purification of the heterodimers away from the homodimers, and optionally in combination with "ablation modifications" and additional Fc modifications discussed herein. A skilled person will understand that any of the amino acid modifications discussed herein with respect to CHI, CH2 and / or CH3 domains and peptide linkers, e.g., hinge variants, can be combined with further amino acid modifications discussed herein or known in the art. For example, any of the skew and pl modifications can independently be combined with ablation, and other Fc modifications. An Fc modification according to the present invention can be an amino acid insertion, addition, deletion, or substitution.

[0283] The Fc modifications discussed herein are defined according to the amino acid modifications that compose them. For example, N434S is an Fc modification with a substitution of serine at position 434 for asparagine relative to parental human IgGl Fc polypeptide and according to EU numbering. The identity of the parental amino acid may be unspecified, in which case the aforementioned variant is referred to as 434S, i.e., the CH2-CH3 region comprising said Fc modification comprises a serine in an amino acid position that corresponds to position 434 in human IgGl according to EU numbering. pl modifications pl modifications increase the isoelectric point (pl) difference between monomers what allows the isoelectric purification of homo- and heteromultimeric proteins. In general, a pl modification either increases the pl of the polypeptide chain (basic change) or decreases the pl of the polypeptide chain (acidic change).

[0284] As discussed herein, a pl difference of at least 0.1, e.g., 0.2, 0.3, 0.4 or 0.5, between two polypeptide chains can allow separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point known in the art. Therefore, inclusion of pl modifications that alter the pl of each of the polypeptide chains interacting with one another, e.g., first and second polypeptide chains described herein, such that each of said polypeptide chains has a different pl and the heteromultimer formed by said polypeptide chains also has a distinct pl facilitates isoelectric purification of binding agents comprising said heteromultimer. These substitutions also aid in the determination and monitoring of any contaminating unwanted homo- or heteromultimer formation. pl modifications can be contained within one or both chains of a heteromultimer comprising heavy chain constant regions, e.g., first and second polypeptide chains of a binding agent described herein, and within CHI , CH2 and / or CH3 and / or within peptide linkers such as linkers connecting the VH and VL domains of an scFv moiety. For example, pl variants can be contained in the first polypeptide chain and / or in the second polypeptide chain of a binding agent described herein to decrease or prevent homomultimer formation. Typically, when contained in both the first and second polypeptide chain, pl variants will be used such that the pl of one polypeptide chain is increased while the pl of the second polypeptide chain is decreased. This can be done, e.g., by replacing a neutral amino acid residue by a positively or negatively charged amino acid residue, or vice versa, or by changing a charged amino acid residue from positive to negative charge or vice versa, as discussed herein. Accordingly, in certain embodiments, of the present invention, a sufficient change in pl in at least one of the polypeptide chains of a binding agent described herein is provided such that heteromultimers, e.g., of the first and second polypeptide chains, can be purified away from homomultimers. In certain embodiments, a pl difference of as little as 0.1 , 0.2, 0.3, 0.4 or 0.5 or greater in pH units is used in the present invention. The amount of pl modifications to be included on one or more polypeptide chains of a binding agent described herein to get good separation will depend in part on the starting pl of the polypeptide chains, the pl of the CH regions, Fv scaffold regions etc. as will be understood by a person skilled in the art. The change in pl can be calculated by any method known in the art, e.g., on the basis of the CH regions, e.g., using the method described by Sillero and Maldonado (Sillero, Maldonado, (2006) Comput. Biol. Med. 36(2): 157-166). Alternatively, the pl of each polypeptide chain can be compared. In addition, heteromultimers can be separated according to their size.

[0285] In certain embodiments, pl modifications, skew modifications, additional Fc modifications or ablation modifications etc. are not included in the variable regions of a binding agent described herein.

[0286] In certain embodiments, pl modifications are derived from different IgG isotypes such that the pl of the respective polypeptide chain is changed without introducing immunogenicity (cf., U.S. 2014 / 0370013). Preferably, pl modifications are derived from human IgG isotypes so as to decrease the risk of introducing immunogenicity. The modifications discussed herein are described in relation to human IgGl, but all IgG isotypes can be altered this way, as well as isotype hybrids. In the case where the heavy chain constant domain is from IgG2-4, R133E and R133Q can also be used.

[0287] IgGl is a common isotype for therapeutic antibodies for a variety of reasons, including high effector function. However, the CH region of IgGl has a higher pl than that of IgG2. By introducing IgG2 derived residues at particular positions into the IgGl backbone, the pl of the resulting monomer is lowered or increased and, additionally, serum half-life may be increased. For example, human IgGl has a glycine (pl approximately 5.97) at position 137 according to EU numbering, and human IgG2 has a glutamic acid (pl approximately 3.22) at the corresponding position; substituting the glutamic acid residue for a glycine residue will affect the pl of the resulting polypeptide. Lowering the pl of an antibody constant region may also increase serum half-life in vivo (see USSN 13 / 194,904; Ghetie and Ward, 1997, Immunol Today. 18(12): 592- 598). Also, variable regions with lower pl may add to longer serum half-lifes (see Igawa et al., (2010) PEDS 23(5): 385-392).

[0288] In a preferred combination of pl modifications, one polypeptide chain (e.g., the first polypeptide chain of a binding agent described herein, e.g. comprising VH(CLDN18.2), CHI, 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 (i.e., N208D / Q295E / N384D / Q418E / N421D with respect to human IgGl) according to EU numbering and another polypeptide chain (e.g., the second polypeptide chain of said binding agent, e.g. comprising VH(CLDN18.2), CHI, VL(CD3), VH(CD3), CH2 and CH3) comprises a positively charged peptide linker connecting VH(CD3) and VL(CD3) ("scFv linker"), such as a polypeptide linker comprising or consisting of the amino acid sequence (GKPGS)4or a functional variant thereof.

[0289] In polypeptide chains that do not comprise a CHI and, thus, do not include an amino acid position that corresponds to position 208 according to EU numbering, the following negative pl modifications can 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 (human IgGl: Q295E / N384D / Q418E / N421D) according to EU numbering.

[0290] In some embodiments, one polypeptide chain, e.g., the first polypeptide chain, contains a set of variants as discussed herein and the polypeptide chain interacting therewith, e.g., the second polypeptide chain, comprises a charged scFv linker, such as a positively charged scFv linker of SEQ ID NO: 2 or a functional variant thereof, or a negatively charged scFv linker.

[0291] Skew modifications

[0292] "Skew modifications" are steric modifications that facilitate interaction of the polypeptide chains comprising such modifications. One strategy making use of steric modifications is referred to in the art as "knobs and holes", referring to amino acid engineering, wherein a protuberance is introduced on a first heavy-chain polypeptide, typically in the Fc region (CH2-CH3), and a corresponding cavity in a second heavy-chain polypeptide, typically in the Fc region (CH2-CH3), such that the protuberance can be positioned in the cavity at the interface of these two heavy chains to promote heterodimer formation and hinder homodimer formation (see USSN 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). "Protuberances" are constructed by replacing small amino-acid side- chains from the interface of the first heavy-chain polypeptide with larger side chains. Compensatory "cavities" of identical or similar size to the protuberances are created in the interface of the second heavy-chain polypeptide by replacing large amino-acid side-chains with smaller ones (US patent 5,731,168). "Knobs and holes" modifications can be combined with disulfide bonds to skew formation to heteromultimerisation, e.g., heterodimerization of said first and second heavy-chain polypeptides (see Merchant et al., (1998) Nature Biotech. 16:677). Useful skew modifications include without limitation the following pairs of double modifications, wherein one part of each pair of double modifications will be present in one polypeptide chain (e.g., the first polypeptide chain described herein) of the binding agent described herein and the second part will be present in another polypeptide chain (e.g., the second polypeptide chain described herein) of the binding agent described herein: S364K / E357Q : L368D / 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 according to EU numbering and with respect to human IgGl. Preferably, L368D / K370S : S364K / E357Q is used in the binding agent described herein.

[0293] Skew modifications described herein may also have an effect on pl (cf., Gunasekaran et al., (2010) J. Biol. Chem. 285(25): 19637), and thus on purification, and, therefore, could also be considered pl variants.

[0294] Ablation modifications

[0295] By "ablation" herein is meant a decrease or removal of activity. "Ablating FcγR binding" means that an Fc region comprising one or more ablation modifications has more than 50% loss in FcγR binding activity as compared to an Fc region not containing the specific modifications. Preferably, the Fc region comprising the one or more ablation modifications has more than 70%, 80%, 90%, 95%, 98% or even more loss of FcγR binding activity. Preferably, the FcγR binding activity of an Fc region comprising the one or more ablation modifications as compared to an Fc region not containing the specific modifications is below the level of detectable binding in a Biacore, SPR or BLI assay.

[0296] As is known, the Fc domain of human IgGl has the highest binding to Fcγ receptors, and thus ablation modifications can be used when the constant domains of a binding agent are derived from IgGl. Alternatively, or in addition to ablation modifications, mutations at the glycosylation position 297 (generally to A or S) can significantly ablate binding to FcγRIIIa, for example. Human IgG2 and IgG4 have naturally lower binding to Fcγ receptors (Farren et al., 1992, J. Clin Invest. 90: 1537-1546; Bruhns et al., 2009, Blood 113: 3716-3725), and thus CHI, CH2 and CH3 domains derived from IgG2 or IgG4 can be used with or without ablation modifications in binding agents described herein. Amino acid modifications that ablate FcγR binding have, e.g., been described in Dall’Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24):12161-12168 (2001).

[0297] Thus, the Fc portion of a binding agent described herein may comprise one or more "FcγR ablation modifications" or "Fc knock out (FcKO or KO) modifications". In certain embodiments, it is desirable to reduce or remove 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 ablate FcγRIIIa binding of binding agents that bind CD3 monovalently such as the binding agents described herein to eliminate or significantly reduce ADCC activity. Thus, in the binding agents described herein, one or more of the polypeptide chains, e.g., the first polypeptide chain and the second polypeptide chain, of a binding agent described herein comprise one or more FcγR ablation variants. In preferred embodiments, the one or more ablation variants are selected from the group consisting of G236R, S239G, S239K, S239Q, S239R, V266D, S267K, S267R, H268K, E269R, 299R, 299K, K322A, A327G, A327L, A327N, A327Q, L328E, L328R, P329A, P329H, P329K, A330L, A330S / P331S, I332K, I332R, V266D / A327Q, V266D / P329K, S267R / A327Q, S267R / P329K, G236R / L328R, E233P / L234V / L235A / G236deVS267K,

[0298] E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del, S239K / S267K, 267K / P329K, E233P / L234V / L235A / G236deVS239K, E233P / L234V / L235A / G236deVS267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236deVS267K / A327G and E233P / L234V / L235A / G236del according to EU numbering and with respect to human IgGl, wherein "del" represents an amino acid deletion at the indicated position. Preferably, the modifications E233P / L234V / L235A / G236del / S267K according to EU numbering and with respect to human IgGl are used in both the first and the second polypeptide chain of the binding agent described herein. It should be noted that the ablation modifications disclosed herein ablate FcγR binding but generally not FcRn binding. However, techniques for reducing or increasing the binding to FcRn in order to reduce or increase serum half-life of the binding agent are known and can be used (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).

[0299] For example, in the binding agent described herein, the first polypeptide chain comprises an amino acid sequence according to SEQ ID NO: 7 and the second polypeptide chain comprises an amino acid sequence according to SEQ ID NO: 8, wherein the first and second polypeptide chains comprise the L368D / K370S : S364K / E357Q set of skew modifications, the first polypeptide chain further comprises theN208D / Q295E / N384D / Q418E / N421D set of pl modifications, and both the first and second polypeptide chain further comprises the E233P / L234V / L235A / G236del / S267K set of ablation modifications, e.g. wherein the first polypeptide chain comprises VH(CLDN18.2) and CHI and the second polypeptide chain comprises scFv(CD3), VH(CLDN18.2) and CHI. Of course, further modifications may be included into the amino acid sequences of a respective binding agent; e.g., a binding agent may comprise additional amino acid modifications such as substitutions in addition to the modifications discussed above.

[0300] Additional Fc modifications

[0301] In addition to other modifications described herein, such as pl, skew and ablation modifications, a number of usefill modifications that alter binding of one or more FcγR receptors, altered binding to FcRn receptors, etc. can be used.

[0302] Accordingly, there is a number of useful amino acid substitutions that can be made to alter binding of the binding agents described herein to one or more FcγR receptors. Substitutions that result in increased binding as well as decreased binding can be usefill. For example, it is known that increased binding to FcγRIIIa results in increased ADCC. Similarly, decreased binding to FcγRIIb can be beneficial as well. Amino acid substitutions that can be used in the present invention include those listed in USSNs 11 / 124,620, 11 / 174,287, 11 / 396,495, 11 / 538,406, all of which are incorporated herein by reference in their entirety. Particular usefill amino acid substitutions that can be incorporated into the binding agents described herein 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.

[0303] In addition, there are additional modifications that are usefill to increase binding to FcRn and increase serum half-life, as disclosed in USSN 12 / 341,769, hereby incorporated by reference in its entirety, including but not limited to 434S, 434A, 428L, 308F, 2591, 428L / 434S, 259I / 308F, 436I / 428L, 436I / 434S, 436V / 434S, 436V / 428L and 259V308F / 428L according to EU numbering.

[0304] In addition, a CH3 on one or both polypeptide chains, preferably on both polypeptide chains, forming an Fc heterodimer may contain the modifications M428L / N434S resulting in longer serum half-life.

[0305] As will be appreciated by those skilled in the art, the modifications discussed herein can be independently combined with other modifications. In some embodiments, one polypeptide chain of a binding agent described herein (e.g., the first polypeptide chain) comprises N208D, Q295E, N384D, Q418E and N481D according to EU numbering, and another polypeptide chain of the binding agent (e.g., the second polypeptide chain) comprises a positively charged scFv linker as described herein. Preferably, the first polypeptide chain of the binding agent further comprises K370S and L368D, 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 and second polypeptide chains further comprise E233P, L234V, L235A, G236del and S267K according to EU numbering. Most preferably, the first polypeptide chain of a binding agent described herein 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 a light chain.

[0306] The term "monoclonal binding agent" as used herein includes a "monoclonal antibody" and refers to a preparation of binding agent molecules of single molecular composition.

[0307] A "monoclonal antibody" displays a single binding specificity and affinity for a particular epitope.

[0308] The term "recombinant binding agent" as used herein includes a "recombinant antibody" and includes all binding agents that are prepared, expressed, created or isolated by recombinant means.

[0309] The term "human binding agent" as used herein includes a "human antibody" 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).

[0310] The term "humanized binding agent" as used herein includes a "humanized antibody" and refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based upon the structure and / or sequence of a human immunoglobulin. This can be achieved, e.g., by grafting of the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see WO92 / 22653 and EP0629240). The antigen binding site may either comprise complete variable domains fused onto constant domains or only the complementarity determining regions (CDR) grafted onto appropriate framework regions in the variable domains. In order to fully reconstitute the binding affinity and specificity of the parental binding agent, the substitution of framework residues from the parental binding agent (i.e. the non-human binding agent, e.g., a murine antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the binding agent. Antigen binding sites may be wild- type or modified by one or more amino acid substitutions, e.g., modified to resemble human immunoglobulins more closely. Some forms of humanized binding agents preserve all CDR sequences (for example a humanized mouse antibody which contains all six CDRs from the mouse antibody). Other forms have one or more CDRs which are altered with respect to the original binding agent, e.g., antibody. Thus, a humanized binding agent may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions.

[0311] The term "chimeric binding agent" as used herein includes a "chimeric antibody" and refers to those binding agents wherein one portion of each of the amino acid sequences of heavy and light chains is homologous to corresponding sequences in binding agents, e.g., antibodies, derived from a particular species or belonging to a particular class, while the remaining segment of the chain is homologous to corresponding sequences in another. Typically, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to sequences of antibodies derived from another. One clear advantage to such chimeric forms is that the variable region can conveniently be derived from presently known sources using readily available B cells or hybridomas from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation and the specificity is not affected by the source, the constant region being human, is less likely to elicit an immune response from a human subject when the binding agents are injected than would the constant region from a non-human source. However, the definition is not limited to this particular example.

[0312] Binding agents or fragments thereof, e.g., variable and / or constant regions, may be derived from different species, including but not limited to mouse, rat, rabbit, guinea pig and human. Immunoglobulins described herein include IgA such as IgAl or IgA2, IgGl (including allotypes with polymorphisms at amino acid position 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 IgGl antibody, more particularly an IgGl, kappa or IgGl, lambda isotype (i.e. IgGl, κ, λ), an IgG2a antibody (e.g. IgG2a, κ, λ), an IgG2b antibody (e.g. IgG2b, κ, λ), an IgG3 antibody (e.g. IgG3, κ, λ) or an IgG4 antibody (e.g. IgG4, κ, λ). Amino acid sequences described herein with respect to IgGl allotype 356D / 358M also include allotype 356E / 358L.

[0313] The term "IgG subclass modification" or "isotype modification" means an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid in a different, aligned IgG isotype. For example, because IgGl comprises a tyrosine and IgG2 a phenylalanine at amino acid position 296 according to EU numbering, a F296Y substitution in IgG2 is considered an IgG subclass modification.

[0314] As used herein, a "heterologous binding agent" includes a "heterologous antibody" and is defined in relation to a transgenic organism producing such a binding agent. This term refers to a binding agent having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic organism, and being generally derived from a species other than the transgenic organism.

[0315] As used herein, a "heterohybrid binding agent" includes a "heterohybrid antibody" and refers to a binding agent having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.

[0316] The binding agents including antibodies described herein are preferably isolated. "Isolated" as used herein, is intended to refer to a binding agent which is substantially free of other agents having different antigenic specificities (e.g., an isolated binding agent that specifically binds to CLDN18.2 and CD3 is substantially free of binding agents that specifically bind antigens other than CLDN18.2 and CD3). An isolated binding agent that specifically binds to an epitope, isoform or variant of human CLDN18.2 may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., CLDN18.2 species homologs). Moreover, an isolated binding agent may be substantially free of other cellular material and / or chemicals. The terms "antigen-binding portion" of a binding agent such as an antibody (or simply "binding portion") or "antigen-binding fragment" of a binding agent such as an antibody (or simply "binding fragment") or similar terms refer to one or more fragments of a binding agent that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of a binding agent such as an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH domains; (ii) F(ab')2fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fab' fragments, derived from a F(ab')2fragment and containing a free sulfhydryl group that may be alkylated or utilized in conjugation with an enzyme, toxin or other protein of interest, wherein the Fab' may contain a small portion of Fc; (iv) Fd fragments consisting of the VH and CH domains; (v) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (vi) dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain; (vii) isolated complementarity determining regions (CDR), and (viii) combinations of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of a binding agent such as an antibody. A further example is binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is 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. The binding-domain immunoglobulin fusion proteins are further disclosed in US 2003 / 0118592 and US 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0317] A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of usually ten to about 30 amino acids such as an scFv linker, e.g., as represented by SEQ ID NO: 2. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the 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 producing a single peptide chain with two VH and two VL domains, yielding tandem scFvs. The invention also includes multispecific molecules comprising more than one scFvs binding domain. One common flexible linking peptide is (G4S)x, wherein x may be 2, 3, 4, 5 or 6. Preferably, a linker connecting the VH and VL domain of an scFv comprises, preferably consists of, the amino acid sequence (GKPGS)xor a functional variant thereof, wherein x may be 2, 3, 4, 5, or 6. Optionally, the association of the VH and VL can be stabilized by one or more intennolecular disulfide bonds.

[0318] Fab (fragment antigen binding) antibody fragments are immunoreactive polypeptides comprising monovalent antigen-binding domains of an antibody composed of a polypeptide consisting of a heavy chain variable region (VH) and heavy chain constant region 1 (CHI) portion and a polypeptide consisting of a light chain variable region (VL) and a light chain constant region (in which the CL and CHI portions are bound together, for example by a disulfide bond between Cys residues). Preferably in the Fab fragments described herein the CHI and the CL are of human origin. In some embodiments, the CL is a kappa-type CL. In some embodiments, the CHI is derived from IgGl, preferably from human IgGl.

[0319] All antibodies and derivatives of antibodies such as antibody fragments as described herein for the purposes of the invention are encompassed by the term "antibody". The term "antibody derivatives" refers to any modified form of an antibody, e.g., a conjugate of the antibody and another agent or antibody, or an antibody fragment. Furthermore, the antibodies and derivatives of antibodies as described herein are useful for producing binding agents described herein.

[0320] Naturally occurring antibodies are generally monospecific, i.e. they bind to a single antigen. The bispecific binding agents described herein bind to a cytotoxic cell such as a T cell (by engaging the CD3 receptor) and a target cell such as a cancer cell (by engaging CLDN18.2). The binding agents described herein bind to at least two different types of antigen and are at least bispecific or multispecific such as trispecific, tetraspecific and so on.

[0321] In some embodiments, a binding agent described herein is at least trivalent. As used herein, "valent", "valence", "valencies", or other grammatical variations thereof, mean the number of antigen binding sites or binding domains in a binding agent. Antigen binding sites binding to the same antigen may recognize different epitopes or preferably the same epitope. A binding agent described herein may also have valencies of 4 or higher.

[0322] A binding agent described herein is preferably an artificial protein (including protein complexes) that may be composed of fragments of at least two different antibodies (said fragments of at least two different antibodies forming at least two different binding domains) and consequently binds to at least two different types of antigen. A binding agent described herein is engineered to simultaneously bind to an immune cell, such as an immune effector cell, in particular a T cell such as a cytotoxic cell (e.g. by binding to CD3) and a target cell like a cancer cell (by binding to the tumor-associated antigen CLDN18.2) to be destroyed.

[0323] In some embodiments, the binding agent described herein is in the format of a Fab2-scFv construct, i.e. a construct comprising two Fab fragments (each comprising VH and CHI domains on one polypeptide chain and corresponding VL und CL domains on another polypeptide chain, wherein an antigen binding domain is formed by interaction of each of said set of polypeptide chains) and an scFv moiety (comprising VH and VL domains connected to one another by a polypeptide linker on the same polypeptide chain, wherein the VH and VL interact to form an antigen binding domain). In some embodiments, the binding agent described herein is a tetramer composed of four polypeptide chains, wherein the first polypeptide chain comprises a VH derived from an immunoglobulin, e.g., an immunoglobulin with a first specificity, the second polypeptide comprises a VH derived from an immunoglobulin, e.g., an immunoglobulin with a first specificity, and an scFv moiety comprising a VH derived from an immunoglobulin, e.g., an immunoglobulin with a second specificity, and a VL derived from an immunoglobulin, e.g., an immunoglobulin with a second specificity, the third polypeptide chain comprises a VL derived from an immunoglobulin, e.g., an immunoglobulin with a first specificity, and the fourth polypeptide chain is identical to the third polypeptide chain. In some embodiments, the first and second polypeptide chains further comprise a CHI derived from an immunoglobulin, e.g., C-terminal to the VH derived from an immunoglobulin with a first specificity, and the third and fourth polypeptide chains further comprise a CL derived from an immunoglobulin. In some embodiments, the first and second polypeptide chains further comprise CH2 and CH3 (CH2-CH3) domains derived from an immunoglobulin, e.g., C-terminal to the Fab fragment and scFv moiety, respectively. Thus, in some embodiments, the binding agent described herein comprises a first polypeptide chain comprising VH-CH1 connected to CH2-CH3, a second polypeptide chain comprising VH-CH1 connected to an scFv moiety connected 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 the third polypeptide chain. In some embodiments, the second polypeptide chain interacts with the fourth polypeptide chain. In some embodiments, the first and second polypeptide chains interact, the first polypeptide chain further interacts with the third polypeptide chain, and the second polypeptide chain further interacts with the fourth polypeptide chain. In some embodiments, the CH2 on the first polypeptide chain interacts with the CH2 on the second polypeptide chain and / or the CH3 on the first polypeptide chain interacts with the CH3 on the second polypeptide chain. In some embodiments, the VH on the first polypeptide chain interacts with the VL on the third polypeptide chain to form a binding domain and / or the CHI on the first polypeptide chain interacts with the CL on the third polypeptide chain. In some embodiments, the VH on the second polypeptide chain (which is not part of the scFv moiety) interacts with the VL on the fourth polypeptide chain to form a binding domain and / or the CHI on the second polypeptide chain interacts with the CL on 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 CHI. One or both polypeptide chains comprising CH2-CH3 may comprise one or more amino acid modifications, e.g., Fc modifications, described herein, such as pl, skew, additional Fc and ablation modifications, e.g., to promote polypeptide chain interaction. According to the invention, the VH and VL of the scFv moiety are preferably connected by a peptide linker ("scFv linker"). In some embodiments, the CHI on the first and / or second polypeptide chain is connected to the CH2 on the same polypeptide chain by a peptide linker. In some embodiments, the scFv is connected to the CH2 by a peptide linker.

[0324] In some embodiments, the VH on the first polypeptide chain and the VL on the third polypeptide chain interact to form a binding domain for CLDN18.2, the additional VH on the second polypeptide chain that is not part of the scFv and the VL on the fourth polypeptide chain interact to form a binding domain for CLDN18.2, and the VH and VL of the scFv on the second polypeptide chain interact to form a binding domain for CD3.

[0325] The term "linker" refers to any means that serves to join two distinct functional units (e.g. domains or regions on a polypeptide chain). Types of linkers include, but are not limited to, chemical linkers, peptide and polypeptide linkers. The sequences of the peptide and polypeptide linkers are not limited. Peptide linkers are preferably non-immunogenic and flexible, such as those comprising serine and glycine sequences. Depending on the particular construct, the linkers may be long or short. In preferred embodiments, the scFv linker, i.e., the linker connecting VH and VL that form the scFv moiety, preferably comprises, and preferably consists of, a flexible peptide linker as described herein, preferably the amino acid sequence (GKPGS)xor a functional variant thereof, wherein x is 2, 3, 4, 5 or 6. In an even more preferred embodiment, the scFv linker comprises, and preferably consists of, the amino acid sequence (GKPGS)4(SEQ ID NO: 2) or a functional variant thereof. Preferably, the scFv moiety is connected to the CH2 on the second polypeptide chain by a peptide linker comprising the amino acid sequence (G4S)2KTHTCPPC (SEQ ID NO: 4) or a functional variant thereof.

[0326] According to the invention, a linker connecting an scFv and a CHI, preferably at the C-terminus of the CHI, preferably comprises, preferably consists of, the amino acid sequence (G4S), or a functional variant thereof, wherein x is 2, 3, 4, 5 or 6, preferably (G4S)2(SEQ ID NO: 5) or a functional variant thereof. According to the invention, a linker connecting CH2 and scFv, preferably at the N-terminus of CH2, preferably comprises, and preferably consists of, the amino acid sequence (G4S)2KTHTCPPC (SEQ ID NO: 4) or a variant thereof. According to the invention, a linker connecting CHI and CH2 preferably comprises, and preferably consists of, the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 6) or a functional variant thereof. According to the invention, CHI and scFv, e.g., on the second polypeptide chain, are connected by a peptide linker preferably comprising, preferably consisting of, the amino acid sequence (G4S)2(SEQ ID NO: 5) or a functional variant thereof. However, other linkers can be used as known in the art.

[0327] In some embodiments, the binding agent described herein comprises a first, a second, a third and a fourth polypeptide chain, wherein i) the first polypeptide chain comprises from N-terminus to C-terminus the following domains: VH(CLDN18.2)-CH1-CH2-CH3, ii) the second polypeptide chain comprises from N-terminus to C-terminus the following domains: VH(CLDN18.2)-CH1-VL(CD3)-VH(CD3)-CH2-CH3, iii) the third polypeptide chain comprises from N-terminus to C-terminus the following domains: VL(CLDN18.2)-CL, and iv) the fourth polypeptide chain is identical to the third polypeptide chain, preferably wherein VH(CLDN 18.2) on the first polypeptide chain and VL(CLDN 18.2) on the third polypeptide chain interact to form a binding domain for CLDN18.2, VH(CLDN18.2) on the second polypeptide chain and VL(CLDN18.2) on the fourth polypeptide chain interact to form a binding domain for CLDN18.2, VH(CD3) and VL(CD3) interact to form a binding domain for CD3, and the domains on the polypeptide chains preferably are connected to one another by peptide linkers as described herein.

[0328] In some embodiments, the binding agent described herein comprises: a) a first polypeptide chain comprising a VH(CLDN18.2), and CHI, CH2 and CH3 comprising 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, 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; b) a second polypeptide chain comprising a VH(CLDN 18.2) and a CH 1 as described herein, VH(CD3) and VL(CD3) connected to one another through a charged scFv linker with the amino acid sequence (GKPGS)4(SEQ ID NO: 2) and CH2 and CH3 comprising 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; c) a third polypeptide chain comprising VL(CLDN18.2) and CL and d) a fourth polypeptide chain identical to the third polypeptide chain.

[0329] In some embodiments, the binding agent described herein comprises a first, a second, a third and a fourth polypeptide chain, wherein i) the first polypeptide chain comprises from N -terminus to C-terminus:

[0330] VH(CLDN 18.2)-CHl-linker-CH2-CH3, ii) the second polypeptide chain comprises from N-terminus to C-terminus: VH(CLDN 18.2)-CHl -linker-VL(CD3)-linker-VH(CD3)-linker-CH2-CH3, iii) the third polypeptide chain comprises from N-terminus to C-terminus the following domains: VL(CLDN18.2)-CL, and iv) the fourth polypeptide chain is identical to the third polypeptide chain, preferably wherein VH(CLDN18.2) on the first polypeptide chain and VL(CLDN18.2) on the third polypeptide chain interact to form a binding domain for CLDN18.2, VH(CLDN18.2) on the second polypeptide chain and VL(CLDN18.2) on the fourth polypeptide chain interact to form a binding domain for CLDN18.2, and VH(CD3) and VL(CD3) interact to form a binding domain for CD3. In some embodiments, the binding agent described herein comprises a first, a second, a third and a fourth polypeptide chain, wherein i) the first polypeptide chain comprises from N-terminus to C-terminus: VH(CLDN1 8.2)-CHl -linker 1-CH2-CH3, ii) the second polypeptide chain comprises from N-terminus to C-terminus: VH(CLDN18.2)-CHl-linker2-VL(CD3)-linker3-VH(CD3)-linker4-CH2-CH3, iii) the third polypeptide chain comprises from N-terminus to C-terminus: VL(CLDN18.2)-CL, and iv) the fourth polypeptide chain is identical to the third polypeptide chain, wherein linker 1 comprises the amino acid sequence EPKSCDKTHTCPPCP or a functional variant thereof, linker2 comprises the amino acid sequence (G4S)xor a functional variant thereof, wherein x is 2, 3, 4, 5 or 6, preferably wherein x is 2, linker3 comprises the amino acid sequence (GKPGS)xor a functional variant thereof, wherein x is 2, 3, 4, 5 or 6, preferably wherein x is 4, and linker4 comprises the amino acid sequence (G4S)2KTHTCPPCP or a functional variant thereof, and preferably wherein VH(CLDN 18.2) on the first polypeptide chain and VL(CLDN 18.2) on the third polypeptide chain interact to form a binding domain for CLDN18.2, VH(CLDN18.2) on the second polypeptide chain and VL(CLDN18.2) on the fourth polypeptide chain interact to form a binding domain for CLDN18.2, and VH(CD3) and VL(CD3) interact to form a binding domain for CD3.

[0331] Binding agents described herein, and / or first, second, third, and fourth polypeptide chains of said binding agents described herein, may also comprise an amino acid sequence for facilitating secretion of the binding agent or polypeptide chain, such as a N-terminal secretion signal, and / or one or more epitope tags facilitating binding, purification or detection of the molecule. Preferably, the secretion signal is a signal sequence that allows a sufficient passage through the secretory pathway and / or secretion of the binding agent or the polypeptide chains thereof into the extracellular environment. Preferably, the secretion signal sequence is cleavable and is removed from the mature binding agent or polypeptide chain. The secretion signal sequence preferably is chosen with respect to the cell or organism wherein the binding agent or polypeptide chain is produced in.

[0332] The amino acid sequence of an epitope tag may be introduced to any position within the amino acid sequence of the binding agent or polypeptide chain, and may take the shape of a loop within the encoded protein structure, or it may be N-terminally or C-terminally fused to the binding agent or polypeptide chain. Preferably, the epitope tag is C-terminally fused to the binding agent or polypeptide chain. The epitope tag may contain a cleavage site that allows a removal of the tag from the binding agent or polypeptide chain. Said epitope tag can be any kind of epitope tag that is functional under native and / or denaturing conditions, preferable a histidine tag, most preferable a tag comprising six histidines.

[0333] The binding agent described herein may contain, in addition to said first, second and third binding domains one or more further binding domains which serve e.g. to enhance selectivity for tumor cells. This can be achieved e.g. by providing binding domains that bind to other antigens expressed on tumor cells.

[0334] The term "posttranslational modification" or similar terms refer to modifications of a protein, such as covalent and enzymatic modifications, that occur following protein biosynthesis. As is commonly known in the art, binding agents such as antibodies that are expressed in cells are often modified after translation. For example, posttranslational modifications of binding agents such as antibodies can occur on the amino acid side chains or at the N- or C-termini of heavy or light chains, e.g., of the first and / or the second polypeptide chain described herein. Examples of posttranslational modifications that may occur in binding agents described herein include, without limitation, cleavage of lysine at the C-terminus of a heavy chain, e.g., of the first and / or second polypeptide chain, e.g., by a carboxypeptidase; modification of glutamine or glutamic acid at the N-terminus of a heavy chain, e.g., of the first and / or second polypeptide chain, to pyroglutamic acid by pyroglutamylation; modification of glutamine or glutamic acid at the N-terminus of a light chain, e.g., of the third and / or fourth polypeptide chain, to pyroglutamic acid by pyroglutamylation; glycosylation; oxidation; deamidation; and glycation. It is known that such posttranslational modifications occur in various binding agents (Liu et al., 2008, J. Pharmacol. Sci. 97(7):2426-2447). Posttranslational modifications due to pyroglutamylation at the N-terminus and deletion of lysine at the C-terminus commonly do not have any influence on the activity of binding agents (Lyubarskaya et al., 2006, Analyt. Biochem. 348(l):24-39).

[0335] Thus, in some embodiments, a binding agent described herein may comprise one or more posttranslational modifications. In some embodiments, the one or more posttranslational modifications comprise pyroglutamylation at the N-terminus of one or more polypeptide chains of the binding agent. In some embodiments, the one or more posttranslational modifications comprise pyroglutamylation at the N-terminus of one or more VH(CLDN18.2). In some embodiments, the one or more posttranslational modifications comprise deletion of lysine at the C-terminus of the first polypeptide chain. In some embodiments, the one or more posttranslational modifications comprise deletion of lysine at the C-terminus of the second polypeptide chain.

[0336] In the context of the present invention, the binding agents described herein are preferably capable of eliciting one or more immune effector functions as described herein. Preferably, said immune effector functions are directed against cells carrying the cancer-associated antigen CLDN18.2 on their surface.

[0337] The term "immune effector functions" in the context of the present invention includes any functions mediated by components of the immune system that result e.g. in the inhibition of cancer growth and / or inhibition of cancer development, including inhibition of cancer dissemination and metastasis. Preferably, immune effector functions result in killing of cancer cells. Immune effector functions comprise complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis in the cells carrying the cancer-associated antigen, cytolysis of the cells carrying the cancer-associated antigen, and / or inhibition of proliferation of the cells carrying the cancer- associated antigen. The binding agents described herein preferably are able to recruit and redirect T cells such as CD4 and / or CDS T cells, in particular CD107a+ T cells, to disease-associated cells such as cancer cells and, thus, act through redirected T cell cytotoxicity (RTCC), i.e., the T cells upon redirection preferably kill the disease-associated cells, e.g., cancer cells. CD107a expression is known to be associated with cytolytic potential of CD4 and CDS T cells. Preferably, said CD107a+ T cells are capable to degranulate, i.e., they are capable to release cytotoxic molecules such as perforines, granzymes, etc., and may also release cytokines such as one or more of Tumor Necrosis Factor a (TNFa), interleukine-2 (IL2), Interferon γ (IFNγ) etc., thereby causing death of target cells, e.g., cancer cells the T cells are redirected to by the binding agents described herein. Binding agents may also exert an effect simply by binding to cancer-associated antigens on the surface of a cancer cell. For example, binding agents may block the function of the cancer- associated antigen or induce apoptosis just by binding to the cancer-associated antigen on the surface of a cancer cell.

[0338] The term "immune effector cell" or "effector cell" in the context of the present invention relates to a cell which exerts effector functions during an immune reaction. For example, immune effector cells comprise 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 (CTLs, CD 8+ T cells) which comprise cytolytic T cells. The term "MHC-dependent T cell" or similar terms relate to a T cell which recognizes an antigen when presented in the context of MHC and preferably exerts effector functions of T cells, e.g., killing of target cells expressing an antigen.

[0339] 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 special receptor on their cell surface called T cell receptor (TCR). The thymus is the principal organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with a distinct function.

[0340] T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages, among other functions. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells usually become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.

[0341] Cytotoxic T cells destroy virally infected cells and cancer cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein on their surface. These cells usually recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body.

[0342] All T cells have a T cell receptor (TCR) existing as a complex of several proteins. The TCR of a T cell is able to interact with immunogenic peptides (epitopes) bound to major histocompatibility complex (MHC) molecules and presented on the surface of target cells. Specific binding of the TCR triggers a signal cascade inside the T cell leading to proliferation and differentiation into a maturated effector T cell. In the majority of T cells, the actual T cell receptor is composed of two separate peptide chains, which are produced from the independent T cell receptor alpha and beta (TCRα and TCRβ) genes and are called a- and β-TCR chains. A much less common (2% of total T cells) group of T cells, the γδ T cells (gamma delta T cells) possess a distinct T cell receptor (TCR) on their surface, which is made up of one y-chain and one 5-chain. All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitors derived from hematopoietic stem cells populate the thymus and expand by cell division to generate a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CDS, and are therefore classed as double-negative (CD4-CD8-) cells. As they progress through their development they become double-positive thymocytes (CD4+CD8+), and finally mature to single- positive (CD4+CD8- or CD4-CD8+) thymocytes that are then released from the thymus to peripheral tissues.

[0343] As used herein, the term "NK cell" or "Natural Killer cell" refers to a subset of peripheral blood lymphocytes defined by the expression of CDS 6 or CD 16 and the absence of the T cell receptor.

[0344] MHC molecules in humans are normally referred to as HLA (human leukocyte antigen) molecules. There are two principal classes of MHC molecules: class I and class II. MHC class I antigens are found on nearly all nucleated cells of the body. The primary function of this class of MHC molecules is to display (or present) peptide fragments of intracellular proteins to CTLs. Based on this display, CTLs will attack those displaying MHC-bound peptides, including disease-associated peptides (antigens) such as cancer antigens. CD8-positive T cells are usually cytotoxic (therefore named cytotoxic T cells = CTL), recognize peptides of 9 to 10 amino acids which are intracellularly processed from proteins of any subcellular localization and which are presented on the cellular surface by MHC class I molecules. Thus, the surface expression of MHC class I molecules plays a crucial role in determining the susceptibility of target cells to CTLs.

[0345] The binding agents described herein may be conjugated to a therapeutic moiety or agent, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radioisotope. A cytotoxin or cytotoxic agent includes any agent that is detrimental to and, in particular, kills cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin 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, fludarabin, 5-fhiorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and anti-mitotic agents (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic agent or a 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.

[0346] Binding agents also can be conjugated to a radioisotope, e.g., iodine-131, yttrium-90 or indium- il l , to generate cytotoxic radiopharmaceuticals.

[0347] Techniques for conjugating such therapeutic moiety to binding agents are well known, see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds. ), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds. ), pp. 475-506 (1985); "Analysis, Results, And Future 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 Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982).

[0348] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.

[0349] As used herein, "isotype switching" refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.

[0350] The term "rearranged" as used herein refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH or VL domain, respectively. A rearranged immunoglobulin (antibody) gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer / nonamer homology element.

[0351] The term "unrearranged" or "germline configuration" as used herein in reference to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.

[0352] In some embodiments, a binding agent described herein has the ability of binding to CLDN18.2, i.e. the ability of binding to, and preferably binds to, an epitope present in CLDN18.2, preferably an epitope located within the extracellular domains of CLDN18.2, in particular the first extracellular loop, preferably amino acid positions 29 to 78 of CLDN18.2. In particular embodiments, an agent having the ability of binding to CLDN18.2 binds to an epitope on CLDN18.2 which is not present on CLDN 18.1.

[0353] An agent having the ability of binding to CLDN18.2 preferably binds to CLDN18.2, preferably of human, mouse and / or cynomolgus, but not to CLDN 18.1, preferably of human, mouse and / or cynomolgus. Preferably, an agent binding to CLDN18.2 does not bind to CLDN9, preferably of human, mouse and / or cynomolgus. Preferably, an agent having the ability of binding to CLDN 18.2 is specific for CLDN 18.2. Preferably, an agent having the ability of binding to CLDN 18.2 binds to CLDN 18.2 expressed on the cell surface. In particular preferred embodiments, an agent having the ability of binding to CLDN18.2 binds to native epitopes of CLDN 18.2 present on the surface of living cells.

[0354] In some embodiments, a binding domain comprises an antibody fragment. 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 of the light chain variable region (VL). In some embodiments, said one or more of the complementarity-determining regions (CDRs) are selected from a 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 of the light chain variable region (VL).

[0355] In some embodiments, a binding domain comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs as described herein comprises said CDRs together with their intervening framework regions. Preferably, the portion will also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of binding agents made by recombinant DNA techniques may result in the introduction of residues N- or C-terminal to the variable regions encoded by linkers introduced to facilitate cloning or other manipulation steps, including the introduction of linkers to join variable regions to further protein sequences including immunoglobulin heavy chains, other variable regions or protein labels.

[0356] In some embodiments, a binding domain comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs as described herein comprises said CDRs in a human antibody framework.

[0357] The exact identification of the CDR regions depends on the calculation method used for determining involved amino acid residues. For example, according to Kabat et al. (supra), variable regions generally encompass amino acid residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the VL and around about 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) in the VH; variable regions may also encompass those residues forming a hypervariable loop (e.g. residues 26-32 (CDR1), 50-52 (CDR2) and 91-96 (CDR3) in the VL and 26-32 (CDR1), 53-55 (CDR2) and 96-101 (CDR3) in the VH (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917)).

[0358] A person skilled in the art will understand that the exact identification of the CDR positions within the sequences disclosed herein may be slightly different depending on the numbering system used as shown in the following Table 1 (see Lafranc et al., Dev. Comp. Immunol. 27(l):55-77 (2003):

[0359] Table 1 Thus, the CDR sequences disclosed herein also comprise variants thereof derived according to different numbering systems. Accordingly, the disclosure of each VH is a disclosure of the CDRs (e.g., CDR1, CDR2 and CDR3) derivable therefrom and the disclosure of each VL is a disclosure of the CDRs (e.g., CDR1, CDR2 and CDR3) derivable therefrom.

[0360] Throughout the present specification, the Kabat numbering system is used when referring to residues in variable regions (approximately, residues 1-107 of the light chain and residues 1-113 of the heavy chain) of binding domains for CD3 or CLDN18.2, and the EU numbering system for CHI and CH2-CH3 (optionally including the hinge) regions, as described herein.

[0361] In some embodiments, a binding domain for CLDN18.2 of a binding agent described herein comprises a VH comprising complementarity determining regions CDR1, CDR2 and / or CDR3 identified within the amino acid sequence of SEQ ID NO: 16.

[0362] In some embodiments, a binding domain for CLDN18.2 of a binding agent described herein comprises a VL comprising complementarity determining regions CDR1, CDR2 and / or CDR3 identified within the amino acid sequence represented by SEQ ID NO: 17.

[0363] In a preferred embodiment, a binding domain for CLDN18.2 of a binding agent described herein comprises the following set of CDRs: the VH comprises a CDR3 comprising the sequence set forth in SEQ ID NO: 12 or a functional variant thereof, and the VL comprises a CDR3 comprising the sequence set forth in SEQ ID NO: 15 or a functional variant thereof.

[0364] In some embodiments, the VH further comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 10 or a functional variant thereof and / or a CDR2 comprising the sequence set forth in SEQ ID NO: 11 or a functional variant thereof, and / or the VL further comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 13 or a functional variant thereof, and / or a CDR2 comprising the sequence set forth in SEQ ID NO: 14 or a functional variant thereof.

[0365] In a preferred embodiment, a binding domain for CLDN18.2 of a binding agent described herein comprises the following set of CDRs: the VH comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 10 or a functional variant thereof, a CDR2 comprising the sequence set forth in SEQ ID NO: 11 or a functional variant thereof and a CDR3 comprising the sequence set forth in SEQ ID NO: 12 or a functional variant thereof and the VL comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 13 or a functional variant thereof, a CDR2 comprising the sequence set forth in SEQ ID NO: 14 or a functional variant thereof and a CDR3 comprising the sequence set forth in SEQ ID NO: 15 or a functional variant thereof. Preferably, the VH comprises CDR1, 2 and 3 of SEQ ID NOs: 10, 11 and 12, and the VL comprises CDR1, 2 and 3 of SEQ ID NOs: 13, 14 and 15.

[0366] In some embodiments, said heavy and light chain variable regions comprise said complementarity determining regions interspersed within framework regions. In some embodiments, each variable region comprises three complementarity determining regions (CDR1, 2, and 3) and four framework regions (FR1, 2, 3, and 4). In some embodiments, said complementarity determining regions and said framework regions are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0367] In a preferred embodiment, a binding domain for CLDN18.2 of a binding agent described herein comprises a VH(CLDN18.2) comprising the amino acid sequences represented by SEQ ID NO: 16 or a functional variant thereof.

[0368] In a preferred embodiment, a binding domain for CLDN18.2 of a binding agent described herein comprises VL(CLDN18.2) comprising the amino acid sequences represented by SEQ ID NO: 17 or a functional variant thereof.

[0369] In a particularly preferred embodiment, a binding domain for CLDN18.2 of a binding agent described herein comprises the following combination of VH(CLDN18.2) and VL(CLDN18.2): the VH(CLDN18.2) comprises an amino acid sequence represented by SEQ ID NO: 16 or a functional variant thereof and the VL(CLDN18.2) comprises an amino acid sequence represented by SEQ ID NO: 17 or a functional variant thereof.

[0370] In a preferred embodiment, the framework regions of VH and VL domains present within a binding agent described herein may comprise amino acid changes but retain at least 80%, 85% or 90% identity to a human germline sequence. In further embodiments, a binding domain for CLDN18.2 of a binding agent described herein comprises heavy and light chain variable regions of an antibody which (i) competes for CLDN 18.2 binding with an antibody comprising heavy and light chain variable regions as described above and / or (ii) has the specificity for CLDN 18.2 of an antibody comprising heavy and light chain variable regions as described above.

[0371] In some embodiments, a binding domain for CLDN 18.2 of a binding agent described herein has the format of a Fab molecule as described herein. In this embodiment, a VH(CLDN18.2) is part of the first and the second polypeptide chains as described herein and a VL(CLDN 18.2) is part of the third polypeptide chain and the fourth polypeptide chain identical to the third polypeptide chain of a binding agent described herein.

[0372] It is to be understood that the binding domains binding to CLDN18.2 of a binding agent described herein in the Fab2-scFv format may be identical or essentially identical and thus may bind to identical or essentially identical epitopes of CLDN18.2. Thus, both binding domains binding to CLDN18.2 of a binding agent described herein in the Fab2-scFv format may correspond or correspond essentially to one of the binding domains binding to CLDN 18.2 described herein.

[0373] Preferably, the binding domain for CD3 is capable of specifically recognizing human CD3 in the context of other TCR subunits as present on activated primary human T cells expressing the TCR in its native configuration.

[0374] In some embodiments, a binding domain for CD3 of a binding agent described herein comprises a VH comprising CDR1 , CDR2 and / or CDR3 identified within the amino acid sequence of SEQ ID NO: 25.

[0375] In some embodiments, a binding domain for CD3 of a binding agent described herein comprises a VH comprising the following set of CDR1, CDR2 and CDR3:

[0376] CDR1: SEQ ID NO: 18 or a functional variant thereof, CDR2: SEQ ID NO: 23 or a functional variant thereof, CDR3: SEQ ID NO: 19 or a functional variant thereof.

[0377] In a preferred embodiment, a binding domain for CD3 of a binding agent described herein comprises a VL comprising CDR1, CDR2 and / or CDR3 identified within the amino acid sequence according to SEQ ID NO: 24. In a preferred embodiment, a binding domain for CD3 of a binding agent described herein comprises a VL comprising the following set of CDR1, CDR2 and CDR3:

[0378] CDR1: SEQ ID NO: 20 or a functional variant thereof, CDR2: SEQ ID NO: 21 or a functional variant thereof, CDR3: SEQ ID NO: 22 or a functional variant thereof.

[0379] In a preferred embodiment, a binding domain for CD3 of a binding agent described herein comprises the following combination of VH and VL each comprising a set of CDR1, CDR2 and CDR3:

[0380] VH: CDR1 : SEQ ID NO: 18 or a functional variant thereof, CDR2: SEQ ID NO: 23 or a functional variant thereof, CDR3: SEQ ID NO: 19 or a functional variant thereof, VL: CDR1: SEQ ID NO: 20 or a functional variant thereof, CDR2: SEQ ID NO: 21 or a functional variant thereof, CDR3: SEQ ID NO: 22 or a functional variant thereof.

[0381] In a preferred embodiment, a binding domain for CD3 of a binding agent described herein comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 25 or a functional variant thereof.

[0382] In a preferred embodiment, a binding domain for CD3 of a binding agent described herein comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 24 or a functional variant thereof.

[0383] In a preferred embodiment, a binding domain for CD3 of a binding agent described herein comprises the following VH and a VL: the VH comprises or consists of an amino acid sequence according to SEQ ID NO: 25 or a functional variant thereof and the VL comprises or consists of an amino acid sequence according to SEQ ID NO: 24 or a functional variant thereof.

[0384] In some embodiments, the binding domain for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 26 or a functional variant thereof.

[0385] In some embodiments, the CHI of the first and / or second polypeptide chain of a binding agent described herein is derived from IgG, preferably IgGl, more preferably human IgGl. In some embodiments, the CH2 and CH3 domains of the first and / or second polypeptide chain of a binding agent described herein are derived from IgG, preferably IgGl, more preferably human IgGl .

[0386] In some embodiments, the CL of the third and / or fourth polypeptide chain of a binding agent described herein is derived from Igκ or Igλ, preferably Igκ, more preferably human Igκ.

[0387] In some embodiments, the first polypeptide chain of a binding agent described herein comprises an amino acid sequence represented by SEQ ID NO: 7 or a functional variant thereof. In some embodiments, the second polypeptide chain of a binding agent described herein comprises an amino acid sequence represented by SEQ ID NO: 8 or a functional variant thereof. In some embodiments, the third and / or fourth polypeptide chain of a binding agent described herein comprises an amino acid sequence represented by SEQ ID NO: 9 or a functional variant thereof.

[0388] In a preferred embodiment, a binding agent described herein comprises first, second, third, and fourth polypeptide chains comprising the following amino acid sequences: the first polypeptide chain comprises SEQ ID NO: 7 or a functional variant thereof, the second polypeptide chain comprises SEQ ID NO: 8 or a functional variant thereof, and the third polypeptide chain comprises SEQ ID NO: 9 or a functional variant thereof, wherein the fourth polypeptide chain is identical to the third polypeptide chain.

[0389] In some embodiments, a binding agent described herein comprises at least two binding domains for CLN 18.2 and at least one binding domain for CD3. In some embodiments, the first polypeptide chain of a binding agent described herein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27 or a functional variant thereof. In some embodiments, the second polypeptide chain of a binding agent described herein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28 or a functional variant thereof. In some embodiments, the third polypeptide chain of a binding agent described herein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29 or a functional variant thereof. In some embodiments, the fourth polypeptide chain of a binding agent described herein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29 or a functional variant thereof. In some embodiments, a binding agent described herein comprising at least two binding domains binding to CLDN18.2 and at least one binding domain binding to CD3 comprises a set of first, second, third and fourth polypeptide chains according to SEQ ID NOs: 27, 28, 29, and 29. It is to be understood that the binding agents described herein may be delivered to a patient by administering a nucleic acid such as RNA encoding the agent and / or by administering a host cell comprising a nucleic acid such as RNA encoding the agent. If the binding agent comprises more than one polypeptide chain, the different polypeptide chains may be encoded on the same nucleic acid or on different nucleic acids, e.g., a set of nucleic acids. Thus, a nucleic acid to be administered may be a mixture of different nucleic acid molecules such as a set of nucleic acids. A nucleic acid or set of nucleic acids encoding a binding agent, e.g., when administered to a subject such as a patient, may be present in naked form or in a suitable delivery vehicle such as in the form of liposomes or nanoparticles or viral particles, or within a host cell. The nucleic acid or set of nucleic acids provided can produce the agent over extended time periods in a sustained manner mitigating the instability at least partially observed for therapeutic antibodies. Nucleic acids or sets of nucleic acids to be delivered to a patient can be produced by recombinant means. If a nucleic acid or set of nucleic acids is administered to a patient without being present within a host cell, it is preferably taken up by cells of the patient for expression of the binding agent encoded by the nucleic acid(s). If a nucleic acid or set of nucleic acids is administered to a patient while being present within a host cell, it is preferably expressed by the host cell within the patient so as to produce the binding agent encoded by the nucleic acid(s).

[0390] The term "recombinant" in the context of the present invention means "made through genetic engineering". Preferably, a "recombinant object" such as a recombinant nucleic acid in the context of the present invention is not occurring naturally.

[0391] 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) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

[0392] The term "nucleic acid", as used herein, is intended to include DNA and RNA such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0393] Nucleic acids or sets of nucleic acids may be comprised in a vector. Sets of nucleic acids may also be comprised in sets of vectors such that each nucleic acid of the set of nucleic acids is comprised in a vector. The term "vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.

[0394] In the context of the present invention, the term "RNA" relates to a molecule which comprises ribonucleotide residues and preferably is entirely or substantially composed of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide with a hydroxyl group at the 2 -position of a 0- 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, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0395] According to the present invention, the term "RNA" includes and preferably relates to "mRNA" which means "messenger RNA" and relates to a "transcript" which may be produced using DNA as template and encodes a peptide or protein. mRNA typically comprises a 5' non translated region (5'-UTR), a protein or peptide coding region and a 3' non translated region (3'-UTR). mRNA has a limited halftime in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In some embodiments, of the invention, the RNA is obtained by in vitro transcription or chemical synthesis. The in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially available.

[0396] In some embodiments, of the present invention, RNA is self-replicating RNA, such as single stranded self-replicating RNA. In some embodiments, the self-replicating RNA is single stranded RNA of positive sense. In some embodiments, the self-replicating RNA is viral RNA or RNA derived from viral RNA. In some embodiments, the self-replicating RNA is alphaviral genomic RNA or is derived from alphaviral genomic RNA. Alphaviral RNA may act as mRNA, as is known in the art. In some embodiments, the self-replicating RNA is a viral gene expression vector. In some embodiments, the virus is Semliki forest virus. In some embodiments, the self-replicating RNA contains one or more transgenes at least one of said transgenes encoding the binding agent described herein. In some embodiments, if the RNA is viral RNA or derived from viral RNA, the transgenes may partially or completely replace viral sequences such as viral sequences encoding structural proteins. In some embodiments, the self-replicating RNA is in vitro transcribed RNA.

[0397] In order to increase expression and / or stability of the RN A used according to the present invention, it may be modified, preferably without altering the sequence of the expressed peptide or protein.

[0398] The term "modification" in the context of RNA as used according to the present invention includes any modification of RNA which is not naturally present in said RNA.

[0399] In some embodiments, of the invention, the RNA used according to the invention does not have uncapped 5 -triphosphates. Removal of such uncapped 5 '-triphosphates can be achieved by treating RNA with a phosphatase.

[0400] The RNA according to the invention may have modified naturally occurring or synthetic ribonucleotides in order to increase its stability and / or decrease cytotoxicity and / or immunogenicity. For example, in some embodiments, in the RNA used according to the invention 5-methylcytidine is substituted partially or completely, preferably completely, for cytidine. Alternatively or additionally, in some embodiments, in the RNA used according to the invention pseudouridine is substituted partially or completely, preferably completely, for uridine.

[0401] In some embodiments, the term "modification" relates to providing an RNA with a 5'-cap or 5'- cap analog. The term "5'-cap" refers to a cap structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via an unusual 5* to 5'- triphosphate linkage. In some embodiments, this guanosine is methylated at the 7-position. The term "conventional 5*-cap" refers to a naturally occurring RNA 5'-cap, preferably to the 7- methylguanosine cap (m7G). In the context of the present invention, the term "5'-cap" includes a 5'-cap analog that resembles the RNA cap structure and is modified to possess the ability to stabilize RNA if attached thereto, preferably in vivo and / or in a cell.

[0402] Providing an RNA with a 5'-eap or 5'-cap analog may be achieved by in vitro transcription of a DNA template in the presence of said 5'-cap or 5'-cap analog, wherein said 5'-cap is co- transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5'-cap may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.

[0403] The RNA may comprise further modifications. For example, a further modification of the RNA used in the present invention may be an extension or truncation of the naturally occurring poly(A) tail or an alteration of the 5'- or 3 '-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA, for example, the insertion of one or more, preferably two copies of a 3 '-UTR derived from a globin gene, such as alpha2-globin, alpha 1- globin, beta-globin, preferably beta-globin, more preferably human beta-globin.

[0404] Therefore, in order to increase stability and / or expression of the RNA used according to the present invention, it may be modified so as to be present in conjunction with a poly- A sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200 and especially 100 to 150 adenosine residues. In an especially preferred embodiment, the poly- A sequence has a length of approximately 120 adenosine residues. In addition, incorporation of two or more 3'-non translated regions (UTR) into the 3'-non translated region of an RNA molecule can result in an enhancement in translation efficiency. In one particular embodiment, the 3 '-UTR is derived from the human β-globin gene.

[0405] Preferably, RNA if delivered to, i.e. transfected into, a cell, in particular a cell present in vivo, expresses the protein, peptide or antigen it encodes.

[0406] The term "transfection" relates to the introduction of nucleic acids, in particular RNA, into a cell. For 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 cell, wherein the cell may be present in a subject, e.g., a patient. Thus, according to the present invention, a cell for transfection of a nucleic acid described herein can be present in "vitro or in vivo, e.g. the cell can form part of an organ, a tissue and / or an organism of a patient. According to the invention, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. RNA can be transfected into cells to transiently express its coded protein.

[0407] The term "stability" of RNA relates to the "half-life" of RNA. "Half-life" relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules. In the context of the present invention, the half-life of an RNA is indicative for the stability of said RNA. The half-life of RNA may influence the "duration of expression" of the RNA. It can be expected that RNA having a long half-life will be expressed for an extended time period.

[0408] In the context of the present invention, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be translated into protein. According to the present invention, the term "transcription" comprises "in vitro transcription", wherein the term "in vitro transcription" relates to a process wherein RNA, in particular mRNA, is in vitro synthesized in a cell-free system, preferably using appropriate cell extracts. Preferably, cloning vectors are applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present invention encompassed by the term "vector".

[0409] The term "translation" according to the invention relates to the process in the ribosomes of a cell by which a strand of messenger RNA directs the assembly of a sequence of amino acids to make a peptide or protein.

[0410] The term "expression" is used according to the invention in its most general meaning and comprises the production of RNA and / or peptides or proteins, e.g. by transcription and / or translation. With respect to RNA, the term "expression" or "translation" relates in particular to the production of peptides or proteins. It also comprises partial expression of nucleic acids. Moreover, expression can be transient or stable. According to the invention, the term expression also includes an "aberrant expression" or "abnormal expression". "Aberrant expression" or "abnormal expression" means according to the 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 certain protein, e.g., a tumor antigen. An increase in expression refers to an increase by at least 10%, in particular at least 20%, at least 50% or at least 100%, or more. In some embodiments, expression is only found in a diseased tissue, while expression in a healthy tissue is repressed.

[0411] The term "specifically expressed" means that a protein is essentially only expressed in a specific tissue or organ. For example, a tumor antigen specifically expressed in gastric mucosa means that said protein is primarily expressed in gastric mucosa and is not expressed in other tissues or is not expressed to a significant extent in other tissue or organ types. Thus, a protein that is exclusively expressed in cells of the gastric mucosa and to a significantly lesser extent in any other tissue, such as testis, is specifically expressed in cells of the gastric mucosa. In some embodiments, a tumor antigen may also be specifically expressed under normal conditions in more than one tissue type or organ, such as in 2 or 3 tissue types or organs, but preferably in not more than 3 different tissue or organ types. In this case, the tumor antigen is then specifically expressed in these organs. For example, if a tumor antigen is expressed under normal conditions preferably to an approximately equal extent in lung and stomach, said tumor antigen is specifically expressed in lung and stomach.

[0412] According to the invention, the term "RNA encoding" means that RNA, if present in the appropriate environment, preferably within a cell, can be expressed to produce a protein or peptide it encodes.

[0413] Some aspects of the invention rely on the adoptive transfer of host cells which are transfected in vitro with a nucleic acid such as RNA encoding a binding agent described herein and transferred to recipients such as patients, preferably after ex vivo expansion from low precursor frequencies to clinically relevant cell numbers. The host cells used for treatment according to the invention may be autologous, allogeneic, or syngeneic to a treated recipient.

[0414] The term "autologous" is used to describe anything that is derived from the same subject For example, "autologous transplant" refers to a transplant of tissue or organs derived from the same subject. Such procedures are advantageous because they overcome the immunological barrier which otherwise results in rejection. The term "allogeneic" is used to describe anything that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.

[0415] The term "syngeneic" is used to describe anything that is derived from individuals or tissues having identical genotypes, i.e., identical twins or animals of the same inbred strain, or their tissues.

[0416] The term "heterologous" is used to describe something consisting of multiple different elements. As an example, the transfer of one individual's bone marrow into a different individual constitutes a heterologous transplant. A heterologous gene is a gene derived from a source other than the subject.

[0417] The term "peptide" according to the invention comprises oligo- and polypeptides and refers to substances comprising two 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 more, preferably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acids joined covalently by peptide bonds. The term "protein" refers to large peptides, preferably to peptides with more than 100 amino acid residues, but in general the terms "peptides" and "proteins" are synonyms and are used interchangeably herein.

[0418] The teaching given herein with respect to specific amino acid sequences, e.g. those shown in the sequence listing, is to be construed so as to also relate to variants of said specific sequences resulting in sequences which are functionally equivalent to said specific sequences, e.g. amino acid sequences exhibiting properties identical or similar to those of the specific amino acid sequences. One important property is to retain binding to a target or to sustain effector functions. Preferably, a sequence which is a variant with respect to a specific sequence, when it replaces the specific sequence in an antibody retains binding of said antibody to CLDN18.2 and / or CD3 and preferably functions of said antibody as described herein. Furthermore, preferably, a sequence which is a variant with respect to a specific sequence, when it replaces the specific sequence in a binding agent retains binding of said binding agent to CLDN18.2 and / or CD3 and preferably functions of said binding agent as described herein, e.g. cytotoxic T-cell mediated lysis.

[0419] For example, the sequences shown in the sequence listing can be modified so as to remove one or more, preferably all free cysteine residues, in particular by replacing the cysteine residues by amino acids other than cysteine, preferably serine, alanine, threonine, glycine, tyrosine, tryptophan, leucine or methionine.

[0420] It will be appreciated by those skilled in the art that in particular the sequences of the CDR, hypervariable and variable regions can be modified without losing the ability to bind CLDN18.2 and / or CD3. For example, CDR regions will be either identical or highly homologous to the regions specified herein. By "highly homologous" it is contemplated that from 1 to 5, preferably from 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 modified so that they show substantial homology with the regions specifically disclosed herein. In some embodiments, the variable region sequences only deviate in the framework sequences from the variable region sequences specifically disclosed herein.

[0421] The binding agents described herein can be produced either intracellularly (e.g. in the cytosol, in the periplasma or in inclusion bodies) and then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g. in the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified. Methods and reagents used for the recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods of induction of protein expression, culture conditions, and the like, are known in the art. Similarly, protein isolation and purification techniques are well known to the skilled person.

[0422] The term "cell" or "host cell" preferably relates to an intact cell, i.e. a cell with an intact membrane that has not released its normal intracellular components such as enzymes, organelles, or genetic material. An intact cell preferably is a viable cell, i.e. a living cell capable of carrying out its normal metabolic functions. Preferably said term relates according to the invention to any cell which can be transfected with an exogenous nucleic acid. Preferably, the cell when transfected with an exogenous nucleic acid and transferred to a recipient can express 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 strains of Escherichia coh, Proteus, and Pseudomonas, and gram-positive bacterial strains such as strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cells include cells from species of Trichoderma, Neurospora, and Aspergillus. Suitable yeast cells include cells from species of Saccharomyces (for example Saccharomyces cerevisiae), Schizosaccharomyces (for example Schizosaccharomyces pombe), Pichia (for example Pichia pastoris and Pichia methanolica), and Hansenula. Suitable mammalian cells include for example CHO cells, BHK cells, HeLa cells, COS cells, 293 HEK and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for the expression of heterologous proteins can be used as well. Mammalian cells are particularly preferred for adoptive transfer, such as cells from humans, mice, hamsters, pigs, goats, and primates. The cells may be derived from a large number of tissue types and include primary cells and cell lines such as cells of the immune system, in particular antigen-presenting cells such as dendritic cells and T cells, stem cells such as hematopoietic stem cells and mesenchymal stem cells and other cell types. An antigen-presenting cell is a cell that displays antigen in the context of major histocompatibility complex on its surface. T cells may recognize this complex using their T-cell receptor (TCR).

[0423] The ability of antibodies and other binding agents to bind an antigen can be determined using standard binding assays (e.g., ELISA, Western Blot, immunofluorescence and flow cytometric analysis).

[0424] Binding agents described herein also can be tested in an in vivo model (e.g. in immunodeficient mice carrying xenografted tumors inoculated with cell lines expressing CLDN18.2) to determine their efficacy in controlling growth of CLDN18.2-expressing tumor cells.

[0425] "Reduce", "decrease" or "inhibit" as used herein means an overall decrease or the ability to cause an overall decrease, preferably of 5% or greater, 10% or greater, 20% or greater, more preferably of 50% or greater, and most preferably of 75% or greater, in the level, e.g. in the level of expression or in the level of proliferation of cells.

[0426] Terms such as "increase" or "enhance" preferably relate to an increase or enhancement by about at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more.

[0427] Antibody-dependent cell-mediated cytotoxicity

[0428] ADCC describes the cell-killing ability of effector cells as described herein, in particular lymphocytes, which preferably requires the target cell being marked by an antibody. ADCC preferably occurs when antibodies bind to antigens on tumor cells and the antibody Fc domains engage Fc receptors (FcR) on the surface of immune effector cells. Several families of Fc receptors have been identified, and specific cell populations characteristically express defined Fc receptors. ADCC can be viewed as a mechanism to directly induce a variable degree of immediate tumor destruction that leads to antigen presentation and the induction of tumor-directed T-cell responses. Preferably, in vivo induction of ADCC will lead to tumor-directed T-cell responses and host-derived antibody responses.

[0429] Antibody-dependent cell-mediated phagocytosis

[0430] ADCP is one mechanism of action of many antibody therapies. It is defined as a highly regulated process by which antibodies eliminate bound targets via connecting their Fc domain to specific receptors on phagocytic cells, and eliciting phagocytosis. ADCP can be mediated by monocytes, macrophages, neutrophils, and dendritic cells, through FcγRIIa, FcγRI, and FcγRIIIa, of which FcγRIIa (CD32a) on macrophages represent the predominant pathway.

[0431] ADCP preferably occurs when non-specific phagocytic cells that express FcγRs recognize antibody that is bond to target cells such as diseased cells including tumor cells and subsequently cause phagocytosis of the target cells such as the diseased cells including tumor cells. ADCP also provides for stimulation of downstream adaptive immune responses by facilitating antigen presentation or by stimulating the secretion of inflammatory mediators. ADCP may be improved in vivo by simultaneous treatment with immunomodulatory agents. The Fc receptor-dependent function of ADCP provides mechanisms for clearance of virus and virus-infected cells, as well as for stimulation of downstream adaptive immune responses by facilitating antigen presentation, or by stimulating the secretion of inflammatory mediators.

[0432] Complement-dependent cytotoxicity

[0433] CDC is yet another cell-killing method that can be directed by antibodies. IgM is the most effective isotype for complement activation. IgGl and IgG3 are also both very effective at directing CDC via the classical complement-activation pathway. Preferably, in this cascade, the formation of antigen-antibody complexes results in the uncloaking of multiple Clq binding sites in close proximity on the CH2 domains of participating antibody molecules such as IgG molecules (Clq is one of three subcomponents of complement Cl). Preferably these uncloaked Clq binding sites convert the previously low-affinity Clq-IgG interaction to one of high avidity, which triggers a cascade of events involving a series of other complement proteins and leads to the proteolytic release of the effector-cell chemotactic / activating agents C3a and C5a. Preferably, the complement cascade ends in the formation of a membrane attack complex, which creates pores in the cell membrane that facilitate free passage of water and solutes into and out of the cell.

[0434] Chimerization

[0435] Non-labeled murine antibodies are highly immunogenic in man when repetitively applied leading to reduction of the therapeutic effect. The main immunogenicity is mediated by the heavy chain constant regions. The immunogenicity of binding agents derived from murine antibodies in man can be reduced or completely avoided if respective binding agents are chimerized or humanized. Chimeric binding agents are binding agents, the different portions of which are derived from different animal species, such as those having a variable region derived from a murine antibody and a human immunoglobulin constant region. Chimerization of binding agents is achieved by joining of the variable regions of the murine antibody heavy and light chain with the constant region of human heavy and light chain (e.g. as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, chimeric binding agents are generated by joining human kappa-light chain constant region to murine light chain variable region. In an also preferred embodiment, chimeric binding agents can be generated by joining human lambda-light chain constant region to murine light chain variable region. The preferred heavy chain constant regions for generation of chimeric binding agents are IgGl, IgG3 and IgG4. Other preferred heavy chain constant regions for generation of chimeric binding agents are IgG2, IgA, IgD and IgM.

[0436] Humanization

[0437] Binding agents such as antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant binding agents, e.g., antibodies, that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U. S. A. 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V (D) J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody at individual evenly across the variable region.

[0438] Agent stabilizing or increasing expression of claudin 18.2 (CLDN18.2)

[0439] It is demonstrated herein that administering an agent stabilizing or increasing expression of CLDN18.2 supports the efficacy of a bispecific binding agent as described herein. In some embodiments, provided herein is a combination therapy for effectively treating and / or preventing cancer diseases comprising administering to a patient a bispecific binding agent as described herein and an agent stabilizing or increasing expression of CLDN18.2. The agent stabilizing or increasing expression of CLDN18.2 may be administered prior to, simultaneously with or following administration of the bispecific binding agent, or a combination thereof.

[0440] Certain chemotherapeutic agents, for example gemcitabine, oxaliplatin, and 5-fluorouracil were shown to upregulate existing CLDN18.2 expression levels in cancer cells (W02013 / 174510; Tiireci 0. et al. (2019) Oncolmmunology, 8:1).

[0441] The term "agent stabilizing or increasing expression of CLDN18.2" refers to an agent or a combination of agents the provision of which to cells results in increased RNA and / or protein levels of CLDN18.2, preferably in increased levels of CLDN18.2 protein on the cell surface, compared to the situation where the cells are not provided with the agent or the combination of agents. Preferably, the cells are cancer cells, in particular cancer cells expressing CLDN18.2, The term "agent stabilizing or increasing expression of CLDN18.2" refers, in particular, to an agent or a combination of agents the provision of which to cells results in a higher density of CLDN18.2 on the surface of said cells compared to the situation where the cells are not provided with the agent or the combination of agents. "Stabilizing expression of CLDN18.2" includes, in particular, the situation where the agent or the combination of agents prevents a decrease or reduces a decrease in expression of CLDN18.2, e.g. expression of CLDN18.2 would decrease without provision of the agent or the combination of agents and provision of the agent or the combination of agents prevents said decrease or reduces said decrease of CLDN18.2 expression. "Increasing expression of CLDN18.2" includes, in particular, the situation where the agent or the combination of agents increases expression of CLDN18.2, e.g. expression of CLDN18.2 would decrease, remain essentially constant or increase without provision of the agent or the combination of agents and provision of the agent or the combination of agents increases CLDN 18.2 expression compared to the situation without provision of the agent or the combination of agents so that the resulting expression is higher compared to the situation where expression of CLDN18.2 would decrease, remain essentially constant or increase without provision of the agent or the combination of agents. In some embodiments, the term "agent stabilizing or increasing expression of CLDN18.2" includes chemotherapeutic agents or combinations of chemotherapeutic agents such as cytostatic agents. In some embodiments, the agent stabilizing or increasing expression of CLDN18.2 may be a cytotoxic and / or cytostatic agent.

[0442] In some embodiments, the term "agent stabilizing or increasing expression of CLDN18.2" relates to an agent or a combination of agents such a cytostatic compound or a combination of cytostatic compounds the provision of which to cells, in particular cancer cells, results in the cells being arrested in or accumulating in one or more phases of the cell cycle, preferably in one or more phases of the cell cycle other than the Gl- and GO-phases, preferably other than the G1 -phase, preferably in one or more of the G2- or S-phase of the cell cycle, or a combination thereof, or a combination of the S-phase or the G2-phase with the Gl-phase such as the G1 / G2-, S / G2-, G2- or S-phase of the cell cycle. The term "cells being arrested in or accumulating in one or more phases of the cell cycle" means that the percentage of cells which are in said one or more phases of the cell cycle increases. Each cell goes through a cycle comprising four phases in order to replicate itself. The first phase called Gl is when the cell prepares to replicate its chromosomes. The second stage is called S, and in this phase DNA synthesis occurs and the DNA is duplicated. The next phase is the G2 phase, when the RNA and protein duplicate. The final stage is the M stage, which is the stage of actual cell division. In this final stage, the duplicated DNA and RNA split and move to separate ends of the cell, and the cell actually divides into two identical, functional cells. Chemotherapeutic agents which are DNA damaging agents usually result in an accumulation of cells in the Gl and / or G2 phase. Chemotherapeutic agents which block cell growth by interfering with DNA synthesis such as antimetabolites usually result in an accumulation of cells in the S- phase. Examples of these drugs are 6-mercaptopurine and 5-fluorouracil.

[0443] In some embodiments, the agent stabilizing or increasing expression of CLDN18.2 may comprise an agent selected from the group consisting of anthracyclines, nucleoside analogs, platinum compounds, camptothecin analogs and taxanes, prodrugs thereof, salts thereof, and combinations thereof. The anthracycline may be selected from the group consisting of epirubicin, doxorubicin, daunorubicin, idarubicin, valrubicin, prodrugs thereof and salts thereof. The anthracycline may be selected from the group consisting of epirubicin, prodrugs thereof and salts thereof. Preferably, the anthracycline is epirubicin. The nucleoside analog may be selected from the group consisting of gemcitabine, 5-fluorouracil, prodrugs thereof and salts thereof. The platinum compound may selected from the group consisting of oxaliplatin, cisplatin, prodrugs thereof and salts thereof. The camptothecin analog may be selected from the group consisting of irinotecan, topotecan, prodrugs thereof and salts thereof. The taxane may be selected from the group consisting of paclitaxel, docetaxel, prodrugs thereof and salts thereof.

[0444] In some embodiments, a reference to an agent stabilizing or increasing expression of CLDN18.2, such as a reference to an anthracycline, a nucleoside analog, a platinum compound, a camptothecin analog or a taxane, for example, a reference to gemcitabine, 5-fhiorouracil, oxaliplatin, irinotecan or paclitaxel is to include any prodrug such as ester, salt or derivative such as conjugate of said agent. Examples are conjugates of said agent with a carrier substance, e.g. protein-bound paclitaxel such as albumin-bound paclitaxel. Preferably, salts of said agent are pharmaceutically acceptable. In some embodiments, an "agent stabilizing or increasing expression of CLDN18.2" comprises an "agent inducing immunogenic cell death".

[0445] In specific circumstances, cancer cells can enter a lethal stress pathway linked to the emission of a spatiotemporally defined combination of signals that is decoded by the immune system to activate tumor-specific immune responses (Zitvogel L. et al. (2010) Cell 140: 798-804). In such scenario cancer cells are triggered to emit signals that are sensed by innate immune effectors such as dendritic cells to trigger a cognate immune response that involves CD8+ T cells and IFN-γ signalling so that tumor cell death may elicit a productive anticancer immune response. These signals include the pre-apoptotic exposure of the endoplasmic reticulum (ER) chaperon calreticulin (CRT) at the cell surface, the pre-apoptotic secretion of ATP, and the post-apoptotic release of the nuclear protein HMGBL Together, these processes constitute the molecular determinants of immunogenic cell death (ICD). Anthracyclines, oxaliplatin, and γ irradiation are able to induce all signals that define ICD, while cisplatin, for example, which is deficient in inducing CRT translocation from the ER to the surface of dying cells - a process requiring ER stress - requires complementation by thapsigargin, an ER stress inducer.

[0446] As used herein, the term "agent inducing immunogenic cell death" refers to an agent or a combination of agents which when provided to cells, in particular cancer cells, is capable of inducing the cells to enter a lethal stress pathway which finally results in tumor-specific immune responses. In particular, an agent inducing immunogenic cell death when provided to cells induces the cells to emit a spatiotemporally defined combination of signals, including, in particular, the pre-apoptotic exposure of the endoplasmic reticulum (ER) chaperon calreticulin (CRT) at the cell surface, the pre-apoptotic secretion of ATP, and the post-apoptotic release of the nuclear protein HMGBL

[0447] As used herein, the term "agent inducing immunogenic cell death" includes anthracyclines such as epirubicin and oxaliplatin. The term "nucleoside analog" refers to a structural analog of a nucleoside, a category that includes both purine analogs and pyrimidine analogs.

[0448] The term "gemcitabine" refers to a compound which is a nucleoside analog of the following formula:

[0449] In particular, the term refers to the compound 4-amino-l-(2-deoxy-2,2-difluoro-β-D-erythro- pentofuranosyl)pyrimidin-2(lH)-one or 4-amino-l-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5- (hydroxymethyl)oxolan-2-yl] - 1 ,2-dihydropyrimidin-2-one.

[0450] The term "nucleoside analog" includes fluoropyrimidine derivatives such as fluorouracil and prodrugs thereof. The term "fluorouracil" or "5-fluorouracil" (5-FU or f5U) (sold under the brand names Adrucil, Carac, Efudix, Efudex and Fluoroplex) is a compound which is a pyrimidine analog of the following formula:

[0451] In particular, the term refers to the compound 5-fluoro-lH-pyrimidine-2, 4-dione.

[0452] The term "capecitabine" (Xeloda, Roche) refers to a chemotherapeutic agent that is a prodrug that is converted into 5-FU in the tissues. Capecitabine which may be orally administered has the following formula:

[0453] In particular, the term refers to the compound pentyl [l-(3,4-dihydroxy-5-methyltetrahydrofuran- 2-yl)-5-fhioro-2-oxo-lH-pyrimidin-4-yl]carbamate.

[0454] "Floxuridine" (5-fhiorodeoxyuridine) is an oncology drug that is rapidly catabolized to 5- fluorouracil, which is the active form of the drug. Floxuridine has the following formula: " Tegafur" (5-fluoro-l-(oxolan-2-yl)pyrimidine-2, 4-dione) is a chemotherapeutic prodrug of 5- fluorouracil. When metabolised, it becomes 5-fluorouracil. Tegafur has the following formula:

[0455] The term "doxifluridine" (5'-deoxy-5-fluorouridine) is a fluoropyrimidine derivative of 5- fluorouracil. This second generation nucleoside analog prodrug is used as a cytostatic agent in chemotherapy in several Asian countries including China and South Korea. Within a cell, pyrimidine nucleoside phosphorylase or thymidine phosphorylase can metabolize doxifluridine into 5-fluorouracil. It is also a metabolite of capecitabine. Doxifluridine which may be orally administered has the following formula:

[0456] The term "Carmofur" (INN) or "HCFU" refers to l-hexylcarbamoyl-5-fluorouracil:

[0457] This compound is a pyrimidine analogue used as an antineoplastic agent. It is a derivative of fluorouracil, being a lypophilic-masked analog of 5-fhiorouracil. Once inside a cell, carmofur prodrug is converted into 5-fluorouracil.

[0458] The term "platinum compound" refers to compounds containing platinum in their structure such as platinum complexes and includes compounds such as cisplatin, carboplatin and oxaliplatin. The term "cisplatin" or "cisplatinum" refers to the compound cw-diamminedichloroplatinum(II) (CDDP) of the following formula:

[0459] The term "carboplatin" refers to the compound cis-diammine( 1,1- cyclobutanedicarboxylato)platmum(II) of the following formula:

[0460] The term "oxaliplatin" refers to a compound which is a platinum compound that is complexed to a diaminocyclohexane carrier ligand of the following formula:

[0461] In particular, the term "oxaliplatin" refers to the compound [(lR,2R)-cyclohexane-l,2- diamine](ethanedioato-O,O')platinum(n). Oxaliplatin for injection is also marketed under the trade name Eloxatine.

[0462] Taxanes are a class of diterpene compounds that were first derived from natural sources such as plants of the genus Taxus, but some have been synthesized artificially. The principal mechanism of action of the taxane class of drugs is the disruption of microtubule function, thereby inhibiting the process of cell division. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0463] The term "docetaxel" refers to a compound having the following formula: In particular, the term "docetaxel" refers to the compound 1,7β,10β-trihydroxy-9-oxo-5β,2O- epoxytax-1 l-ene-2a,4,13α-triyl 4-acetate 2 -benzoate 13-{(2R,3S)-3-[(tert-butoxycarbonyl)- amino]-2-hydroxy-3 -phenylpropanoate} .

[0464] The term "paclitaxel" refers to a compound having the following formula:

[0465] In particular, the term "paclitaxel" refers to the compound (2a,4a,5P,7β,10p,13α)-4,10-bis- (acetyloxy)- 13 - { [(2R,3 S)- 3 -(benzoylamino)-2-hydroxy-3 -phenylpropanoyl] oxy} - 1,7- dihydroxy-9-oxo-5,20-epoxytax-l l-en-2-yl benzoate.

[0466] The term "camptothecin analog" refers to derivatives of the compound camptothecin (CPT; (S)-4- ethyl-4-hydroxy-lH-pyrano[3',4':6,7]indolizino[l,2-b] quinoline-3,14-(4H,12H)-dione). Preferably, the term "camptothecin analog" refers to compounds comprising the following structure:

[0467] Preferred camptothecin analogs are inhibitors of DNA enzyme topoisomerase I (topo I). Preferred camptothecin analogs are irinotecan and topotecan.

[0468] Irinotecan is a drug preventing DNA from unwinding by inhibition of topoisomerase I. In chemical terms, it is a semisynthetic analogue of the natural alkaloid camptothecin having the following formula:

[0469] In particular, the term "irinotecan" refers to the compound (S)-4, 11 -diethyl-3 ,4, 12,14-tetrahydro- 4-hydroxy-3 , 14-dioxo 1 H-pyrano [3 ’ ,4 ’ : 6,7]-indolizino[ 1 ,2-b] quinolin-9-yl- [ 1 ,4’ -bipiperidine]- 1 ’ - carboxylate.

[0470] Topotecan is a topoisomerase inhibitor of the formula:

[0471] In particular, the term "topotecan" refers to the compound (S)-10-[(dimethylamino)methyl]-4- ethyl-4,9-dihydroxy- lH-pyrano[3 ',4': 6,7]indolizino [ 1 ,2-b] quinoline-3 , 14(4H, 12H)-dione monohydrochloride.

[0472] Anthracyclines are a class of drugs commonly used in cancer chemotherapy that are also antibiotics. Structurally, all anthracyclines share a common four-ringed 7,8,9,10- tetrahydrotetracene-5,12-quinone structure and usually require glycosylation at specific sites.

[0473] Anthracyclines preferably bring about one or more of the following mechanisms of action: 1) Inhibiting DNA and RNA synthesis by intercalating between base pairs of the DNA / RNA strand, thus preventing the replication of rapidly-growing cancer cells; 2) Inhibiting topoisomerase II enzyme, preventing the relaxing of supercoiled DNA and thus blocking DNA transcription and replication; and 3) Creating iron-mediated free oxygen radicals that damage the DNA and cell membranes.

[0474] The term "anthracycline" preferably relates to an agent, preferably an anticancer agent for inducing apoptosis, preferably by inhibiting the rebinding of DNA in topoisomerase II.

[0475] Preferably, the term "anthracycline" generally refers to a class of compounds having the following ring structure including analogs and derivatives, pharmaceutical salts, hydrates, esters, conjugates and prodrugs thereof.

[0476] Examples of anthracyclines and anthracycline analogs include, but are not limited to, daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin, rhodomycin, pyrarubicin, valrubicin, N-trifluoro-acetyl doxorubicin-14-valerate, aclacinomycin, morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin (cyano-morpholino-DOX), 2-pyrrolino- doxorubicin (2-PDOX), 5-iminodaunomycin, mitoxantrone and aclacinomycin A (aclarubicin). Mitoxantrone is a member of the anthracendione class of compounds, which are anthracycline analogs that lack the sugar moiety of the anthracyclines but retain the planar polycylic aromatic ring structure that permits intercalation into DNA. Particularly preferred as anthracyline is a compound of the following formula: wherein

[0477] R1is selected from the group consisting of H and OH, R2is selected from the group consisting of H and OMe, R3is selected from the group consisting of H and OH, and R4is selected from the group consisting of H and OH.

[0478] In one embodiment, R1is H, R2is OMe, R3is H, and R4is OH. In another embodiment, R1is OH, R2is OMe, R3is H, and R4is OH. In another embodiment, R1is OH, R2is OMe, R3is OH, and R4is H. In another embodiment, R1is H, R2is H, R3is H, and R4is OH.

[0479] Specifically contemplated as anthracycline is epirubicin. Epirubicin is an anthracycline drug which has the following formula: and is marketed under the trade name Ellence in the US and Pharmorubicin or Epirubicin Ebewe elsewhere. In particular, the term "epirubicin" refers to the compound (8R,10S)-10- [(2S,4S,5R,6S)-4-amino-5 -hydroxy-6-methyl-oxan-2-yl]oxy-6, 11 -dihydroxy-8 -(2- hydroxyacetyl)-l-methoxy-8-methyl-9,10-dihydro-7H-tetracen-5,12-dion. Epirubicin is favoured over doxorubicin, the most popular anthracycline, in some chemotherapy regimens as it appears to cause fewer side-effects.

[0480] A bispecific binding agent described herein may be administered to a subject simultaneously, or sequentially in any order with an agent stabilizing or increasing expression of CLDN18.2. For example, a bispecific binding agent may be administered prior to administration of an agent stabilizing or increasing expression of CLDN18.2 or a bispecific binding agent may be administered after administration of an agent stabilizing or increasing expression of CLDN18.2. The compositions described herein may comprise further agents and the methods described herein may comprise administration of further agents. In some embodiments, such agents are usefid in the treatments described herein. In some embodiments, such agents are useful in preventing blood vessel formation. In some embodiments, such agents comprise an antibody that binds to human VEGFR2. In some embodiments, such agents are useful in enhancing T cell function. In some embodiments, such agents comprise an immune checkpoint inhibitor.

[0481] Antibody that binds to human VEGFR2

[0482] Tumor blood vessels transport nutrients for the occurrence and development of tumors and provide escape channels for tumor cells. Drugs that target angiogenesis can block the nutrient supply of tumors, thereby achieving the effect of "starving" the tumor. However, tumor angiogenesis is comprehensively regulated by a variety of growth factors, receptors and downstream signaling pathways in the tumor microenvironment. Among them, vascular endothelial growth factor (vascular endothelial growth factor, VEGF) binds to its receptor (vascular endothelial growth factor receptor, VEGFR), and plays an important role in the process of physiological and pathological blood vessel formation.

[0483] VEGFR2 (also known as FLK-1, or KDR), is the main member of the VEGFR family, is a type III receptor tyrosine kinase, and is mainly distributed on the surface of the endothelial cell membrane of blood vessels and lymphatic vessels. The extracellular domain of VEGFR2 contains 7 immunoglobulin-like domains (i.e., D1-D7), including the ligand VEGF binding domain (D2- D3) and dimerization domain (D4-D7), and its intracellular domain contains a tyrosine kinase domain. When the high concentration of VEGF dimer in the tumor microenvironment binds to VEGFR2, it induces VEGFR2 receptors to form homodimers (primary), and heterodimers with VEGFR1 or VEGFR3 (secondary), causing VEGFR2 intracellular autophosphorylation of tyrosine residues in the region, ultimately causing tumor angiogenesis.

[0484] In some embodiments, as used herein, the term "VEGFR2" refers to a polypeptide comprising the amino acid sequence given in SEQ ID NO: 30 or a variant thereof, e.g., an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, as used herein, the term "VEGFR2" refers to a polypeptide comprising the amino acid sequence given in SEQ ID NO: 30. In some embodiments, as used herein, the term "VEGFR2" refers to the polypeptide whose amino acid sequence is that given in SEQ ID NO: 30. VEGFR2 is also known as kinase domain receptor (KDR). As used herein, the term "extracellular domain of VEGFR2" or "VEGFR2-ECD" means the protein beginning and ending at amino acids 1 and 744, respectively, of SEQ ID NO: 30. (VEGFR2 ; SEQ ID NO : 30 )

[0485] ASVGLPSVSLDLPRLSIQKDILTIKANTTLQITCRGQRDLDWLWPNNQSGSEQRVEVTEC SDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYVQDYRSPFIASVSDQHGWYIT ENKNKTWIPCLGSISNLNVSLCARYPEKRFVPDGNRISWDSKKGFTIPSYMISYAGMVF CEAKINDESYQSIMYIVWVGYRIYDWLSPSHGIELSVGEKLVLNCTARTELNVGIDFN WEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNS TFVRVHEKPFVAFGSGMESLVEATVGERVRI PAKYLGYPPPEIKWYKNGI PLESNHTIKA GHVLTIMEVSERDTGNYTVILTNPISKEKQSHWSLWYVPPQIGEKSLISPVDSYQYGT TQTLTCTVYAIPPPHHIHWYWQLEEECANEPSQAVSVTNPYPCEEWRSVEDFQGGNKIEV NKNQFALIEGKNKTVSTLVIQAANVSALYKCEAVNKVGRGERVISFHVTRGPEITLQPDM QPTEQESVSLWCTADRSTFENLTWYKLGPQPLPIHVGELPTPVCKNLDTLWKLNATMFSN STNDILIMELKNASLQDQGDYVCLAQDRKTKKRHCWRQLTVLERVAPTITGNLENQTTS IGESIEVSCTASGNPPPQIMWFKDNETLVEDSGIVLKDGNRNLTIRRVRKEDEGLYTCQA CSVLGCAKVEAFFI IEGAQEKTNLEI I ILVGTAVIAMFFWLLLVI ILRTVKRANGGELKT GYLSIVMDPDELPLDEHCERLPYDASKWEFPRDRLKLGKPLGRGAFGQVIEADAFGIDKT ATCRTVAVKMLKEGATHSEHRALMSELKILIHIGHHLNWNLLGACTKPGGPLMVIVEFC KFGNLSTYLRSKRNEFVPYKTKGARFRQGKDYVGAIPVDLKRRLDSITSSQSSASSGFVE EKSLSDVEEEEAPEDLYKDFLTLEHLICYSFQVAKGMEFLASRKCIHRDLAARNILLSEK NWKICDFGLARDIYKDPDYVRKGDARLPLKWMAPETIFDRVYTIQSDVWSFGVLLWEIF SLGASPYPGVKIDEEFCRRLKEGTRMRAPDYTTPEMYQTMLDCWHGEPSQRPTFSELVEH LGNLLQANAQQDGKDYIVLPISETLSMEEDSGLSLPTSPVSCMEEEEVCDPKFHYDNTAG ISQYLQNSKRKSRPVSVKTFEDIPLEEPEVKVIPDDNQTDSGMVLASEELKTLEDRTKLS PSFGGMVPSKSRESVASEGSNQTSGYQSGYHSDDTDTTVYSSEEAELLKLIEIGVQTGST AQILQPDSGTTLSSPPV

[0486] VEGFR2 antibody drugs can block the binding of VEGFR2 and VEGF.

[0487] As used herein, the term "VEGFR2 antibody" or "anti-VEGFR2 antibody" or "anti-VEGFR2 Ab" refers to an antibody that binds to VEGFR2, preferably to the extracellular domain of VEGFR2.

[0488] The antibody will have a sufficiently strong binding affinity for VEGFR2. In some embodiments, the antibody binds VEGFR2 with a Kd value of between about 100 nM and about 1 pM. Antibody affinities may be determined by a surface plasmon resonance based assay (such as the BIAcore assay is described in PCT Application Publication No. W02005 / 012359); Enzyme-Linked

[0489] Immunoabsorbent Assay (ELISA); and competition assays (e.g. a radiolabeled antigen binding assay (RIA)), for example. In one embodiment, Kj is measured by a RIA performed with an anti-

[0490] VEGFR2 Ab, preferably ramucirumab.

[0491] In some embodiments, an anti-VEGFR2 Ab is an antibody comprising a variable region of a heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 31, and a variable region of a light chain (VL) comprising the amino acid sequence of SEQ ID NO: 32.

[0492] In some embodiments, an anti-VEGFR2 Ab is an antibody comprising a variable region of a heavy chain (VH) whose amino acid sequence is that given in SEQ ID NO: 31, and a variable region of a light chain (VL) whose amino acid sequence is that given in SEQ ID NO: 32.

[0493] (VH ; SEQ ID NO : 31)

[0494] EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTI

[0495] SRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQGTMVTVSS

[0496] (VL; SEQ ID NO : 32 ) DIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLIYDASNLDTGVPSRFSGSGSGTY

[0497] FTLTISSLQAEDFAVYFCQQAKAFPPTFGGGTKVDIK

[0498] In some embodiments, an anti-VEGFR2 Ab is an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 33, and a light chain comprising the amino acid sequence of SEQ ID NO: 34.

[0499] In some embodiments, an anti-VEGFR2 Ab is an antibody comprising a heavy chain whose amino acid sequence is that given in SEQ ID NO: 33, and a light chain whose amino acid sequence is that given in SEQ ID NO: 34.

[0500] In some embodiments, the anti-VEGFR2 Ab is ramucirumab.

[0501] As used herein, the term "ramucirumab" also known as Cyramza®, IMC-1121b, CAS registry number 947687-13-0, refers to an anti-VEGFR2 Ab comprising two heavy chains, each of whose amino acid sequence is that given in SEQ ID NO: 33 and two light chains, each of whose amino acid sequence is that given in SEQ ID NO: 34.

[0502] (Heavy chain; SEQ ID NO : 33 )

[0503] EVQLVQSGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYY

[0504] ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVT DAFDIWGQGT MVTVSSASTK

[0505] GPSVLPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS

[0506] LSSWTVPSS SLGTQTYICN VNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PELLGGPSVF

[0507] LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR

[0508] WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN

[0509] QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN

[0510] VFSCSVMHEA LHNHYTQKSL SLSPGK

[0511] (Light chain; SEQ ID NO : 34 )

[0512] DIQMTQSPSS VSASIGDRVT ITCRASQGID NWLGWYQQKP GKAPKLLIYD ASNLDTGVPS

[0513] RFSGSGSGTY FTLTISSLQA EDFAVYFCQQ AKAFPPTFGG GTKVDIKRTV AAPSVFIFPP

[0514] SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT

[0515] LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

[0516] Ramucirumab is a fully human monoclonal antibody directed against the human vascular endothelial growth factor receptor 2 (VEGFR2). Ramucirumab and methods of making and using this compound including for the treatment of neoplastic diseases such as solid and non-solid tumors are disclosed in W02003 / 075840 . Furthermore, clinical activity for ramucirumab has also been reported in patients with several cancer types.

[0517] As used herein, the term "DC101" refers to a rat monoclonal antibody directed against mouse

[0518] VEGFR2 that may be used in experiments as a surrogate in mice for an anti-VEGFR2 Ab, preferably ramucirumab. See, for example, Witte L„ et al. Monoclonal antibodies targeting the

[0519] VEGF receptor-2 (Flkl / KDR) as an anti-angiogenic therapeutic strategy. Cancer Metastasis Rev., 17: 155-161, 1998; and / or PrewettM., et al., Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors. Cancer Res., 59: 5209-5218, 1999.

[0520] An anti-VEGFR2 Ab, preferably ramucirumab, is generally effective over a wide dosage range in the combination of the present invention. For example, dosages per three- week cycle normally fall within the range of about 6 to 10 mg / kg, preferably about 8 to about 10 mg / kg, and most preferably about 10 mg / kg. In some embodiments, an anti-VEGFR2 Ab, preferably ramucirumab, is administered on day one within the range of about 6 to 10 mg / kg. In some embodiments, an anti- VEGFR2 Ab, preferably ramucirumab, is administered on day one at about 10 mg / kg.

[0521] As used herein, the phrase "in combination with" refers to the administration of two agents simultaneously, or sequentially in any order.

[0522] A bispecific binding agent described herein may be administered to a subject simultaneously, or sequentially in any order with an antibody that binds to human VEGFR2. For example, a bispecific binding agent may be administered prior to administration of an antibody that binds to human VEGFR2 or a bispecific binding agent may be administered after administration of an antibody that binds to human VEGFR2.

[0523] Immune checkpoint inhibitor

[0524] As used herein, "immune checkpoint" refers to regulators of the immune system, and, in particular, co-stimulatory and inhibitory signals that regulate the amplitude and quality of T cell receptor recognition of an antigen. In certain embodiments, the immune checkpoint is an inhibitory signal. In certain embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1 and / or PD-L2. In certain embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86 to displace CD28 binding. In certain embodiments the inhibitory signal is the interaction between LAG-3 and MHC class II molecules. In certain embodiments, the inhibitory signal is the interaction between TIM-3 and one or more of its ligands, such as galectin 9, PtdSer, HMGB1 and CEACAM1 . In certain embodiments, the inhibitory signal is the interaction between one or several KIRs and their ligands. In certain embodiments, the inhibitory signal is the interaction between TIGIT and one or more of its ligands, PVR, PVRL2 and PVRL3. In certain embodiments, the inhibitory signal is the interaction between CD94 / NKG2A and HLA-E. In certain embodiments, the inhibitory signal is the interaction between VISTA and its binding partners). In certain embodiments, the inhibitory signal is the interaction between one or more Siglecs and their ligands. In certain embodiments, the inhibitory signal is the interaction between GARP and one or more of it ligands. In certain embodiments, the inhibitory signal is the interaction between CD47 and SIRPa. In certain embodiments, the inhibitory signal is the interaction between PVRIG and PVRL2. In certain embodiments, the inhibitory signal is the interaction between CSF1R and CSF1. In certain embodiments, the inhibitory signal is the interaction between BTLA and HVEM. In certain embodiments, the inhibitory signal is part of the adenosinergic pathway, e.g., the interaction between A2AR and / or A2BR and adenosine, produced by CD39 and CD73. In certain embodiments, the inhibitory signal is the interaction between B7-H3 and its receptor and / or B7- H4 and its receptor. In certain embodiments, the inhibitory signal is mediated by IDO, CD20, NOX or TDO.

[0525] The "Programmed Death- 1 (PD-1)" receptor refers to an immuno-inhibitory receptor belonging to the CD28 family. PD-1 is expressed predominantly on previously activated T cells in vivo, and binds to two ligands, PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273). The term "PD-1" as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. "Programmed Death Ligand- 1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1" as used herein includes human PD-L1 (hPD-Ll), variants, isoforms, and species homologs of hPD-Ll, and analogs having at least one common epitope with hPD-Ll. The term "PD-L2" as used herein includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, and analogs having at least one common epitope with hPD-L2. The ligands of PD-1 (PD-L1 and PD-L2) are expressed on the surface of antigen-presenting cells, such as dendritic cells or macrophages, and other immune cells. Binding of PD-1 to PD-L1 or PD-L2 results in downregulation of T cell activation. Cancer cells expressing PD-L1 and / or PD-L2 are able to switch off T cells expressing PD-1 what results in suppression of the anticancer immune response. The interaction between PD-1 and its ligands results in a decrease in tumor infiltrating lymphocytes, a decrease in T cell receptor mediated proliferation, and immune evasion by the cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well. "Cytotoxic T Lymphocyte Associated Antigen-4 (CTLA-4)" (also known as CD152) is a T cell surface molecule and is a member of the immunoglobulin superfamily. This protein downregulates the immune system by binding to CD80 (B7-1) and CD86 (B7-2). The term "CTLA-4" as used herein includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA- 4, and analogs having at least one common epitope with hCTLA-4. CTLA-4 is a homolog of the stimulatory checkpoint protein CD28 with much higher binding affinity for CD80 and CD86. CTLA4 is expressed on the surface of activated T cells and its ligands are expressed on the surface of professional antigen-presenting cells. Binding of CTLA-4 to its ligands prevents the co- stimulatory signal of CD28 and produces an inhibitory signal. Thus, CTLA-4 downregulates T cell activation.

[0526] "T cell Immunoreceptor with Ig and ITIM domains" (TIGIT, also known as WUCAM or Vstm3) is an immune receptor on T cells and Natural Killer (NK) cells and binds to PVR (CD 155) on DCs, macrophages etc., and PVRL2 (CD112; nectin-2) and PVRL3 (CD113; nectin-3) and regulates T cell-mediated immunity. The term "TIGIT" as used herein includes human TIGIT (hTIGIT), variants, isoforms, and species homologs of hTIGIT, and analogs having at least one common epitope with hTIGIT. The term "PVR" as used herein includes human PVR (hPVR), variants, isoforms, and species homologs of hPVR, and analogs having at least one common epitope with hPVR. The term "PVRL2" as used herein includes human PVRL2 (hPVRL2), variants, isoforms, and species homologs of hPVRL2, and analogs having at least one common epitope with hPVRL2. The term "PVRL3" as used herein includes human PVRL3 (hPVRL3), variants, isoforms, and species homologs of hPVRL3, and analogs having at least one common epitope with hPVRL3.

[0527] The "B7 family" refers to inhibitory ligands with undefined receptors. The B7 family encompasses B7-H3 and B7-H4, both upregulated on tumor cells and tumor infiltrating cells. The terms "B7- H3" and "B7-H4" as used herein include human B7-H3 (hB7-H3) and human B7-H4 (hB7-H4), variants, isoforms, and species homologs thereof, and analogs having at least one common epitope with B7-H3 and B7-H4, respectively.

[0528] "B and T Lymphocyte Attenuator" (BTLA, also known as CD272) is a TNFR family member expressed in Thl but not Th2 cells. BTLA expression is induced during activation of T cells and is in particular expressed on surfaces of CD8+ T cells. The term "BTLA" as used herein includes human BTLA (hBTLA), variants, isoforms, and species homologs of hBTLA, and analogs having at least one common epitope with hBTLA. BTLA expression is gradually downregulated during differentiation of human CD8+ T cells to effector cell phenotype. Tumor-specific human CD8+ T cells express high levels of BTLA. BTLA binds to "Herpesvirus entry mediator" (HVEM, also known as TNFRSF14 or CD270) and is involved in T cell inhibition. The term "HVEM" as used herein includes human HVEM (hHVEM), variants, isoforms, and species homologs of hHVEM, and analogs having at least one common epitope with hHVEM. BTLA-HVEM complexes negatively regulate T cell immune responses.

[0529] "Killer-cell Immunoglobulin-like Receptors" (KIRs) are receptors for MHC Class I molecules on NK T cells and NK cells that are involved in differentiation between healthy and diseased cells. KIRs bind to human leukocyte antigen (HLA) A, B and C, what suppresses normal immune cell activation. The term "KIRs" as used herein includes human KIRs (hKIRs), variants, isofonns, and species homologs of hKIRs, and analogs having at least one common epitope with a hKIR. The term "HLA" as used herein includes variants, isoforms, and species homologs of HLA, and analogs having at least one common epitope with a HLA. KIR as used herein in particular refers to KIR2DL1, KIR2DL2, and / or KIR2DL3.

[0530] "Lymphocyte Activation Gene-3 (LAG-3)" (also known as CD223) is an inhibitory receptor associated with inhibition of lymphocyte activity by binding to MHC class II molecules. This receptor enhances the function of Treg cells and inhibits CD8+ effector T cell function leading to immune response suppression. LAG-3 is expressed on activated T cells, NK cells, B cells and DCs. The term "LAG-3" as used herein includes human LAG-3 (hLAG-3), variants, isofonns, and species homologs of hLAG-3, and analogs having at least one common epitope.

[0531] "T Cell Membrane Protein-3 (TIM-3)" (also known as HAVcr-2) is an inhibitory receptor involved in the inhibition of lymphocyte activity by inhibition of Thl cell responses. Its ligand is galectin 9 (GAL9), which is upregulated in various types of cancers. Other TIM-3 ligands include phosphatidyl serine (PtdSer), High Mobility Group Protein 1 (HMGB1) and Carcinoembryonic Antigen Related Cell Adhesion Molecule 1 (CEACAM1). The term "TIM-3" as used herein includes human TIM3 (hTIM-3), variants, isoforms, and species homologs of hTIM-3, and analogs having at least one common epitope. The term "GAL9" as used herein includes human GAL9 (hGAL9), variants, isoforms, and species homologs of hGAL9, and analogs having at least one common epitope. The term "PdtSer" as used herein includes variants and analogs having at least one common epitope. The term "HMGB1" as used herein includes human HMGB1 (hHMGBl), variants, isoforms, and species homologs of hHMGBl, and analogs having at least one common epitope. The term "CEACAM1" as used herein includes human CEACAM1 (hCEACAMl), variants, isofonns, and species homologs of hCEACAMl, and analogs having at least one common epitope.

[0532] "CD94 / NKG2A" is an inhibitory receptor predominantly expressed on the surface of natural killer cells and of CD8+ T cells. The term "CD94 / NKG2A" as used herein includes human CD94 / NKG2A (hCD94 / NKG2A), variants, isofonns, and species homologs of hCD94 / NKG2A, and analogs having at least one common epitope. The CD94 / NKG2A receptor is a heterodimer comprising CD94 and NKG2A. It suppresses NK cell activation and CD8+ T cell function, probably by binding to ligands such as HLA-E. CD94 / NKG2A restricts cytokine release and cytotoxic response of natural killer cells (NK cells), Natural Killer T cells (NK-T cells) and T cells ( α / β and γ / δ). NKG2A is frequently expressed in tumor infiltrating cells and HLA-E is overexpressed in several cancers. "Indoleamine 2,3-dioxygenase" (IDO) is a tryptophan catabolic enzyme with immune-inhibitory properties. The term "IDO" as used herein includes human IDO (hIDO), variants, isoforms, and species homologs of hIDO, and analogs having at least one common epitope. IDO is the rate limiting enzyme in tryptophan degradation catalyzing its conversion to kynurenine. Therefore, IDO is involved in depletion of essential amino acids. It is known to be involved in suppression of T and NK cells, generation and activation of Tregs and myeloid-derived suppressor cells, and promotion of tumor angiogenesis. IDO is overexpressed in many cancers and was shown to promote immune system escape of tumor cells and to facilitate chronic tumor progression when induced by local inflammation.

[0533] In the "adenosinergic pathway" or "adenosine signaling pathway" as used herein ATP is converted to adenosine by the ectonucleotidases CD39 and CD73 resulting in inhibitory signaling through adenosine binding by one or more of the inhibitory adenosine receptors "Adenosine A2A Receptor" (A2AR, also known as ADORA2A) and "Adenosine A2B Receptor" (A2BR, also known as ADORA2B). Adenosine is a nucleoside with immunosuppressive properties and is present in high concentrations in the tumor microenvironment restricting immune cell infiltration, cytotoxicity and cytokine production. Thus, adenosine signaling is a strategy of cancer cells to avoid host immune system clearance. Adenosine signaling through A2AR and A2BR is an important checkpoint in cancer therapy that is activated by high adenosine concentrations typically present in the tumor microenvironment. CD39, CD73, A2AR and A2BR are expressed by most immune cells, including T cells, invariant natural killer cells, B cells, platelets, mast cells and eosinophils. Adenosine signaling through A2AR and A2BR counteracts T cell receptor mediated activation of immune cells and results in increased numbers of Tregs and decreased activation of DCs and effector T cells. The term "CD39" as used herein includes human CD39 (hCD39), variants, isoforms, and species homologs of hCD39, and analogs having at least one common epitope. The term "CD73" as used herein includes human CD73 (hCD73), variants, isoforms, and species homologs of hCD73, and analogs having at least one common epitope. The term "A2AR" as used herein includes human A2AR (hA2AR), variants, isoforms, and species homologs of hA2AR, and analogs having at least one common epitope. The term "A2BR" as used herein includes human A2BR (hA2BR), variants, isoforms, and species homologs of hA2BR, and analogs having at least one common epitope.

[0534] "V-domain Ig suppressor of T cell activation" (VISTA, also known as C10orf54) bears homology to PD-L1 but displays a unique expression pattern restricted to the hematopoietic compartment. The term "VISTA" as used herein includes human VISTA (hVISTA), variants, isoforms, and species homologs of h VISTA, and analogs having at least one common epitope. VISTA induces T cell suppression and is expressed by leukocytes within tumors.

[0535] The "Sialic acid binding immunoglobulin type lectin" (Siglec) family members recognize sialic acids and are involved in distinction between "self and "non-self. The term "Siglecs" as used herein includes human Siglecs (hSiglecs), variants, isoforms, and species homologs of hSiglecs, and analogs having at least one common epitope with one or more hSiglecs. The human genome contains 14 Siglecs of which several are involved in immunosuppression, including, without limitation, Siglec-2, Siglec-3, Siglec-7 and Siglec-9. Siglec receptors bind glycans containing sialic acid, but differ in their recognition of the linkage regiochemistry and spatial distribution of sialic residues. The members of the family also have distinct expression patterns. A broad range of malignancies overexpress one or more Siglecs.

[0536] "CD20" is an antigen expressed on the surface of B and T cells. High expression of CD20 can be found in cancers, such as B cell lymphomas, hairy cell leukemia, B cell chronic lymphocytic leukemia, and melanoma cancer stem cells. The term "CD20" as used herein includes human CD20 (hCD20), variants, isoforms, and species homologs of hCD20, and analogs having at least one common epitope.

[0537] "Glycoprotein A repetitions predominant" (GARP) plays a role in immune tolerance and the ability of tumors to escape the patient's immune system. The term "GARP" as used herein includes human GARP (hGARP), variants, isoforms, and species homologs of hGARP, and analogs having at least one common epitope. GARP is expressed on lymphocytes including Treg cells in peripheral blood and tumor infiltrating T cells at tumor sites. It probably binds to latent "transforming growth factor P" (TGF-P). Disruption of GARP signaling in Tregs results in decreased tolerance and inhibits migration of Tregs to the gut and increased proliferation of cytotoxic T cells.

[0538] "CD47" is a transmembrane protein that binds to the ligand "signal-regulatory protein alpha" (SIRPa). The term "CD47" as used herein includes human CD47 (hCD47), variants, isoforms, and species homologs of hCD47, and analogs having at least one common epitope with hCD47. The term "SIRPa" as used herein includes human SIRPa (hSIRPa), variants, isoforms, and species homologs of hSIRPa, and analogs having at least one common epitope with hSIRPa. CD47 signaling is involved in a range of cellular processes including apoptosis, proliferation, adhesion and migration. CD47 is overexpressed in many cancers and functions as "don’t eat me" signal to macrophages. Blocking CD47 signaling through inhibitory anti-CD47 or anti-SIRPa antibodies enables macrophage phagocytosis of cancer cells and fosters the activation of cancer-specific T lymphocytes. "Poliovirus receptor related immunoglobulin domain containing" (PVRIG, also known as CD112R) binds to "Poliovirus receptor-related 2" (PVRL2). PVRIG and PVRL2 are overexpressed in a number of cancers. PVRIG expression also induces TIGIT and PD- 1 expression and PVRL2 and PVR (a TIGIT ligand) are co-overexpressed in several cancers. Blockade of the PVRIG signaling pathway results in increased T cell function and CD8+ T cell responses and, therefore, reduced immune suppression and elevated interferon responses. The term "PVRIG" as used herein includes human PVRIG (hPVRIG), variants, isoforms, and species homologs of hPVRIG, and analogs having at least one common epitope with hPVRIG. "PVRL2" as used herein includes hPVRL2, as defined above.

[0539] The "colony-stimulating factor 1 " pathway is another checkpoint that can be targeted according to the disclosure. CSF1R is a myeloid growth factor receptor that binds CSF1. Blockade of the CSF1R signaling can functionally reprogram macrophage responses, thereby enhancing antigen presentation and anti-tumor T cell responses. The term "CSF1R" as used herein includes human CSF1R (hCSFIR), variants, isoforms, and species homologs of hCSFIR, and analogs having at least one common epitope with hCSFIR. The term "CSF1" as used herein includes human CSF1 (hCSFl), variants, isoforms, and species homologs of hCSFl, and analogs having at least one common epitope with hCSFl .

[0540] "Nicotinamide adenine dinucleotide phosphate NADPH oxidase" refers to an enzyme of the NOX family of enzymes of myeloid cells that generate immunosuppressive reactive oxygen species (ROS). Five NOX enzymes (NOXI to NOX5) have been found to be involved in cancer development and immunosuppression. Elevated ROS levels have been detected in almost all cancers and promote many aspects of tumor development and progression. NOX produced ROS dampens NK and T cell functions and inhibition of NOX in myeloid cells improves anti-tumor functions of adjacent NK cells and T cells. The term "NOX" as used herein includes human NOX (hNOX), variants, isoforms, and species homologs of hNOX, and analogs having at least one common epitope with hNOX.

[0541] Another immune checkpoint that can be targeted according to the disclosure is the signal mediated by "tryptophan-2, 3 -dioxygenase" (TDO). TDO represents an alternative route to IDO in tryptophan degradation and is involved in immune suppression. Since tumor cells may catabolize tryptophan via TDO instead of IDO, TDO may represent an additional target for checkpoint blockade. Indeed, several cancer cell lines have been found to upregulate TDO and TDO may complement IDO inhibition. The term "TDO" as used herein includes human TDO (hTDO), variants, isoforms, and species homologs of hTDO, and analogs having at least one common epitope with hTDO. Many of the immune checkpoints are regulated by interactions between specific receptor and ligand pairs, such as those described above. Thus, immune checkpoint proteins mediate immune checkpoint signaling. For example, checkpoint proteins directly or indirectly regulate T cell activation, T cell proliferation and / or T cell function. Cancer cells often exploit these checkpoint pathways to protect themselves from being attacked by the immune system. Hence, the function of checkpoint proteins, which is modulated according to the present disclosure is typically the regulation of T cell activation, T cell proliferation and / or T cell function. Immune checkpoint proteins thus regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Many of the immune checkpoint proteins belong to the B7:CD28 family or to the tumor necrosis factor receptor (TNFR) super family and, by binding to specific ligands, activate signaling molecules that are recruited to the cytoplasmic domain (Suzuki et al., 2016, Jap J Clin One, 46:191-203).

[0542] As used herein, the term "immune checkpoint modulator" or "checkpoint modulator" refers to a molecule or to a compound that modulates the function of one or more checkpoint proteins. Immune checkpoint modulators are typically able to modulate self-tolerance and / or the amplitude and / or the duration of the immune response. Preferably, the immune checkpoint modulator used according to the present disclosure modulates the function of one or more human checkpoint proteins and is, thus, a "human checkpoint modulator". In a preferred embodiment, the human checkpoint modulator as used herein is an immune checkpoint inhibitor.

[0543] As used herein, "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that totally or partially reduces, inhibits, interferes with or negatively modulates one or more checkpoint proteins or that totally or partially reduces, inhibits, interferes with or negatively modulates expression of one or more checkpoint proteins. In certain embodiments, the immune checkpoint inhibitor binds to one or more checkpoint proteins. In certain embodiments, the immune checkpoint inhibitor binds to one or more molecules regulating checkpoint proteins. In certain embodiments, the immune checkpoint inhibitor binds to precursors of one or more checkpoint proteins e.g., on DNA- or RNA-level. Any agent that functions as a checkpoint inhibitor according to the present disclosure can be used.

[0544] The term "partially" as used herein means at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% in the level, e.g., in the level of inhibition of a checkpoint protein.

[0545] In certain embodiments, the immune checkpoint inhibitor suitable for use herein, is an antagonist of inhibitory signals, e.g., an antibody which targets, for example, PD-1, PD-L1, CTLA-4, LAG- 3, B7-H3, B7-H4, or TIM-3. These ligands and receptors are reviewed in Pardoll, D., Nature. 12: 252-264, 2012. Further immune checkpoint proteins that can be targeted according the disclosure are described herein.

[0546] In certain embodiments, the immune checkpoint inhibitor prevents inhibitory signals associated with the immune checkpoint. In certain embodiments, the immune checkpoint inhibitor is an antibody, or fragment thereof that disrupts inhibitory signaling associated with the immune checkpoint. In certain embodiments, the immune checkpoint inhibitor is a small molecule inhibitor that disrupts inhibitory signaling. In certain embodiments, the immune checkpoint inhibitor is a peptide-based inhibitor that disrupts inhibitory signaling. In certain embodiments, the immune checkpoint inhibitor is an inhibitory nucleic acid molecule that disrupts inhibitory signaling.

[0547] In certain embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimic, that prevents the interaction between checkpoint blocker proteins, e.g., an antibody, or fragment thereof that prevents the interaction between PD-1 and PD-L1 or PD-L2. In certain embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimic, that prevents the interaction between CTLA-4 and CD80 or CD86. In certain embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimic, that prevents the interaction between LAG-3 and its ligands, or TIM-3 and its ligands. In certain embodiments, the immune checkpoint inhibitor prevents inhibitory signaling through CD39 and / or CD73 and / or the interaction of A2AR and / or A2BR with adenosine. In certain embodiments, the immune checkpoint inhibitor prevents interaction of B7-H3 with its receptor and / or of B7-H4 with its receptor. In certain embodiments, the immune checkpoint inhibitor prevents the interaction of BTLA with its ligand HVEM. In certain embodiments, the immune checkpoint inhibitor prevents the interaction of one or more KIRs with their respective ligands. In certain embodiments, the immune checkpoint inhibitor prevents the interaction of LAG-3 with one or more of its ligands. In certain embodiments, the immune checkpoint inhibitor prevents the interaction of TIM-3 with one or more of its ligands Galectin-9, PtdSer, HMGB1 and CEACAM1. In certain embodiments, the immune checkpoint inhibitor prevents the interaction of TIGIT with one or more of its ligands PVR, PVRL2 and PVRL3. In certain embodiments, the immune checkpoint inhibitor prevents the interaction of CD94 / NKG2A with HLA-E. In certain embodiments, the immune checkpoint inhibitor prevents the interaction of VISTA with one or more of its binding partners. In certain embodiments, the immune checkpoint inhibitor prevents the interaction of one or more Siglecs and their respective ligands. In certain embodiments, the immune checkpoint inhibitor prevents CD20 signaling. In certain embodiments, the immune checkpoint inhibitor prevents the interaction of GARP with one or more of its ligands. In certain embodiments, the immune checkpoint inhibitor prevents the interaction of CD47 with SIRPa. Tn certain embodiments, the immune checkpoint inhibitor prevents the interaction of PVRIG with PVRL2. In certain embodiments, the immune checkpoint inhibitor prevents the interaction of CSF1R with CSF1. In certain embodiments, the immune checkpoint inhibitor prevents NOX signaling. In certain embodiments, the immune checkpoint inhibitor prevents IDO and / or TDO signaling.

[0548] Inhibiting or blocking of inhibitory immune checkpoint signaling, as described herein, results in preventing or reversing immune-suppression and establishment or enhancement of T cell immunity against cancer cells. In one embodiment, inhibition of immune checkpoint signaling, as described herein, reduces or inhibits dysfunction of the immune system. In one embodiment, inhibition of immune checkpoint signaling, as described herein, renders dysfunctional immune cells less dysfunctional. In one embodiment, inhibition of immune checkpoint signaling, as described herein, renders a dysfunctional T cell less dysfunctional.

[0549] The term "dysfunction", as used herein, refers to a state of reduced immune responsiveness to antigenic stimulation. The term includes the common elements of both exhaustion and / or anergy in which antigen recognition may occur, but the ensuing immune response is ineffective to control infection or tumor growth. Dysfunction also includes a state in which antigen recognitinn is retarded due to dysfunctional immune cells.

[0550] The term "dysfunctional", as used herein, refers to an immune cell that is in a state of reduced immune responsiveness to antigen stimulation. Dysfunctional includes unresponsive to antigen recognition and impaired capacity to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2) and / or target cell killing.

[0551] The term "anergy", as used herein, refers to the state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signals delivered through the T cell receptor (TCR). T cell anergy can also result upon stimulation with antigen in the absence of co-stimulation, resulting in the cell becoming refractory to subsequent activation by the antigen even in the context of co- stimulation. The unresponsive state can often be overridden by the presence of IL-2. Anergic T cells do not undergo clonal expansion and / or acquire effector functions.

[0552] The term "exhaustion", as used herein, refers to immune cell exhaustion, such as T cell exhaustion as a state of T cell dysfunction that arises from sustained TCR signaling that occurs during many chronic infections and cancer. It is distinguished from anergy in that it arises not through incomplete or deficient signaling, but from sustained signaling. Exhaustion is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of diseases (e.g., infection and tumors). Exhaustion can result from both extrinsic negative regulatory pathways (e.g., immunoregulatory cytokines) as well as cell intrinsic negative regulatory pathways (inhibitory immune checkpoint pathways, such as described herein).

[0553] "Enhancing T cell function" means to induce, cause or stimulate a T cell to have a sustained or amplified biological function, or renew or reactivate exhausted or inactive T cells. Examples of enhancing T cell function include increased secretion of - / -interferon from CD8+ T cells, increased proliferation, increased antigen responsiveness (e.g., tumor clearance) relative to such levels before the intervention. In one embodiment, the level of enhancement is as least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, or more. Manners of measuring this enhancement are known to one of ordinary skill in the art.

[0554] The immune checkpoint inhibitor may be an inhibitory nucleic acid molecule. The term "inhibitory nucleic acid" or "inhibitory nucleic acid molecule" as used herein refers to a nucleic acid molecule, e.g., DNA or RNA, that totally or partially reduces, inhibits, interferes with or negatively modulates one or more checkpoint proteins. Inhibitory nucleic acid molecules include, without limitation, oligonucleotides, siRNA, shRNA, antisense DNA or RNA molecules, and aptamers (e.g., DNA or RNA aptamers).

[0555] The term "oligonucleotide" as used herein refers to a nucleic acid molecule that is able to decrease protein expression, in particular expression of a checkpoint protein, such as the checkpoint proteins described herein. Oligonucleotides are short DNA or RNA molecules, typically comprising from 2 to 50 nucleotides. Oligonucleotides maybe single-stranded or double-stranded. A checkpoint inhibitor oligonucleotide may be an antisense-oligonucleotide. Antisense-oligonucleotides are single-stranded DNA or RNA molecules that are complementary to a given sequence, in particular to a sequence of the nucleic acid sequence (or a fragment thereof) of a checkpoint protein. Antisense RNA is typically used to prevent protein translation of mRNA, e.g., of mRNA encoding a checkpoint protein, by binding to said mRNA. Antisense DNA is typically used to target a specific, complementary (coding or non-coding) RNA. If binding takes place, such a DNA / RNA hybrid can be degraded by the enzyme RNase H. Moreover, morpholino antisense oligonucleotides can be used for gene knockdowns in vertebrates. For example, Kryczek et al., 2006 (J Exp Med, 203:871-81) designed B7-H4-specific morpholinos that specifically blocked B7-H4 expression in macrophages, resulting in increased T cell proliferation and reduced tumor volumes in mice with tumor associated antigen (TAA)-specific T cells.

[0556] The terms "siRNA" or "small interfering RNA" or "small inhibitory RNA" are used interchangeably herein and refer to a double-stranded RNA molecule with a typical length of 20- 25 base pairs that interferes with expression of a specific gene, such as a gene coding for a checkpoint protein, with a complementary nucleotide sequence. In one embodiment, siRNA interferes with mRNA therefore blocking translation, e.g., translation of an immune checkpoint protein. Transfection of exogenous siRNA may be used for gene knockdown, however, the effect maybe only transient, especially in rapidly dividing cells. Stable transfection may be achieved, e.g., by RNA modification or by using an expression vector. Usefill modifications and vectors for stable transfection of cells with siRNA are known in the art. siRNA sequences may also be modified to introduce a short loop between the two strands resulting in a "small hairpin RNA" or "shRNA". shRNA can be processed into a functional siRNA by Dicer. shRNA has a relatively low rate of degradation and turnover. Accordingly, the immune checkpoint inhibitor may be a shRNA. The term "aptamer" as used herein refers to a single-stranded nucleic acid molecule, such as DNA or RNA, typically in a length of 25-70 nucleotides that is capable of binding to a target molecule, such as a polypeptide. In one embodiment, the aptamer binds to an immune checkpoint protein such as the immune checkpoint proteins described herein. For example, an aptamer according to the disclosure can specifically bind to an immune checkpoint protein or polypeptide, or to a molecule in a signaling pathway that modulates the expression of an immune checkpoint protein or polypeptide. The generation and therapeutic use of aptamers is well known in the art (see, e.g., US 5,475,096).

[0557] The terms "small molecule inhibitor" or "small molecule" are used interchangeably herein and refer to a low molecular weight organic compound, usually up to 1000 daltons, that totally or partially reduces, inhibits, interferes with, or negatively modulates one or more checkpoint proteins as described above. Such small molecular inhibitors are usually synthesized by organic chemistry, but may also be isolated from natural sources, such as plants, fungi, and microbes. The small molecular weight allows a small molecule inhibitor to rapidly diffuse across cell membranes. For example, various A2AR antagonists known in the art are organic compounds having a molecular weight below 500 daltons.

[0558] The immune checkpoint inhibitor may be an antibody, an antigen-binding fragment thereof, an antibody mimic or a fusion protein comprising an antibody portion with an antigen-binding fragment of the required specificity. Antibodies or antigen-binding fragments thereof are as described herein. Antibodies or antigen-binding fragments thereof that are immune checkpoint inhibitors include in particular antibodies or antigen-binding fragments thereof that bind to immune checkpoint proteins, such as immune checkpoint receptors or immune checkpoint receptor ligands. Antibodies or antigen-binding fragments may also be conjugated to further moieties, as described herein. In particular, antibodies or antigen-binding fragments thereof are chimerized, humanized or human antibodies. Preferably, immune checkpoint inhibitor antibodies or antigen- binding fragments thereof are antagonists of immune checkpoint receptors or of immune checkpoint receptor ligands.

[0559] In a preferred embodiment, an antibody that is an immune checkpoint inhibitor, is an isolated antibody.

[0560] The antibody that is an immune checkpoint inhibitor or the antigen-binding fragment thereof according to the present disclosure may also be an antibody that cross-competes for antigen binding with any known immune checkpoint inhibitor antibody. In certain embodiments, an immune checkpoint inhibitor antibody cross-competes with one or more of the immune checkpoint inhibitor antibodies described herein. The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies may bind to the same epitope region of the antigen or when binding to another epitope sterically hinder the binding of known immune checkpoint inhibitor antibodies to that particular epitope region. These cross-competing antibodies may have functional properties very similar to those they are cross-competing with as they are expected to block binding of the immune checkpoint to its ligand either by binding to the same epitope or by sterically hindering the binding of the ligand. Cross-competing antibodies can be readily identified based on their ability to cross-compete with one or more of known antibodies in standard binding assays such as Surface Plasmon Resonance (SPR) analysis, ELISA assays or flow cytometry (see, e.g., WO 2013 / 173223).

[0561] In certain embodiments, antibodies or antigen binding fragments thereof that cross-compete for binding to a given antigen with, or bind to the same epitope region of a given antigen as, one or more known antibodies are monoclonal antibodies. For administration to human patients, these cross-competing antibodies can be chimeric antibodies, or humanized or human antibodies. Such chimeric, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0562] The checkpoint inhibitor may also be in the form of the soluble form of the molecules (or variants thereof) themselves, e.g., a soluble PD-L1 or PD-L1 fusion.

[0563] In the context of the disclosure, more than one checkpoint inhibitor can be used, wherein the more than one checkpoint inhibitors are targeting distinct checkpoint pathways or the same checkpoint pathway. Preferably, the more than one checkpoint inhibitors are distinct checkpoint inhibitors. Preferably, if more than one distinct checkpoint inhibitor is used, in particular at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 distinct checkpoint inhibitors are used, preferably 2, 3, 4 or 5 distinct checkpoint inhibitors are used, more preferably 2, 3 or 4 distinct checkpoint inhibitors are used, even more preferably 2 or 3 distinct checkpoint inhibitors are used and most preferably 2 distinct checkpoint inhibitors are used. Preferred examples of combinations of distinct checkpoint inhibitors include combination of an inhibitor of PD-1 signaling and an inhibitor of CTLA-4 signaling, an inhibitor of PD-1 signaling and an inhibitor of TIGIT signaling, an inhibitor of PD-1 signaling and an inhibitor of B7-H3 and / or B7-H4 signaling, an inhibitor of PD-1 signaling and an inhibitor of BTLA signaling, an inhibitor of PD-1 signaling and an inhibitor of KIR signaling, an inhibitor of PD-1 signaling and an inhibitor of LAG-3 signaling, an inhibitor of PD- 1 signaling and an inhibitor of TIM-3 signaling, an inhibitor of PD-1 signaling and an inhibitor of CD94 / NKG2A signaling, an inhibitor of PD-1 signaling and an inhibitor of IDO signaling, an inhibitor of PD-1 signaling and an inhibitor of adenosine signaling, an inhibitor of PD-1 signaling and an inhibitor of VISTA signaling, an inhibitor of PD-1 signaling and an inhibitor of Siglec signaling, an inhibitor of PD-1 signaling and an inhibitor of CD20 signaling, an inhibitor of PD-1 signaling and an inhibitor of GARP signaling, an inhibitor of PD-1 signaling and an inhibitor of CD47 signaling, an inhibitor of PD-1 signaling and an inhibitor of PVRIG signaling, an inhibitor of PD-1 signaling and an inhibitor of CSF1R signaling, an inhibitor of PD-1 signaling and an inhibitor of NOX signaling, and an inhibitor of PD-1 signaling and an inhibitor of TDO signaling.

[0564] In certain embodiments, the inhibitory immunoregulator (immune checkpoint blocker) is a component of the PD-1ZPD-L1 or PD-1 / PD-L2 signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the PD-1 signaling pathway. In certain embodiments, the checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 inhibitor. In certain embodiments, the checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 ligand inhibitor, such as a PD-L1 inhibitor or a PD-L2 inhibitor. In a preferred embodiment, the checkpoint inhibitor of the PD-1 signaling pathway is an antibody or an antigen-binding portion thereof that disrupts the interaction between the PD-1 receptor and one or more of its ligands, PD-L1 and / or PD-L2. Antibodies which bind to PD-1 and disrupt the interaction between PD-1 and one or more of its ligands are known in the art. In certain embodiments, the antibody or antigen-binding portion thereof binds specifically to PD-1. In certain embodiments, the antibody or antigen-binding portion thereof binds specifically to PD-L1 and inhibits its interaction with PD- 1 , thereby increasing immune activity. In certain embodiments, the antibody or antigen-binding portion thereof binds specifically to PD-L2 and inhibits its interaction with PD-1, thereby increasing immune activity.

[0565] In certain embodiments, the inhibitory immunoregulator is a component of the CTLA-4 signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the CTLA-4 signaling pathway. In certain embodiments, the checkpoint inhibitor of the CTLA-4 signaling pathway is a CTLA-4 inhibitor. In certain embodiments, the checkpoint inhibitor of the CTLA-4 signaling pathway is a CTLA-4 ligand inhibitor.

[0566] In certain embodiments, the inhibitory immunoregulator is a component of the TIGIT signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the TIGIT signaling pathway. In certain embodiments, the checkpoint inhibitor of the TIGIT signaling pathway is a TIGIT inhibitor. In certain embodiments, the checkpoint inhibitor of the TIGIT signaling pathway is a TIGIT ligand inhibitor.

[0567] In certain embodiments, the inhibitory immunoregulator is a component of the B7 family signaling pathway. In certain embodiments, the B7 family members are B7-H3 and B7-H4. Certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of B7- H3 and / or B7-4. Accordingly, certain embodiments of the disclosure provide for administering to a subject an antibody or an antigen-binding portion thereof that targets B7-H3 or B7-H4. The B7 family does not have any defined receptors but these ligands are upregulated on tumor cells or tumor-infiltrating cells. Preclinical mouse models have shown that blockade of these ligands can enhance anti-tumor immunity.

[0568] In certain embodiments, the inhibitory immunoregulator is a component of the BTLA signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the BTLA signaling pathway. In certain embodiments, the checkpoint inhibitor of the BTLA signaling pathway is a BTLA inhibitor. In certain embodiments, the checkpoint inhibitor of the BTLA signaling pathway is a HVEM inhibitor.

[0569] In certain embodiments, the inhibitory immunoregulator is a component of one or more KIR signaling pathways. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of one or more KIR signaling pathways. In certain embodiments, the checkpoint inhibitor of one or more KIR signaling pathways is a KIR inhibitor. In certain embodiments, the checkpoint inhibitor one or more KIR signaling pathways is a KIR ligand inhibitor. For example, the KIR inhibitor according to the present disclosure may be an anti-KIR antibody that binds to KIR2DL1, KIR2DL2, and / or KIR2DL3.

[0570] In certain embodiments, the inhibitory immunoregulator is a component of the LAG-3 signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of LAG-3 signaling. In certain embodiments, the checkpoint inhibitor of the LAG-3 signaling pathway is a LAG-3 inhibitor. In certain embodiments, the checkpoint inhibitor of the LAG-3 signaling pathway is a LAG-3 ligand inhibitor.

[0571] In certain embodiments, the inhibitory immunoregulator is a component of the TIM-3 signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the TIM-3 signaling pathway. In certain embodiments, the checkpoint inhibitor of the TIM-3 signaling pathway is a TIM-3 inhibitor. In certain embodiments, the checkpoint inhibitor of the TIM-3 signaling pathway is a TIM-3 ligand inhibitor.

[0572] In certain embodiments, the inhibitory immunoregulator is a component of the CD94 / NKG2A signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the CD94 / NKG2A signaling pathway. In certain embodiments, the checkpoint inhibitor of the CD94 / NKG2A signaling pathway is a CD94 / NKG2A inhibitor. In certain embodiments, the checkpoint inhibitor of the CD94 / NKG2A signaling pathway is a CD94ZNKG2A ligand inhibitor.

[0573] In certain embodiments, the inhibitory immunoregulator is a component of the IDO signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the IDO signaling pathway, e.g., an IDO inhibitor.

[0574] In certain embodiments, the inhibitory immunoregulator is a component of the adenosine signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the adenosine signaling pathway. In certain embodiments, the checkpoint inhibitor of the adenosine signaling pathway is a CD39 inhibitor. In certain embodiments, the checkpoint inhibitor of the adenosine signaling pathway is a CD73 inhibitor. In certain embodiments, the checkpoint inhibitor of the adenosine signaling pathway is an A2AR inhibitor. In certain embodiments, the checkpoint inhibitor of the adenosine signaling pathway is an A2BR inhibitor.

[0575] In certain embodiments, the inhibitory immunoregulator is a component of the VISTA signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the VISTA signaling pathway. In certain embodiments, the checkpoint inhibitor of the VISTA signaling pathway is a VISTA inhibitor.

[0576] In certain embodiments, the inhibitory immunoregulator is a component of one or more Siglec signaling pathways. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of one or more Siglec signaling pathways. In certain embodiments, the checkpoint inhibitor of one or more Siglec signaling pathways is a Siglec inhibitor. In certain embodiments, the checkpoint inhibitor of one or more Siglec signaling pathways is a Siglec ligand inhibitor.

[0577] In certain embodiments, the inhibitory immunoregulator is a component of the CD20 signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the CD20 signaling pathway. In certain embodiments, the checkpoint inhibitor of the CD20 signaling pathway is a CD20 inhibitor. In certain embodiments, the inhibitory immunoregulator is a component of the GARP signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the GARP signaling pathway. In certain embodiments, the checkpoint inhibitor of the GARP signaling pathway is a GARP inhibitor.

[0578] In certain embodiments, the inhibitory immunoregulator is a component of the CD47 signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the CD47 signaling pathway. In certain embodiments, the checkpoint inhibitor of the CD47 signaling pathway is a CD47 inhibitor. In certain embodiments, the checkpoint inhibitor of the CD47 signaling pathway is a SIRPa inhibitor.

[0579] In certain embodiments, the inhibitory immunoregulator is a component of the PVRIG signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the PVRIG signaling pathway. In certain embodiments, the checkpoint inhibitor of the PVRIG signaling pathway is a PVRIG inhibitor. In certain embodiments, the checkpoint inhibitor of the PVRIG signaling pathway is a PVRIG ligand inhibitor.

[0580] In certain embodiments, the inhibitory immunoregulator is a component of the CSF1R signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the CSF1R signaling pathway. In certain embodiments, the checkpoint inhibitor of the CSF1R signaling pathway is a CSF1R inhibitor. In certain embodiments, the checkpoint inhibitor of the CSF1R signaling pathway is a CSF1 inhibitor.

[0581] In certain embodiments, the inhibitory immunoregulator is a component of the NOX signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the NOX signaling pathway, e.g., a NOX inhibitor.

[0582] In certain embodiments, the inhibitory immunoregulator is a component of the TDO signaling pathway. Accordingly, certain embodiments of the disclosure provide for administering to a subject a checkpoint inhibitor of the TDO signaling pathway, e.g., a TDO inhibitor.

[0583] Exemplary PD-1 inhibitors include, without limitation, anti-PD-1 antibodies such as BGB-A317 (BeiGene; see US 8,735,553, WO 2015 / 35606 and US 2015 / 0079109), cemiplimab (Regeneron; see WO 2015 / 112800) and lambrolizumab (e.g., disclosed as hPD109A and its humanized derivatives h409Al, h409A16 and h409A17 in WO2008 / 156712), AB137132 (Abeam), EH12.2H7 and RMP1-14 (#BE0146; Bioxcell Lifesciences Pvt. LTD.), MIH4 (Aflymetrix eBioscience), nivolumab (OPDIVO, BMS-936558; Bristol Myers Squibb; see WO 2006 / 121168), pembrolizumab (KEYTRUDA; MK-3475; Merck; see WO 2008 / 156712), pidilizumab (CT-011; CureTech; see Hardy et al., 1994, Cancer Res., 54(22):5793-6 and WO 2009 / 101611), PDR001 (Novartis; see WO 2015 / 112900), MEDI0680 (AMP-514; AstraZeneca; see WO 2012 / 145493), TSR-042 (see WO 2014 / 179664), REGN-2810 (H4H7798N; cf. US 2015 / 0203579), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., 2007, J. Hematol. Oncol. 70: 136), AMP- 224 (GSK-2661380; cf. Li et al., 2016, Int J Mol Sci 17(7): 1151 and WO 2010 / 027827 and WO 2011 / 066342), PF-06801591 (Pfizer), BGB-A317 (BeiGene; see WO 2015 / 35606 and US 2015 / 0079109), BI 754091, sintilimab (IBI308), camrelizumab (SHR-1210) (see WO2015 / 085847), and antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 as described in WO 2006 / 121168, INCSHR1210 (Jiangsu Hengrui Medicine; also known as camrelizumab (SHR- 1210); see WO 2015 / 085847), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see W02014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang et al., 2017, J. Hematol. Oncol. 70: 136), STI-1110 (Sorrento Therapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics; see WO 2017 / 19846), IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540), anti-PD-1 antibodies as described, e.g., in US 7,488,802, US 8,008,449, US 8,168,757, WO 03 / 042402, WO 2010 / 089411 (further disclosing anti-PD-Ll antibodies), WO 2010 / 036959, WO 2011 / 159877 (further disclosing antibodies against TIM-3), WO 2011 / 082400, WO 2011 / 161699, WO 2009 / 014708, WO 03 / 099196, WO 2009 / 114335, WO 2012 / 145493 (further disclosing antibodies against PD-L1), WO 2015 / 035606, WO 2014 / 055648 (further disclosing anti-KIR antibodies), US 2018 / 0185482 (further disclosing anti-PD-Ll and anti-TIGIT antibodies), US 8,008,449, US 8,779,105, US 6,808,710, US 8,168,757, US 2016 / 0272708, and US 8,354,509, small molecule antagonists to the PD-1 signaling pathway as disclosed, e.g., in Shaabani et al., 2018, Expert Op Ther Pat, 28(9):665-678 and Sasikumar and Ramachandra, 2018, BioDrugs, 32(5):481-497, siRNAs directed to PD-1 as disclosed, e.g., in WO 2019 / 000146 and WO 2018 / 103501, soluble PD-1 proteins as disclosed in WO 2018 / 222711 and oncolytic viruses comprising a soluble form of PD-1 as described, e.g., in WO 2018 / 022831.

[0584] In a certain embodiment, the PD-1 inhibitor is nivolumab (OPDIVO; BMS-936558), pembrolizumab (KEYTRUDA; MK-3475), pidilizumab (CT-011), PDR001, MEDI0680 (AMP- 514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF-06801591, BGB-A317, BI 754091, sintilimab (IBI308), or camrelizumab (SHR-1210).

[0585] Exemplary PD-1 ligand inhibitors are PD-L1 inhibitors and PD-L2 inhibitors and include, without limitation, anti-PD-Ll antibodies such as MEDI4736 (durvalumab; AstraZeneca; see WO 2011 / 066389), MSB-0010718C (see US 2014 / 0341917), YW243.55.S70 (see SEQ ID NO: 20 of WO 2010 / 077634 and US 8,217,149), MIH1 (Afiymetrix eBioscience; cf. EP 3 230 319), MDX- 1105 (Roche / Genentech; see W02013019906 and US 8,217,149) STI-1014 (Sorrento; see W02013 / 181634), CK-301 (Checkpoint Therapeutics), KN035 (3D Med / Alphamab; see Zhang et al., 2017, Cell Discov. 3:17004), atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267; see US 9,724,413), BMS-936559 (Bristol Myers Squibb; see US 7,943,743, WO 2013 / 173223), avelumab (bavencio; cf. US 2014 / 0341917), LY3300054 (Eli Lilly Co.), CX-072 (Proclaim-CX-072; also called CytomX; see W02016 / 149201), FAZ053, KN035 (see W02017020801 and W02017020802), sugemalimab (CS1001), MDX-1105 (see US 2015 / 0320859), anti-PD-Ll antibodies disclosed in US 7,943,743, including 3G10, 12A4 (also referred to as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, anti-PD-Ll antibodies as described in WO 201...

Claims

Claims1. A composition or medical preparation comprising:(a) a bispecific binding agent comprising a first binding domain that binds to human CLDN 18.2, a second binding domain that binds to human CLDN 18.2, and a third binding domain that binds to human CD3, wherein the binding agent comprises four polypeptide chains, wherein(i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 27;(ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 28;(iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and(iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and(b) an agent stabilizing or increasing expression of CLDN 18.2.

2. The composition or medical preparation of claim 1, wherein the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 27.

3. The composition or medical preparation of claim 1 or 2, wherein the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 28.

4. The composition or medical preparation of any one of claims 1 to 3, wherein the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29.

5. The composition or medical preparation of any one of claims 1 to 4, wherein the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29.

6. The composition or medical preparation of any one of claims 1 to 5, wherein(i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: T1 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 27;(ii) the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 28;(iii) the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and(iv) the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29.

7. A composition or medical preparation comprising:(a) a bispecific binding agent comprising a first binding domain that binds to human CLDN18.2, a second binding domain that binds to human CLDN18.2, and a third binding domain that binds to human CD3, wherein the binding agent comprises four polypeptide chains, wherein(i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27;(ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28;(iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and(iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and(b) an agent stabilizing or increasing expression of CLDN18.2.

8. The composition or medical preparation of claim 7, wherein the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27.

9. The composition or medical preparation of claim 7 or 8, wherein the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28.

10. The composition or medical preparation of any one of claims 7 to 9, wherein the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29.

11. The composition or medical preparation of any one of claims 7 to 10, wherein the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29.

12. The composition or medical preparation of any one of claims 7 to 11, wherein(i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27;(ii) the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28;(iii) the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29; and(iv) the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29.

13. A composition or medical preparation comprising:(a) a bispecific binding agent comprising a first binding domain that binds to human CLDNl 8.2, a second binding domain that binds to human CLDN18.2, and a third binding domain that binds to human CD3, wherein the binding agent is encoded by one or more nucleic acid molecules comprising:(i) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 27;(ii) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 28; and(iii) a third nucleic acid sequence encoding a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 29; and(b) an agent stabilizing or increasing expression of CLDNl 8.2.

14. The composition or medical preparation of claim 13, wherein the one or more nucleic acid molecules is a set of nucleic acids.

15. The composition or medical preparation of claim 13 or 14, wherein the bispecific binding agent comprises four polypeptide chains, wherein(i) the first polypeptide chain is encoded by the first nucleic acid sequence;(ii) the second polypeptide chain is encoded by the second nucleic acid sequence;(iii) the third polypeptide chain is encoded by the third nucleic acid sequence; and(iv) the fourth polypeptide chain is encoded by the third nucleic acid sequence.

16. The composition or medical preparation of claim 15, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncation variant thereof,wherein the C-terminal truncation variant of SEQ ID NO: 27 comprises a deletion of lysine at position 447 of SEQ ID NO: 27.

17. The composition or medical preparation of claim 15 or 16, wherein the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 28 comprises a deletion of lysine at position 720 of SEQ ID NO: 28.

18. The composition or medical preparation of any one of claims 15 to 17, wherein the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29.

19. The composition or medical preparation of any one of claims 15 to 18, wherein the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29.

20. The composition or medical preparation of any one of claims 15 to 19, wherein(i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO:27 comprises a deletion of lysine at position 447 of SEQ ID NO: 27;(ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO:28 comprises a deletion of lysine at position 720 of SEQ ID NO: 28;(iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and(iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29.

21. The composition or medical preparation of any one of claims 15 to 20, wherein(i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 27 comprises a deletion of lysine at position 447 of SEQ ID NO: 27;(ii) the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 28 comprises a deletion of lysine at position 720 of SEQ ID NO: 28;(iii) the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29; and(iv) the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29.

22. The composition or medical preparation of any one of claims 1 to 21, wherein the first polypeptide chain interacts with the second polypeptide chain and with the third polypeptide chain.

23. The composition or medical preparation of any one of claims 1 to 12, and 15 to 22, wherein the second polypeptide chain interacts with the fourth polypeptide chain.

24. The composition or medical preparation of any one of claims 1 to 12, and 15 to 23, wherein the first polypeptide chain interacts with the second polypeptide chain and with the third polypeptide chain and the second polypeptide chain interacts with the fourth polypeptide chain.

25. The composition or medical preparation of any one of claims 1 to 24, wherein the first polypeptide chain comprises, from N-terminus to C-terminus,(i) a variable region of a heavy chain (VH) derived from an immunoglobulin that binds to human CLDN18.2 (VH(CLDN18.2)),(ii) a constant region 1 of a heavy chain (CHI) derived from an immunoglobulin or a functional variant thereof,(iii) a constant region 2 of a heavy chain (CH2) derived from an immunoglobulin or a functional variant thereof, and(iv) a constant region 3 of a heavy chain (CH3) derived from an immunoglobulin or a functional variant thereof.

26. The composition or medical preparation of any one of claims 1 to 25, wherein the second polypeptide chain comprises, from N-terminus to C-terminus,(i) a variable region of a heavy chain (VH) derived from an immunoglobulin that binds to human CLDN18.2 (VH(CLDN18.2)),(ii) a constant region 1 of a heavy chain (CHI) derived from an immunoglobulin or a functional variant thereof,(iii) a variable region of a light chain (VL) derived from an immunoglobulin that binds to human CD3 (VL(CD3)),(iv) a variable region of a heavy chain (VH) derived from an immunoglobulin that binds to human CD3 (VH(CD3)),(v) a constant region 2 of a heavy chain (CH2) derived from an immunoglobulin or a functional variant thereof, and(vi) a constant region 3 of a heavy chain (CH3) derived from an immunoglobulin or a functional variant thereof.

27. The composition or medical preparation of any one of claims 1 to 26, wherein the third polypeptide chain comprises, from N-terminus to C-terminus,(i) a variable region of a light chain (VL) derived from an immunoglobulin that binds to human CLDN18.2 (VL(CLDN18.2)), and(ii) a constant region of a light chain (CL) derived from an immunoglobulin or a functional variant thereof.

28. The composition or medical preparation of any one of claims 1 to 12, and 15 to 27, wherein the fourth polypeptide chain comprises, from N-terminus to C-terminus,(i) a variable region of a light chain (VL) derived from an immunoglobulin that binds to human CLDN18.2 (VL(CLDN18.2)), and(ii) a constant region of a light chain (CL) derived from an immunoglobulin or a functional variant thereof.

29. The composition or medical preparation of claim 27 or 28, wherein the VH(CLDN18.2) on the first polypeptide chain and the VL(CLDN18.2) on the third polypeptide chain interact to form a binding domain that binds to human CLDN18.2.

30. The composition or medical preparation of claim 28 or 29, wherein the VH(CLDN 18.2) on the second polypeptide chain and the VL(CLDN18.2) on the fourth polypeptide chain interact to form a binding domain that binds to human CLDN18.2.

31. The composition or medical preparation of any one of claims 26 to 30, wherein theVH(CD3) and the VL(CD3) interact to form a binding domain that binds to human CD3.

32. The composition or medical preparation of any one of claims 25 to 31, wherein the VH(CLDN18.2) comprises a CDR1 comprising the amino acid sequence SYWIN (SEQ ID NO: 10), a CDR2 comprising the amino acid sequence NIYPSDSYTNYNQKFQG (SEQ ID NO: 11), and a CDR3 comprising the amino acid sequence SWRGNSFDY (SEQ ID NO: 12).

33. The composition or medical preparation of any one of claims 27 to 32, wherein the VL(CLDN18.2) comprises a CDR1 comprising the amino acid sequence KSSQSLLNSGNQKNYLT (SEQ ID NO: 13), a CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO: 14), and a CDR3 comprising the amino acid sequence QNDYSYPFT (SEQ ID NO: 15).

34. The composition or medical preparation of any one of claims 26 to 33, wherein theVH(CD3) comprises a CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 18), a CDR2 comprising the amino acid sequence RIRSKANNYATYYADSVKG (SEQ ID NO: 23), and a CDR3 comprising the amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO: 19).

35. The composition or medical preparation of any one of claims 26 to 34, wherein theVL(CD3) comprises a CDR1 comprising the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 20), a CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 21), and a CDR3 comprising the amino acid sequence ALWYSNHWV (SEQ ID NO: 22).

36. The composition or medical preparation of any one of claims 26 to 35, wherein the CH2 on the first polypeptide chain interacts with the CH2 on the second polypeptide chain and / or the CH3 on the first polypeptide chain interacts with the CH3 on the second polypeptide chain.

37. The composition or medical preparation of any one of claims 27 to 36, wherein the CHI on the first polypeptide chain interacts with the CL on the third polypeptide chain.

38. The composition or medical preparation of any one of claims 28 to 37, wherein the CHI on the second polypeptide chain interacts with the CL on the fourth polypeptide chain.

39. The composition or medical preparation of any one of claims 25 to 38, wherein the immunoglobulin is IgGl.

40. The composition or medical preparation of claim 39, wherein the IgGl is human IgGl.

41. The composition or medical preparation of any one of claims 25 to 40, wherein theVH(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 16.

42. The composition or medical preparation of any one of claims 27 to 41, wherein the VL(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 17.

43. The composition or medical preparation of any one of claims 26 to 42, wherein the VL(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 24.

44. The composition or medical preparation of any one of claims 26 to 43, wherein the VH(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 25.

45. The composition or medical preparation of any one of claims 27 to 44, wherein the VH(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ IDNO: 16, the VL(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 17, the VH(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 25, and the VL(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 24.

46. The composition or medical preparation of any one of claims 25 to 45, wherein the VH(CLDN18.2), VL(CLDN18.2), VH(CD3) and / or VL(CD3) are humanized.

47. The composition or medical preparation of any one of claims 25 to 46, wherein on the first polypeptide chain the CHI is connected to the CH2 by a peptide linker LI.

48. The composition or medical preparation of claim 47, wherein the peptide linker LI comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 6) or a functional variant thereof.

49. The composition or medical preparation of any one of claims 26 to 48, wherein the VL(CD3) is connected to the CHI by a peptide linker L2.

50. The composition or medical preparation of claim 49, wherein the peptide linker L2 comprises the amino acid sequence (G4S)xor a functional variant thereof, wherein x is 2, 3, 4, 5 or 6.

51. The composition or medical preparation of claim 49 or 50, wherein the peptide linker L2 comprises the amino acid sequence (G4S)2(SEQ ID NO: 5) or a functional variant thereof.

52. The composition or medical preparation of any one of claims 26 to 51, wherein the VL(CD3) and the VH(CD3) are connected to one another by a peptide linker L3.

53. The composition or medical preparation of claim 52, wherein the peptide linker L3 comprises the amino acid sequence (GKPGS)xor a functional variant thereof, wherein x is 2, 3, 4, 5 or 6.

54. The composition or medical preparation of claim 52 or 53, wherein the peptide linker L3 comprises the amino acid sequence (GKPGS)4(SEQ ID NO: 2) or a functional variant thereof.

55. The composition or medical preparation of any one of claims 26 to 54, wherein the VH(CD3) is connected to the CH2 by a peptide linker LA.

56. The composition or medical preparation of claim 55, wherein the peptide linker L4 comprises the amino acid sequence (G4S)xor a functional variant thereof, wherein x is 2, 3, 4, 5 or 6.

57. The composition or medical preparation of claim 55 or 56, wherein the peptide linker L4 comprises the amino acid sequence (G4S)2(SEQ ID NO: 5) or a functional variant thereof.

58. The composition or medical preparation of any one of claims 1 to 57, wherein the first polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 7.

59. The composition or medical preparation of any one of claims 1 to 58, wherein the second polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 8.

60. The composition or medical preparation of any one of claims 1 to 59, wherein the third and / or fourth polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 9.

61. The composition or medical preparation of any one of claims 1 to 60, wherein the bispecific binding agent binds to an extracellular portion of human CLDN18.2.

62. The composition or medical preparation of any one of claims 1 to 61, wherein binding of the bispecific binding agent to CD3 on T cells results in proliferation and / or activation of the T cells.

63. The composition or medical preparation of any one of claims 1 to 62, wherein the bispecific binding agent induces T cell-mediated cytotoxicity against cancer cells expressing CLDN18.2.

64. The composition or medical preparation of any one of claims 1 to 63, wherein the agent stabilizing or increasing expression of CLDN18.2 comprises a chemotherapeutic agent.

65. The composition or medical preparation of any one of claims 1 to 64, wherein the agent stabilizing or increasing expression of CLDN18.2 comprises a cytotoxic and / or cytostatic agent.

66. The composition or medical preparation of any one of claims 1 to 65, wherein the agent stabilizing or increasing expression of CLDN18.2 comprises an agent selected from the group consisting of anthracyclines, nucleoside analogs, platinum compounds, camptothecin analogs and taxanes, prodrugs thereof, salts thereof, and combinations thereof.

67. The composition or medical preparation of any one of claims 1 to 66, wherein the agent stabilizing or increasing expression of CLDN18.2 comprises a nucleoside analog, a prodrug thereof, or a salt thereof.

68. The composition or medical preparation of any one of claims 1 to 67, wherein the agent stabilizing or increasing expression of CLDN18.2 comprises a taxane, a prodrug thereof, or a salt thereof.

69. The composition or medical preparation of any one of claims 1 to 68, wherein the agent stabilizing or increasing expression of CLDN18.2 comprises (i) gemcitabine, a prodrug thereof, or a salt thereof, (ii) paclitaxel, a prodrug thereof, or a salt thereof, or (iii) a combination of gemcitabine, a prodrug thereof, or a salt thereof, and paclitaxel, a prodrug thereof, or a salt thereof.

70. The composition or medical preparation of any one of claims 1 to 66 comprising:(i) the bispecific binding agent;(ii) oxaliplatin;(iii) fluorouracil or a precursor thereof; and optionally(iv) folinic acid.

71. The composition or medical preparation of claim 70 further comprising pembrolizumab.

72. The composition or medical preparation of claim 70 further comprising irinothecan.

73. The composition or medical preparation of any one of claims 1 to 66 comprising:(i) the bispecific binding agent;(ii) paclitaxel; and(iii) ramucirumab.

74. The composition or medical preparation of any one of claims 1 to 73, which is a pharmaceutical composition.

75. The composition or medical preparation of claim 74, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

76. The composition or medical preparation of any one of claims 1 to 73, which is a kit.

77. The composition or medical preparation of claim 76, wherein the bispecific binding agent and the agent stabilizing or increasing expression of CLDN18.2 are in separate vials.

78. The composition or medical preparation of claim 76 or 77, further comprising instructions for use of the bispecific binding agent and the agent stabilizing or increasing expression of CLDN18.2 for treating or preventing cancer.

79. The composition or medical preparation of any one of claims 1 to 78 for pharmaceutical use.

80. The composition or medical preparation of claim 79, wherein the pharmaceutical use comprises a therapeutic or prophylactic treatment of a disease or disorder.

81. The composition or medical preparation of claim 80, wherein the therapeutic or prophylactic treatment of a disease or disorder comprises treating or preventing cancer.

82. The composition or medical preparation of any one of claims 78 to 81, wherein the cancer involves cancer cells expressing CLDN18.2.

83. The composition or medical preparation of any one of claims 78 to 82, wherein the cancer is selected from the group consisting of gastric cancer, particularly gastric adenocarcinoma, esophageal cancer, cancer of the gastroesophageal junction (GEJ), particularly GEJ adenocarcinoma, pancreatic cancer, particularly pancreatic adenocarcinoma, lung cancer such as non small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, rectal cancer, colorectal cancer, hepatic cancer, head-neck cancer, bile duct cancer, cancer of the gallbladder and the metastasis thereof, a Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis.

84. The composition or medical preparation of any one of claims 1 to 83, which is for administration to a human.

85. A method of treating or preventing cancer in a subject comprising administering to the subject:(a) a bispecific binding agent comprising a first binding domain that binds to human CLDN18.2, a second binding domain that binds to human CLDN18.2, and a third binding domain that binds to human CD3, wherein the binding agent comprises four polypeptide chains, wherein(i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 27;(ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 28;(iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and(iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and(b) an agent stabilizing or increasing expression of CLDN18.2.

86. The method of claim 85, wherein the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 27.

87. The method of claim 85 or 86, wherein the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 28.

88. The method of any one of claims 85 to 87, wherein the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29.

89. The method of any one of claims 85 to 88, wherein the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29.

90. The method of any one of claims 85 to 89, wherein(i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 27;(ii) the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 28;(iii) the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and(iv) the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence which has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29.

91. A method of treating or preventing cancer in a subject comprising administering to the subject:(a) a bispecific binding agent comprising a first binding domain that binds to human CLDN18.2, a second binding domain that binds to human CLDN18.2, and a third binding domain that binds to human CD3, wherein the binding agent comprises four polypeptide chains, wherein(i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27;(ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28;(iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and(iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and(b) an agent stabilizing or increasing expression of CLDN18.2.

92. The method of claim 91, wherein the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27.

93. The method of claim 91 or 92, wherein the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28.

94. The method of any one of claims 91 to 93 , wherein the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29.

95. The method of any one of claims 91 to 94, wherein the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29.

96. The method of any one of claims 91 to 95, wherein(i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27;(ii) the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28;(iii) the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29; and(iv) the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29.

97. A method of treating or preventing cancer in a subject comprising administering to the subject:(a) a bispecific binding agent comprising a first binding domain that binds to human CLDN18.2, a second binding domain that binds to human CLDN18.2, and a third binding domain that binds to human CD3, wherein the binding agent is encoded by one or more nucleic acid molecules comprising:(i) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 27;(ii) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 28; and(iii) a third nucleic acid sequence encoding a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 29; and(b) an agent stabilizing or increasing expression of CLDN18.2.

98. The method of claim 97, wherein the one or more nucleic acid molecules is a set of nucleic acids.

99. The method of claim 97 or 98, wherein the bispecific binding agent comprises four polypeptide chains, wherein(i) the first polypeptide chain is encoded by the first nucleic acid sequence;(ii) the second polypeptide chain is encoded by the second nucleic acid sequence;(iii) the third polypeptide chain is encoded by the third nucleic acid sequence; and(iv) the fourth polypeptide chain is encoded by the third nucleic acid sequence.

100. The method of claim 99, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 27 comprises a deletion of lysine at position 447 of SEQ ID NO: 27.

101. The method of claim 99 or 100, wherein the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 28 comprises a deletion of lysine at position 720 of SEQ ID NO: 28.

102. The method of any one of claims 99 to 101, wherein the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29.

103. The method of any one of claims 99 to 102, wherein the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29.

104. The method of any one of claims 99 to 103, wherein(i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO:27 comprises a deletion of lysine at position 447 of SEQ ID NO: 27;(ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO:28 comprises a deletion of lysine at position 720 of SEQ ID NO: 28;(iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and(iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29.

105. The method of any one of claims 99 to 104, wherein(i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 27 comprises a deletion of lysine at position 447 of SEQ ID NO: 27;(ii) the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncation variant thereof, wherein the C-terminal truncation variant of SEQ ID NO: 28 comprises a deletion of lysine at position 720 of SEQ ID NO: 28;(iii) the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29; and(iv) the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29.

106. The method of any one of claims 85 to 105, wherein the first polypeptide chain interacts with the second polypeptide chain and with the third polypeptide chain.

107. The method of any one of claims 85 to 96, and 99 to 106, wherein the second polypeptide chain interacts with the fourth polypeptide chain.

108. The method of any one of claims 85 to 96, and 99 to 107, wherein the first polypeptide chain interacts with the second polypeptide chain and with the third polypeptide chain and the second polypeptide chain interacts with the fourth polypeptide chain.

109. The method of any one of claims 85 to 108, wherein the first polypeptide chain comprises, from N-terminus to C-terminus,(i) a variable region of a heavy chain (VH) derived from an immunoglobulin that binds to human CLDN18.2 (VH(CLDN18.2)),(ii) a constant region 1 of a heavy chain (CHI) derived from an immunoglobulin or a functional variant thereof,(iii) a constant region 2 of a heavy chain (CH2) derived from an immunoglobulin or a functional variant thereof, and(iv) a constant region 3 of a heavy chain (CH3) derived from an immunoglobulin or a functional variant thereof.

110. The method of any one of claims 85 to 109, wherein the second polypeptide chain comprises, from N-terminus to C-terminus,(i) a variable region of a heavy chain (VH) derived from an immunoglobulin that binds to human CLDN18.2 (VH(CLDN18.2)),(ii) a constant region 1 of a heavy chain (CHI) derived from an immunoglobulin or a functional variant thereof,(iii) a variable region of a light chain (VL) derived from an immunoglobulin that binds to human CD3 (VL(CD3)),(iv) a variable region of a heavy chain (VH) derived from an immunoglobulin that binds to human CD3 (VH(CD3)),(v) a constant region 2 of a heavy chain (CH2) derived from an immunoglobulin or a functional variant thereof, and(vi) a constant region 3 of a heavy chain (CH3) derived from an immunoglobulin or a functional variant thereof.

111. The method of any one of claims 85 to 110, wherein the third polypeptide chain comprises, from N-terminus to C-terminus,(i) a variable region of a light chain (VL) derived from an immunoglobulin that binds to human CLDN18.2 (VL(CLDN18.2)), and(ii) a constant region of a light chain (CL) derived from an immunoglobulin or a functional variant thereof.

112. The method of any one of claims 85 to 96, and 99 to 111, wherein the fourth polypeptide chain comprises, from N-terminus to C-terminus,(i) a variable region of a light chain (VL) derived from an immunoglobulin that binds to human CLDN18.2 (VL(CLDN18.2)), and(ii) a constant region of a light chain (CL) derived from an immunoglobulin or a functional variant thereof.

113. The method of claim 111 or 112, wherein the VH(CLDN18.2) on the first polypeptide chain and the VL(CLDN18.2) on the third polypeptide chain interact to form a binding domain that binds to human CLDN18.2.

114. The method of claim 112 or 113, wherein the VH(CLDN18.2) on the second polypeptide chain and the VL(CLDN18.2) on the fourth polypeptide chain interact to form a binding domain that binds to human CLDN18.2.

115. The method of any one of claims 110 to 114, wherein the VH(CD3) and the VL(CD3) interact to form a binding domain that binds to human CD3.

116. The method of any one of claims 109 to 115, wherein the VH(CLDN18.2) comprises a CDR1 comprising the amino acid sequence SYWIN (SEQ ID NO: 10), a CDR2 comprising the amino acid sequence NIYPSDSYTNYNQKFQG (SEQ ID NO: 11), and a CDR3 comprising the amino acid sequence SWRGNSFDY (SEQ ID NO: 12).

117. The method of any one of claims 111 to 116, wherein the VL(CLDN 18.2) comprises a CDR1 comprising the amino acid sequence KSSQSLLNSGNQKNYLT (SEQ ID NO: 13), a CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO: 14), and a CDR3 comprising the amino acid sequence QNDYSYPFT (SEQ ID NO: 15).

118. The method of any one of claims 110 to 117, wherein the VH(CD3) comprises a CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 18), a CDR2 comprising the amino acid sequence RIRSKANNYATYYADSVKG (SEQ ID NO: 23), and a CDR3 comprising the amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO: 19).

119. The method of any one of claims 110 to 118, wherein the VL(CD3) comprises a CDR1 comprising the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 20), a CDR2 comprising the amino acid sequence GINKRAP (SEQ ID NO: 21), and a CDR3 comprising the amino acid sequence ALWYSNHWV (SEQ ID NO: 22).

120. The method of any one of claims 110 to 119, wherein the CH2 on the first polypeptide chain interacts with the CH2 on the second polypeptide chain and / or the CH3 on the first polypeptide chain interacts with the CH3 on the second polypeptide chain.

121. The method of any one of claims 111 to 120, wherein the CHI on the first polypeptide chain interacts with the CL on the third polypeptide chain.

122. The method of any one of claims 112 to 121, wherein the CHI on the second polypeptide chain interacts with the CL on the fourth polypeptide chain.

123. The method of any one of claims 109 to 122, wherein the immunoglobulin is IgGl .

124. The method of claim 123, wherein the IgGl is human IgGl.

125. The method of any one of claims 109 to 124, wherein the VH(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 16.

126. The method of any one of claims 111 to 125, wherein the VL(CLDN 18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 17.

127. The method of any one of claims 110 to 126, wherein the VL(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 24.

128. The method of any one of claims 110 to 127, wherein the VH(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 25.

129. The method of any one of claims 111 to 128, wherein the VH(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 16, the VL(CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 17, the VH(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 25, and the VL(CD3) comprises or consists of the amino acid sequence represented by SEQ ID NO: 24.

130. The method of any one of claims 109 to 129, wherein the VH(CLDN18.2), VL(CLDN18.2), VH(CD3) and / or VL(CD3) are humanized.

131. The method of any one of claims 109 to 130, wherein on the first polypeptide chain the CHI is connected to the CH2 by a peptide linker LI .

132. The method of claim 131, wherein the peptide linker LI comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 6) or a functional variant thereof.

133. The method of any one of claims 110 to 132, wherein the VL(CD3) is connected to the CHI by a peptide linker L2.

134. The method of claim 133, wherein the peptide linker L2 comprises the amino acid sequence (G4S)xor a functional variant thereof, wherein x is 2, 3, 4, 5 or 6.

135. The method of claim 133 or 134, wherein the peptide linker L2 comprises the amino acid sequence (G4S)2(SEQ ID NO: 5) or a functional variant thereof.

136. The method of any one of claims 110 to 135, wherein the VL(CD3) and the VH(CD3) are connected to one another by a peptide linker L3.

137. The method of claim 136, wherein the peptide linker L3 comprises the amino acid sequence (GKPGS)xor a functional variant thereof, wherein x is 2, 3, 4, 5 or 6.

138. The method of claim 136 or 137, wherein the peptide linker L3 comprises the amino acid sequence (GKPGS)4(SEQ ID NO: 2) or a functional variant thereof.

139. The method of any one of claims 110 to 138, wherein the VH(CD3) is connected to the CH2 by a peptide linker L4.

140. The method of claim 139, wherein the peptide linker L4 comprises the amino acid sequence (G4S)% or a functional variant thereof, wherein x is 2, 3, 4, 5 or 6.

141. The method of claim 139 or 140, wherein the peptide linker L4 comprises the amino acid sequence (G4S)2(SEQ ID NO: 5) or a functional variant thereof.

142. The method of any one of claims 85 to 141, wherein the first polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 7.

143. The method of any one of claims 85 to 142, wherein the second polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 8.

144. The method of any one of claims 85 to 143, wherein the third and / or fourth polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 9.

145. The method of any one of claims 85 to 144, wherein the bispecific binding agent binds to an extracellular portion of human CLDN18.2.

146. The method of any one of claims 85 to 145, wherein binding of the bispecific binding agent to CD3 on T cells results in proliferation and / or activation of the T cells.

147. The method of any one of claims 85 to 146, wherein the bispecific binding agent induces T cell-mediated cytotoxicity against cancer cells expressing CLDN18.2.

148. The method of any one of claims 85 to 147, wherein the agent stabilizing or increasing expression of CLDN18.2 comprises a chemotherapeutic agent.

149. The method of any one of claims 85 to 148, wherein the agent stabilizing or increasing expression of CLDN18.2 comprises a cytotoxic and / or cytostatic agent.

150. The method of any one of claims 85 to 149, wherein the agent stabilizing or increasing expression of CLDN18.2 comprises an agent selected from the group consisting of anthracyclines, nucleoside analogs, platinum compounds, camptothecin analogs and taxanes, prodrugs thereof, salts thereof, and combinations thereof .

151. The method of any one of claims 85 to 150, wherein the agent stabilizing or increasing expression of CLDN18.2 comprises a nucleoside analog, a prodrug thereof, or a salt thereof.

152. The method of any one of claims 85 to 151, wherein the agent stabilizing or increasing expression of CLDN18.2 comprises a taxane, a prodrug thereof, or a salt thereof .

153. The method of any one of claims 85 to 152, wherein the agent stabilizing or increasing expression of CLDN18.2 comprises (i) gemcitabine, a prodrug thereof, or a salt thereof, (ii) paclitaxel, a prodrug thereof, or a salt thereof, or (iii) a combination of gemcitabine, a prodrug thereof, or a salt thereof, and paclitaxel, a prodrug thereof, or a salt thereof.

154. The method of any one of claims 85 to 150 comprising administering to the subject:(i) the bispecific binding agent;(ii) oxaliplatin;(iii) fluorouracil or a precursor thereof; and optionally(iv) folinic acid.

155. The method of claim 154 further comprising administering to the subject pembrolizumab.

156. The method of claim 154 further comprising administering to the subject irinothecan.

157. The method of any one of claims 85 to 150 comprising administering to the subject:(i) the bispecific binding agent;(ii) paclitaxel; and(iii) ramucirumab.

158. The method of any one of claims 85 to 157, wherein the bispecific binding agent and the agent stabilizing or increasing expression of CLDN18.2 are present in the same pharmaceutical composition or in separate pharmaceutical compositions.

159. The method of claim 158, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

160. The method of any one of claims 85 to 159, wherein the cancer involves cancer cells expressing CLDN18.2.

161. The method of any one of claims 85 to 160, wherein the cancer is selected from the group consisting of gastric cancer, particularly gastric adenocarcinoma, esophageal cancer, cancer of the gastroesophageal junction (GEJ), particularly GEJ adenocarcinoma, pancreatic cancer, particularly pancreatic adenocarcinoma, lung cancer such as non small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, rectal cancer, colorectal cancer, hepatic cancer, head-neck cancer, bile duct cancer, cancer of the gallbladder and the metastasis thereof, a Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis.

162. The method of claim 155 or 157, wherein the cancer is stomach cancer and / or gastroesophageal junction cancer.

163. The method of claim 156, wherein the cancer is pancreatic cancer.

164. The method of any one of claims 85 to 163, wherein the subject is a human.

165. The composition or medical preparation of any one of claims 1 to 84 for use in the method of any one of claims 85 to 164.