Combination therapy comprising a bispecific binding agent that binds CLDN18.2 and CD3 and an immune checkpoint inhibitor

Abstract

The present invention provides a combination therapy comprising a bispecific binding agent comprising two binding domains that bind to CLDN18.2 and one that binds to CD3, and an immune checkpoint inhibitor, which is effective in treating cancers involving cancer cells that express CLDN18.2.

Description

[Technical Field]

[0001] The present invention provides combination therapies comprising a bispecific binding agent comprising two binding domains that bind to CLDN18.2 and a binding domain that binds to CD3, and an immune checkpoint inhibitor. [Background technology]

[0002] Cancer is the second leading cause of death worldwide, and is expected to be responsible for an estimated 9.6 million deaths in 2018 (Non-Patent Document 1). In general, once solid tumors metastasize, the 5-year survival rate is approximately 25% or less, with exceptions such as germ cell and certain carcinoid tumors. The poor prognosis of certain cancers highlights the need for additional therapeutic approaches. The tight junction molecule claudin 18 (CLDN18) is an integral transmembrane protein (tetraspanin) with four transmembrane hydrophobic regions and two extracellular loops (loop 1 encompassed by hydrophobic regions 1 and 2; loop 2 encompassed by hydrophobic regions 3 and 4). CLDN18 exists in two different splicing variants reported in mice and humans (Non-Patent Document 2). The molecular weights of the splicing variants (Genbank accession numbers: splicing variant 1 (CLDN18.1): NP_057453, NM_016369, and splicing variant 2 (CLDN18.2): NM_001002026, NP_001002026) are approximately 27.9 / 27.72 kD. The splice variants CLDN18.1 and CLDN18.2 differ in the N-terminal portion, including the first transmembrane (TM) region and loop 1, but have identical primary protein sequences at the C-terminus.

[0003] In normal tissues, there is no detectable CLDN18.2 expression, except in the stomach, where it is expressed exclusively on short-lived differentiated gastric epithelial cells. CLDN18.2 is maintained during malignant transformation and is therefore frequently displayed on the surface of human gastric cancer cells. Furthermore, this pan-tumor antigen is ectopically activated at significant levels in adenocarcinomas of the esophagus, pancreas, and lung. CLDN18.2 protein is also localized in lymph node metastases of gastric adenocarcinoma and distant metastases, particularly to the ovary (so-called Krukenberg tumors).

[0004] The differential expression of claudins such as CLDN18.2 between cancer and normal cells, their membrane localization and absence in most toxicity-associated normal tissues may make these molecules attractive targets for cancer immunotherapy, and the use of antibody-based therapeutics to target CLDN18.2 in cancer therapy promises a high level of therapeutic specificity.

[0005] IMAB362 (zolbetuximab, formerly known as claudiximab), a chimeric IgG1 against CLDN18.2, was developed by Ganymed Pharmaceuticals AG. IMAB362 recognizes the first extracellular domain (ECD1) of CLDN18.2 with high affinity and specificity. IMAB362 does not bind to any other claudin family members, including the closely related splicing variant 1 of claudin 18 (CLDN18.1). IMAB362 exhibits precise tumor cell specificity and combines two independent and highly potent mechanisms of action. Upon target binding, IMAB362 primarily mediates cell killing by ADCC and CDC. Accordingly, IMAB362 efficiently lyses CLDN18.2-positive cells, including human gastric cancer cell lines, in vitro and in vivo. The antitumor effects of IMAB362 have been demonstrated in mice bearing xenograft tumors inoculated with CLDN18.2-positive cancer cell lines.

[0006] IgG1 antibodies typically participate in the cellular immune system through interaction of their Fc domains with Fcγ receptors (FcγRs) expressed on various immune cells, including natural killer cells, which are the primary effectors of ADCC. However, IgG1 monoclonal antibodies (mAbs) that induce ADCC have certain limitations, including the widespread distribution of low-affinity Fc receptor variants in the population (up to 80%) and in vivo IgG1 modifications that reduce mAb efficacy (Non-Patent Document 3). Therapeutic antibodies must also compete with the patient's IgG, and high in vivo mAb doses are required. Furthermore, therapeutic antibodies can interact with FcγRIIb (an inhibitory FcγR expressed by B cells, macrophages, dendritic cells, and neutrophils), resulting in negative signaling that reduces their efficacy. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Bray, F. et al., CA:A Cancer Journal for Clinicians, 68:394-424, 2018 [Non-patent document 2] Niimi, Mol. Cell. Biol. 21:7380-90, 2001 [Non-patent document 3] Chames et al., (2009) Br J Pharmacol, 157(2):220-233 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a novel method for treating cancer diseases.

[0009] The present invention generally encompasses treatment of a subject comprising administration of a bispecific binding agent described herein comprising two CLDN18.2-binding domains in Fab format specific for cancer cells and a CD3-binding domain in scFv format specific for the T cell-specific antigen CD3, where the CD3-binding domain can bind to and recruit T cells into a complex, thereby targeting the cytotoxic effects of the T cells to cancer cells. Once formed, the complex can induce signaling in cytotoxic T cells, either by itself or in combination with accessory cells, resulting in the release of cytotoxic mediators. Treatment with the bispecific binding agents disclosed herein is combined with an additional treatment comprising administration of one or more immune checkpoint inhibitors. It is demonstrated herein that treatments comprising the bispecific binding agents disclosed herein and immune checkpoint inhibitors can induce potent anti-tumor effects. [Means for solving the problem]

[0010] Summary of the Invention The present invention generally provides combination therapies comprising a bispecific binding agent that binds CLDN18.2 and CD3 and an immune checkpoint inhibitor. In one embodiment, the bispecific binding agent is a bispecific tetrameric binding agent. In one embodiment, the immune checkpoint inhibitor is a PD-1 antibody.

[0011] The present invention provides the following: (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 said bispecific binding agent comprises four polypeptide chains, wherein: (i) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence represented by SEQ ID NO: 27; (ii) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 28; and (iii) a third nucleic acid sequence encoding a third polypeptide chain comprising the amino acid sequence represented by SEQ ID NO: 29; and (b) an immune checkpoint inhibitor; In one embodiment, the first polypeptide chain consists of the amino acid sequence of SEQ ID NO:27 or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:27. In one embodiment, the second polypeptide chain consists of the amino acid sequence of SEQ ID NO:28 or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:28. In one embodiment, the third polypeptide chain consists of the amino acid sequence of SEQ ID NO:29 or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:29. In one embodiment, the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO:29 or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:29.

[0012] In one embodiment, (i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO:27, or an amino acid sequence that 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 that 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 that 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 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:29.

[0013] The present invention further provides the following: (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 said bispecific binding agent comprises four polypeptide chains, wherein: (i) the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:27; (ii) the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 28; (iii) the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 29; and (iv) the fourth polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:29; and (b) an immune checkpoint inhibitor. In one embodiment, the first polypeptide chain consists of the amino acid sequence of SEQ ID NO:27. In one embodiment, the second polypeptide chain consists of the amino acid sequence of SEQ ID NO:28. In one embodiment, the third polypeptide chain consists of the amino acid sequence of SEQ ID NO:29. In one embodiment, the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO:29. In one embodiment, (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.

[0014] The present invention further provides the following: (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 said bispecific binding agent is encoded by one or more nucleic acid molecules, wherein: (i) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence represented by SEQ ID NO: 27; (ii) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 28; and (iii) a third nucleic acid sequence encoding a third polypeptide chain comprising the amino acid sequence represented by SEQ ID NO: 29; and (b) an immune checkpoint inhibitor. In certain embodiments, the one or more nucleic acid molecules is a set of nucleic acids.

[0015] In one embodiment, the bispecific binding agent comprises four polypeptide chains, wherein: (i) a first polypeptide chain is encoded by a first nucleic acid sequence; (ii) the second polypeptide chain is encoded by a second nucleic acid sequence; (iii) the third polypeptide chain is encoded by a third nucleic acid sequence; and (iv) a fourth polypeptide chain is encoded by a third nucleic acid sequence.

[0016] In one embodiment, the first polypeptide chain comprises the amino acid sequence set forth in 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. In one embodiment, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:28 or a C-terminal truncation mutant thereof, wherein the C-terminal truncation mutant of SEQ ID NO:28 comprises a deletion of lysine at position 720 of SEQ ID NO:28. In one embodiment, the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:29. In one embodiment, the fourth polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:29.

[0017] In one embodiment, (i) the first polypeptide chain comprises the amino acid sequence set forth in 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 set forth in 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 set forth in SEQ ID NO:29; and (iv) the fourth polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:29.

[0018] In one embodiment, (i) the first polypeptide chain consists of the amino acid sequence set forth in 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 set forth in 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 represented by SEQ ID NO: 29; and (iv) The fourth polypeptide chain consists of the amino acid sequence shown in SEQ ID NO:29.

[0019] In one embodiment, the first polypeptide chain interacts with the second polypeptide chain and the third polypeptide chain. In one embodiment, the second polypeptide chain interacts with the fourth polypeptide chain. In one embodiment, the first polypeptide chain interacts with the second polypeptide chain and the third polypeptide chain, and the second polypeptide chain interacts with the fourth polypeptide chain.

[0020] In one embodiment, the first polypeptide chain has, 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 heavy chain constant region 1 (CH1) derived from an immunoglobulin or a functional variant thereof; (iii) a heavy chain constant region 2 (CH2) derived from an immunoglobulin or a functional variant thereof; and (iv) comprises a heavy chain constant region 3 (CH3) derived from an immunoglobulin or a functional variant thereof. In one embodiment, the second polypeptide chain has, from N-terminus to C-terminus, the following: (i) a variable region of a heavy chain (VH) derived from an immunoglobulin that binds to human CLDN18.2 (VH(CLDN18.2)); (ii) a heavy chain constant region 1 (CH1) 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 heavy chain constant region 2 (CH2) derived from an immunoglobulin or a functional variant thereof; and (vi) Contains a heavy chain constant region 3 (CH3) derived from an immunoglobulin or a functional variant thereof. In one embodiment, the third polypeptide chain has, from N-terminus to C-terminus, the following: (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. In one embodiment, the fourth polypeptide chain has, from N-terminus to C-terminus, the following: (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 light chain constant region (CL) derived from an immunoglobulin or a functional variant thereof.

[0021] In one embodiment, VH(CLDN18.2) on the first polypeptide chain and VL(CLDN18.2) on the third polypeptide chain interact to form a binding domain that binds to human CLDN18.2. In one embodiment, VH(CLDN18.2) on the second polypeptide chain and VL(CLDN18.2) on the fourth polypeptide chain interact to form a binding domain that binds to human CLDN18.2. In one embodiment, the VH(CD3) and the VL(CD3) interact to form a binding domain that binds to human CD3.

[0022] In one embodiment, the VH(CLDN18.2) comprises a CDR1 comprising the amino acids SYWIN (SEQ ID NO: 10), a CDR2 comprising the amino acids NIYPSDSYTNYNQKFQG (SEQ ID NO: 11), and a CDR3 comprising the amino acids SWRGNSFDY (SEQ ID NO: 12). In one embodiment, the VL(CLDN18.2) comprises a CDR1 comprising the amino acids KSSQSLLNSGNQKNYLT (SEQ ID NO: 13), a CDR2 comprising the amino acids WASTRES (SEQ ID NO: 14), and a CDR3 comprising the amino acids QNDYSYPFT (SEQ ID NO: 15). In one embodiment, the VH(CD3) comprises a CDR1 comprising the amino acids TYAMN (SEQ ID NO: 18), a CDR2 comprising the amino acids RIRSKANNYATYYADSVKG (SEQ ID NO: 23), and a CDR3 comprising the amino acids HGNFGDSYVSWFAY (SEQ ID NO: 19). In one embodiment, the VL(CD3) comprises a CDR1 comprising the amino acid sequence GSSTGAVTSTNYAN (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). In one embodiment, CH2 on the first polypeptide chain interacts with CH2 on the second polypeptide chain, and / or CH3 on the first polypeptide chain interacts with CH3 on the second polypeptide chain. In one embodiment, CH1 on the first polypeptide chain interacts with CL on the third polypeptide chain. In one embodiment, CH1 on the second polypeptide chain interacts with CL on the fourth polypeptide chain.

[0023] In some embodiments, the immunoglobulin is IgG1. In some embodiments, the IgG1 is human IgG1.

[0024] In one embodiment, the VH(CLDN18.2) comprises or consists of the amino acid sequence set forth in SEQ ID NO:16. In one embodiment, the VL(CLDN18.2) comprises or consists of the amino acid sequence set forth in SEQ ID NO:17. In one embodiment, the VL(CD3) comprises or consists of the amino acid sequence set forth in SEQ ID NO:24. In one embodiment, the VH(CD3) comprises or consists of the amino acid sequence set forth in SEQ ID NO:25.

[0025] In one embodiment, 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 set forth in SEQ ID NO: 25; and The VL(CD3) comprises or consists of the amino acid sequence shown in SEQ ID NO:24. In one embodiment, the VH(CLDN18.2), the VL(CLDN18.2), the VH(CD3) and / or the VL(CD3) are humanized.

[0026] In one embodiment, on the first polypeptide chain, the CH1 is connected to the CH2 by a peptide linker, L1, hi one embodiment, on the first polypeptide chain, the peptide linker, L1, comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 6) or a functional variant thereof. In one embodiment, the VL(CD3) is connected to the CH1 by a peptide linker, L2. In one embodiment, the peptide linker, L2, comprises the amino acid sequence (G4S)x or a functional variant thereof, where x is 2, 3, 4, 5, or 6. In one embodiment, the peptide linker, L2, comprises the amino acid sequence (G4S)2 (SEQ ID NO: 5) or a functional variant thereof. In one embodiment, the VL(CD3) and the VH(CD3) are linked to each other by a peptide linker L3. In one embodiment, the peptide linker L3 comprises the amino acid sequence (GKPGS)x or a functional variant thereof, where x is 2, 3, 4, 5, or 6. In one embodiment, the peptide linker L3 comprises the amino acid sequence (GKPGS)4 (SEQ ID NO: 2) or a functional variant thereof. In one embodiment, the VH(CD3) is connected to the CH2 by a peptide linker L4. In one embodiment, the peptide linker L4 comprises the amino acid sequence (G4S)x or a functional variant thereof, where x is 2, 3, 4, 5, or 6. In one embodiment, the peptide linker L4 comprises the amino acid sequence (G4S)z (SEQ ID NO: 5) or a functional variant thereof.

[0027] In certain embodiments, the CH1, CH2, and / or CH3 domains of the bispecific binding agents disclosed herein comprise one or more amino acid modifications, particularly substitutions and / or deletions, at positions corresponding to positions in human IgG1 according to EU numbering. In certain embodiments, CH1 on the first and / or second polypeptide chain comprises an amino acid sequence comprising an aspartic acid residue at position 208 according to EU numbering. In certain embodiments, CH1 on the first polypeptide chain comprises an amino acid sequence comprising an aspartic acid residue at position 208 according to EU numbering, and CH1 on the second polypeptide chain comprises an amino acid sequence comprising an asparagine residue at position 208 according to EU numbering.

[0028] Furthermore, in certain embodiments, the binding agents disclosed herein do not substantially bind, e.g., do not detectably bind, to human FcγRI, IIa, IIb, and / or IIIa. In certain embodiments, CH2 on the first and / or second polypeptide chain comprises an amino acid sequence that includes one or more of the following: a proline residue at position 233, a valine residue at position 234, an alanine residue at position 235, a deletion at position 236, a lysine residue at position 267, and a glutamic acid residue at position 295, according to EU numbering. In one embodiment, CH2 on the first and second polypeptide chains comprises an amino acid sequence comprising a proline residue at position 233, a valine residue at position 234, an alanine residue at position 235, a deletion at position 236, and a lysine residue at position 267 according to EU numbering, and CH2 on the first polypeptide chain further comprises a glutamic acid residue at position 295 according to EU numbering, and CH2 on the second polypeptide chain further comprises a glutamine residue at position 295 according to EU numbering.

[0029] In one embodiment, CH3 on the first and / or second polypeptide chain comprises an amino acid sequence comprising one or more of the following: a glutamine residue at position 357, a lysine residue at position 364, an aspartic acid residue at position 368, a serine residue at position 370, an aspartic acid residue at position 384, a glutamic acid residue at position 418, and an aspartic acid residue at position 421 according to EU numbering. In one embodiment, CH3 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 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. In one embodiment, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:7. In one embodiment, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:8. In one embodiment, the third and / or fourth polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:9.

[0030] In some embodiments, CD3 is expressed on the surface of T cells. In some embodiments, the bispecific binding agents described herein bind 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 T cells. In some embodiments, proliferation and / or activation of T cells includes proliferation and / or activation of CD4 and / or CD8 T cells, preferably CD107a T cells. + In one embodiment, the proliferated and / or activated T cells can degranulate. In one embodiment, the activated T cells release cytotoxic factors, such as perforin and granzymes, to initiate cytolysis and / or apoptosis of cancer cells.

[0031] 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 the extracellular portion of CLDN18.2. In some embodiments, the binding agent induces T cell-mediated cytotoxicity against cancer cells expressing CLDN18.2.

[0032] In one embodiment, the cancer cells are selected from the group consisting of gastric cancer, particularly gastric adenocarcinoma, esophageal cancer, cancer of the gastroesophageal junction (GEJ), GEJ adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, lung cancer, non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, rectal cancer, colorectal cancer, liver cancer, head and neck cancer, bile duct cancer, gallbladder cancer and metastases thereof, Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis.

[0033] In some embodiments, the bispecific binding agent comprises one or more post-translational modifications. In some embodiments, the bispecific binding agent is derived from one or more post-translational modifications of a binding agent described herein. In some embodiments, the one or more post-translational modifications are selected from pyroglutamylation at the N-terminus of one or more VH(CLDN18.2), deletion of a lysine at the C-terminus of the first polypeptide chain, and deletion of a lysine at the C-terminus of the second polypeptide chain.

[0034] In some embodiments, the bispecific binding agent is provided as a nucleic acid encoding the binding agent. In some embodiments, the bispecific binding agent is provided as a set of nucleic acids that together encode the binding agent. In some embodiments, the nucleic acid or set of nucleic acids is capable of expressing the bispecific binding agent.

[0035] In some embodiments, a vector comprises a nucleic acid or a set of nucleic acids. In some embodiments, a set of vectors comprises a set of nucleic acids. In some embodiments, each nucleic acid of the set of nucleic acids is comprised in a vector of the set of vectors. In some embodiments, the vector or set of vectors is capable of expressing a bispecific binding agent. For example, expression of a subject nucleic acid or set of nucleic acids, or a vector or set of vectors can provide the bispecific binding agent.

[0036] In some embodiments, the nucleic acids are operably linked to any number of regulatory elements (promoter, origin of replication, selectable marker, ribosome binding site, inducer, etc.). The vector can be an expression vector, and can be extrachromosomal or integrating. In some embodiments, the nucleic acids encoding the polypeptide chains of the binding agents disclosed herein are each contained within a single expression vector. The nucleic acids can be under the control of different promoters or the same promoter. In such embodiments, different vector ratios can be used to form the binding agents disclosed herein.

[0037] In certain embodiments, the immune checkpoint inhibitor comprises one or more immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor comprises an antibody selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, and a combination thereof. In certain embodiments, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

[0038] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of cemiplimab (LIBTAYO, REGN2810), nivolumab (Opdivo; BMS-936558), pembrolizumab (KEYTRUDA; MK-3475), pidilizumab (CT-011), spartalizumab (PDR001), MEDI0680 (AMP-514), dostarlimab (TSR-042), cetrelimab (JNJ 63723283), toripalimab (JSOO1), AMP-224 (GSK-2661380), PF-06801591, tislelizumab (BGB-A317), ABBV-181, Bl 754091, including sintilimab (IBI308) or karelizumab (5HR-1210). In some embodiments, the immune checkpoint inhibitor comprises an anti-PD-L1 antibody. In some embodiments, immune checkpoint inhibitors comprise atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267), durvalumab (MEDI4736), BMS-936559, avelumab (BAVENCIO), lodapolimab (LY3300054), CX-072 (Proclaim-CX-072), FAZ053, KN035, sugemalimab (CS1001), or MDX-1105.

[0039] In certain embodiments, the composition or pharmaceutical preparation is a pharmaceutical composition, hi certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients. In some embodiments, the composition or pharmaceutical preparation is a kit. In some embodiments, the bispecific binding agent and the immune checkpoint inhibitor are in separate vials.

[0040] In certain embodiments, the composition or pharmaceutical preparation further comprises instructions for using the bispecific binding agent and the immune checkpoint inhibitor to treat or prevent cancer. The present invention further provides a composition or pharmaceutical preparation as described herein for pharmaceutical use. In certain embodiments, medical uses include therapeutic or prophylactic treatment of a disease or disorder. In certain embodiments, therapeutic or prophylactic treatment of a disease or disorder includes treating or preventing cancer.

[0041] In some embodiments, the cancer comprises cancer cells that express CLDN18.2. In one embodiment, the cancer is selected from the group consisting of gastric cancer, particularly gastric adenocarcinoma, esophageal cancer, cancer of the gastroesophageal junction (GEJ), GEJ adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, lung cancer, non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, rectal cancer, colorectal cancer, liver cancer, head and neck cancer, bile duct cancer, gallbladder cancer and metastases thereof, Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis.

[0042] In certain embodiments, the compositions or pharmaceutical preparations described herein are for administration to a human. The present invention further provides a method of treating or preventing cancer in a subject, comprising administering to said subject: (a) a bispecific binding agent disclosed herein; and (b) immune checkpoint inhibitors, The method includes administering In some embodiments, the bispecific binding agent is a bispecific binding agent described in the context of a composition or pharmaceutical preparation described herein. In some embodiments, the immune checkpoint inhibitor is an immune checkpoint inhibitor disclosed herein, e.g., described in the context of a composition or pharmaceutical preparation disclosed herein. In certain embodiments, the subject is a human.

[0043] The invention further provides a composition or pharmaceutical preparation as described herein for use in the methods described herein. According to the present invention, CLDN18.2 preferably has the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the first polypeptide chain does not include a VL(CLDN18.2). In some embodiments, the second polypeptide chain does not include a VL(CLDN18.2). In some embodiments, the third polypeptide chain does not include a VH(CLDN18.2). In some embodiments, the fourth polypeptide chain does not include a VH(CLDN18.2).

[0044] In certain embodiments, the CLDN18.2 binding domain of the bispecific binding agent is in the form of a Fab fragment. In certain embodiments, the CD3 binding domain of the bispecific binding agent is in the form of an scFv portion. In some embodiments, the bispecific binding agent does not bind to CLDN18.1. In some embodiments, the bispecific binding agent does not bind to human, mouse, or cynomolgus monkey CLDN18.1. In some embodiments, the bispecific binding agent does not bind to CLDN9, such as human CLDN9. In certain embodiments, the bispecific binding agent binds to CLDN18.2 from more than one species, such as human, mouse, and cynomolgus monkey CLDN18.2.

[0045] In some embodiments, treating a subject, such as a patient, with a bispecific binding agent and an immune checkpoint inhibitor disclosed herein extends the survival of the subject. In some embodiments, treating a patient with a bispecific binding agent and an immune checkpoint inhibitor disclosed herein reduces, preferably significantly reduces, the growth and / or volume of a tumor in a subject, such as a patient. In certain embodiments, the bispecific binding agents disclosed herein are capable of redirecting T cells to attack cancer cells, thus acting via redirected T cell cytotoxicity (RTCC). In certain embodiments, the bispecific binding agent is unable or substantially unable to induce ADCC. In certain embodiments, the binding agent is unable or substantially unable to induce CDC.

[0046] In some embodiments, the bispecific binding agent is produced by a method comprising transfecting a host cell with a nucleic acid, set of nucleic acids, vector, or set of vectors encoding the polypeptide chains of the bispecific binding agent. In some embodiments, the host cell expresses the nucleic acid, set of nucleic acids, vector, or set of vectors. In some embodiments, the host cell co-expresses a nucleic acid encoding a first polypeptide chain of the bispecific binding agent, a nucleic acid encoding a second polypeptide chain of the bispecific binding agent, a nucleic acid encoding a third polypeptide chain of the bispecific binding agent, and a nucleic acid encoding a fourth polypeptide chain of the bispecific binding agent. In some embodiments, the nucleic acids are contained in a vector or set of vectors. In some embodiments, the host cell expresses all polypeptide chains of the bispecific binding agent. In some embodiments, the transfected host cell preferably produces the bispecific binding agent when grown under conditions appropriate for binding agent production, such as those disclosed herein or known in the art. In some embodiments, the bispecific binding agent is obtainable from the host cell.

[0047] Thus, in one embodiment, a method for producing a bispecific binding agent includes transfecting a host cell with a nucleic acid encoding a first polypeptide chain of the bispecific binding agent, a nucleic acid encoding a second polypeptide chain of the bispecific binding agent, a nucleic acid encoding a third polypeptide chain of the bispecific binding agent, and a nucleic acid encoding a fourth polypeptide chain of the bispecific binding agent, expressing the nucleic acids in the host cell, and obtaining the bispecific binding agent. In one embodiment, 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, etc. In one embodiment, the host cell is a bacterial cell, yeast cell, fungal cell, plant cell, or insect cell. In one embodiment, the binding agent is produced in vitro. In one embodiment, the binding agent is produced in vivo, e.g., in a subject to be treated, such as a subject with a disease, particularly a disease associated with cells expressing CLDN18.2, e.g., cancer. In one embodiment, the different polypeptide chains of the bispecific binding agent are produced in two or more different host cells. In certain embodiments, all polypeptide chains of the bispecific binding agent are produced in the same host cell.

[0048] In some embodiments, the polypeptide chains of the bispecific binding agent are linked, e.g., covalently linked, to one another. In some embodiments, the polypeptide chains of the bispecific binding agent are produced as a single polypeptide comprising all of the polypeptide chains of the bispecific binding agent. In some embodiments, at least two polypeptide chains of the bispecific binding agent are linked together and produced as a single polypeptide. In some embodiments, the polypeptide chains of the bispecific binding agents disclosed herein are produced separately, i.e., as separate polypeptides, e.g., in the same cell or different cells, and interact during or after production to form the bispecific binding agent, e.g., intracellularly or extracellularly. In some embodiments, the polypeptide chains are produced as separate polypeptides, i.e., the first polypeptide chain is produced as a single polypeptide, the second polypeptide chain is produced as a single polypeptide, the third polypeptide chain is produced as a single polypeptide, and the fourth polypeptide chain is produced as a single polypeptide, and the polypeptide chains interact to form the bispecific binding agent. [Effects of the Invention]

[0049] Other features and advantages of the invention will be apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0050] [Figure 1] FIG. 1 shows the "Fab2-scFv" format of a bispecific binding agent disclosed herein, comprising a VH recombinantly fused to one side of a heterodimeric Fc (a first polypeptide chain disclosed herein), a VH recombinantly fused to an scFv fused to the other side of a heterodimeric Fc (a second polypeptide chain disclosed herein), and an LC (a third and fourth polypeptide chain disclosed herein) that forms a Fab domain with the VH of the first polypeptide chain and a further VH of the second polypeptide chain. [Figure 2]Figure 1 shows the antitumor effect of ASP2138 monotherapy in a hCD3εKI mouse model harboring hCLDN18.2-expressing MC38 tumor cells. MC38_hCLDN18.2 cells were inoculated subcutaneously into the flank of mice at 2.0 × 10 cells on day -5. hCD3εKI mice were intraperitoneally administered PBS or ASP2138 on days 0 and 7. Top: Tumor volumes for each group are plotted as mean ± SEM (n = 8) at each time point. Bottom: Scatter plots show individual tumor volumes on day 13; short horizontal lines and error bars represent mean ± SEM (n = 8). Statistical analysis was performed on values ​​on day 13. **: P < 0.01 compared with the PBS control group (Dunnett's multiple comparison test). CLDN18.2: Claudin 18.2; hCD3εKI: Human CD3ε knock-in; MC38_hCLDN18.2: Human CLDN18.2-expressing MC38 [Figure 3] This figure shows the effect of the combination of ASP2138 and anti-mPD-1 antibody in a hCD3ε KI mouse model harboring hCLDN18.2-expressing MC38 tumor cells. MC38_hCLDN18.2 cells were inoculated subcutaneously into the flank of mice at 2.0 × 10 cells on day -6. hCD3ε KI mice were intraperitoneally administered PBS or ASP2138 (0.1 mg / kg) on ​​days 0 and 7, and anti-mPD-1 antibody (anti-mPD-1 Ab and combination group) or isotype control antibody (control and ASP2138 group) at a dose of 100 μg / mouse on days 0, 4, 7, and 11. Top: Tumor volume for each group is plotted as mean ± SEM (n = 15) for each time point. Bottom: Scatter plot shows individual tumor volumes on day 14. The short horizontal line and error bars represent mean ± SEM (n = 15). Statistical analysis was performed on the values ​​on day 14. Compared with the values ​​of the combination group on day 14, *: P<0.05, **: P<0.01 (unpaired Student's t-test). CLDN18.2: Claudin 18.2; hCD3εKI: human CD3ε knock-in; MC38_hCLDN18.2: human CLDN18.2-expressing MC38; anti-mPD-1 antibody: anti-mouse programmed death-1 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0051] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the particular methodology, protocols, and reagents disclosed herein, as these may vary. It should also 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 is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. A description of each element of the present invention follows. While the elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining any number of the disclosed and / or preferred elements with the explicitly described embodiment. Furthermore, all permutations and combinations of elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise. Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," H.G. W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Throughout this specification and claims, unless the context requires otherwise, the terms "comprise," variations thereof, "comprises," and the like, are understood to mean the inclusion of a stated number, integer, or step, or group of numbers, integers, or steps, but not the exclusion of other numbers, integers, or steps, or groups of numbers, integers, or steps; in some embodiments, such other numbers, integers, or steps, or groups of numbers, integers, or steps, may be excluded, i.e., the subject matter comprises a stated number, integer, or step, or group of numbers, integers, or steps. For certain embodiments of the present disclosure, the term "comprising" is intended to encompass the possibility that no additional elements are present; i.e., for purposes of this embodiment, "comprising" should be understood to mean "consisting of" or "consisting essentially of." The terms "a," "an," and "the" and similar references used in the context of describing the invention (particularly in the context of the claims) are to be construed as including both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if individually referred to. All methods disclosed herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any and all examples or exemplary language (e.g., "etc.") provided herein are intended merely to better illustrate the invention and do not impose limitations on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. The term "about" means approximately or approximately, and in the context of numerical values ​​or ranges disclosed herein, means, in certain embodiments, ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range. Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0052] Bispecific Binding Agents The primary target molecule for the bispecific binding agents disclosed herein is CLDN18.2. Claudins are a family of proteins that are essential components of tight junctions, which establish a paracellular barrier that regulates the flow of molecules across the intercellular space between epithelial cells. Claudins are transmembrane proteins that span the membrane four times, with both their N- and C-termini located in the cytoplasm. The first extracellular loop, designated EC1 or ECL1, consists of an average of 53 amino acids, while the second extracellular loop, designated EC2 or ECL2, consists of approximately 24 amino acids. Cell surface proteins of the claudin family, such as CLDN18.2, are expressed in tumors of various origins. Their selective expression (not expressed in toxicity-associated normal tissues) and localization to the plasma membrane make them particularly suitable as target structures for antibody-mediated cancer immunotherapy.

[0053] CLDN18.2 has been identified as being differentially expressed in tumor tissues, and the only normal tissue expressing CLDN18.2 is the stomach. In normal tissues, CLDN18.2 is selectively expressed in differentiated epithelial cells of the gastric mucosa. CLDN18.2 is expressed in cancers of various origins, such as pancreatic cancer, esophageal cancer, gastric cancer, bronchial cancer, breast cancer, and ENT tumors. CLDN18.2 is a valuable target for the prevention and / or treatment of primary tumors, such as gastric cancer, particularly gastric adenocarcinoma, esophageal cancer, gastroesophageal junction (GEJ) cancer, GEJ adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, lung cancer, non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, rectal cancer, colorectal cancer, liver cancer, head and neck cancer, bile duct cancer, gallbladder cancer and its metastasis, Krukenberg tumor, peritoneal metastasis, and / or lymph node metastasis. The term "claudin 18" or "CLDN18" refers to claudin 18 and includes any variant, including claudin 18 splicing variant 1 (claudin 18.1 (CLDN18.1)) and claudin 18 splicing variant 2 (claudin 18.2 (CLDN18.2)). The term "claudin 18.2" or "CLDN18.2" preferably relates to human CLDN18.2, and in particular to a protein comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 1 in the Sequence Listing or a variant of said amino acid sequence. The first extracellular loop of CLDN18.2 preferably comprises amino acids 27 to 81, more preferably amino acids 29 to 78, of the amino acid sequence shown in SEQ ID NO: 1. The second extracellular loop of CLDN18.2 preferably comprises amino acids 140 to 180 or 144 to 167 of the amino acid sequence shown in SEQ ID NO: 1. The first and second extracellular loops preferably form the extracellular portion of CLDN18.2.

[0054] The second target molecule of the bispecific binding agents disclosed herein is CD3 (cluster of differentiation 3). The CD3 complex refers to an antigen expressed as part of the multimolecular T cell receptor (TCR) complex on mature human T cells, thymocytes, and a subset of natural killer cells. The T cell coreceptor is a protein complex composed of four different chains. In mammals, the complex includes the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with a molecule known as the T cell receptor (TCR) and a ζ chain to generate an activation signal in T lymphocytes. The TCR, ζ chain, and CD3 molecule together comprise the TCR complex.

[0055] Human CD3ε is designated GenBank Accession No. NM_000733. Human CD3γ is designated GenBank Accession No. NM_000073. Human CD3δ is designated GenBank Accession No. NM_000732. CD3 is responsible for TCR signaling. As described in Lin and Weiss, Journal of Cell Science 114, 243-244 (2001), activation of the TCR complex by binding of an MHC-presented specific antigen epitope results in phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) by Src family kinases, triggering the recruitment of additional kinases that result in T cell activation, including Ca2+ release. For example, clustering of CD3 on T cells by immobilized anti-CD3 antibodies mimics T cell receptor engagement but activates T cells independent of the specificity typical of their clone.

[0056] As used herein, the term "CD3" includes human CD3 and refers to an antigen expressed on human T cells as part of the multimolecular T cell receptor complex. With respect to CD3, the binding agent disclosed herein preferably recognizes the epsilon chain of CD3. In one embodiment, it recognizes an epitope corresponding to the first 27 N-terminal amino acids of CD3ε or a functional fragment of this 27 amino acid stretch.

[0057] According to the present invention, the term "CLDN18.2-positive cancer" or similar terms refers to a cancer with cancer cells that express CLDN18.2, preferably on the surface of the cancer cells. "Cell surface" is used in accordance with its normal meaning in the art and includes the outside of a cell that is accessible to binding by proteins and other molecules.

[0058] CLDN18.2 is expressed on the surface of a cell when it is located on the surface of the cell and is accessible for binding by a CLDN18.2-specific antibody added to the cell. CD3 is expressed on the surface of a cell if it is located on the surface of the cell and is accessible for binding by a CD3-specific antibody added to the cell.

[0059] The term "extracellular portion" in the context of the present invention refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from the outside of the cell, for example, to an antigen-binding molecule, such as an antibody, that is located on the outside of the cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0060] The terms "portion" and "fragment" are used interchangeably herein and refer to a contiguous element. For example, a portion of a structure, such as an amino acid sequence or protein, refers to a contiguous element of the structure. A portion, site, or fragment of a structure preferably comprises one or more functional properties of the structure. A portion or fragment of an amino acid sequence preferably comprises a sequence of at least 4, particularly at least 6, at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 contiguous amino acids of the protein sequence. In reference to an amino acid sequence (peptide or protein), a "fragment" refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A fragment truncated at the C-terminus (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame from which the 3' end of the open reading frame has been deleted. A fragment truncated at the N-terminus (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame from which the 5' end of the open reading frame has been deleted, as long as the truncated open reading frame contains an initiation codon serving to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. As used herein, a "variant" of an amino acid sequence or similar expressions refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or may be a modified version of 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., 1 to about 20 amino acid modifications compared to the parent, preferably 1 to about 10 or 1 to about 5 amino acid modifications. The terms "wild-type" or "WT" or "native" as used herein with respect to an amino acid sequence refer to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or protein is an amino acid sequence that has not been intentionally modified.

[0061] For the purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splicing variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that occur naturally. The term "variant" particularly includes fragments of an amino acid sequence. Amino acid insertion variants involve the insertion of one or more amino acids into a particular amino acid sequence. In the case of insertional amino acid sequence variants, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertion with appropriate screening of the resulting products is also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more. The deletion can occur at any position in the protein. Amino acid deletion variants containing deletions at the N-terminus and / or C-terminus of the protein are also referred to as N- and / or C-terminal truncation variants.

[0062] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or the substitution of an amino acid with another amino acid with similar properties are preferred. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one member of a family of amino acids that are related in their side chains. Conservative substitutions in the context of the present invention may be defined by substitutions within the classes of amino acids reflected in the table below.

[0063] [Table 1] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably achieved over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. In one embodiment, for example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is provided for consecutive amino acids, preferably 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 one embodiment, the degree of similarity or identity is provided for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, and Gap Extend 0.5.

[0064] The term "sequence similarity" refers to the proportion of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the proportion of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the proportion of nucleotides that are identical between the sequences. The terms "% identical", "% identity" or similar terms are intended to refer in particular to the proportion of nucleotides or amino acids that are identical in optimal alignment between the compared sequences. Said proportion is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the compared sequences. Comparison of two sequences usually involves, after optimal alignment, comparing the sequences over a segment or "window of comparison" to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or using the algorithm described in Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or using computer programs that use the above algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In one embodiment, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available at the National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In one embodiment, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) expectation threshold set to 10, (ii) word size set to 28, (iii) maximum matches within the query range set to 0, (iv) match / mismatch scores set to 1, -2, (v) gap cost set to linear, and (vi) a low-complexity region filter used. In one embodiment, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) expectation threshold set to 10, (ii) word size set to 3, (iii) maximum match within query set to 0, (iv) Matrix set to BLOSUM62, (v) gap costs set to Existence:11 Extension:1, and (vi) conditional composition score matrix adjustment. The percent identity is obtained by determining the number of corresponding identical positions in the compared sequences, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0065] In some embodiments, the degree of similarity or identity is provided over a region that 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 provided over 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, consecutive nucleotides. In some embodiments, the degree of similarity or identity is provided over the entire length of the reference sequence. According to the present invention, homologous amino acid sequences exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98% or at least 99% of the amino acid residues.

[0066] Amino acid sequence variants disclosed herein can be readily prepared by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants disclosed herein can be readily prepared using known peptide synthesis techniques, such as, for example, solid-phase synthesis and similar methods.

[0067] In certain 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 refers to any fragment or variant that exhibits one or more functional properties identical to or similar to the amino acid sequence from which it is derived, i.e., is functionally equivalent. For antigen-binding domains comprising functional VH and VL variants, one specific function is to retain the binding of said binding domain. As used herein, the term "functional fragment" or "functional variant" specifically refers to a variant molecule or sequence comprising an amino acid sequence in which one or more amino acids have been changed compared to the amino acid sequence of the parent molecule or sequence and which can still fulfill one or all of the functions of the parent molecule or sequence, e.g., form a binding domain with specificity 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 properties compared to the parent molecule. In certain embodiments, 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, the characteristics of a molecule comprising a functional fragment or functional variant, e.g., a binding characteristic such as the binding strength of a binding domain, may be reduced but still significantly present; for example, the binding characteristic such as the binding strength of a binding domain comprising a functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. For example, a functional variant may contain one, two, three, four, five, or more amino acid insertions, additions, substitutions, and / or deletions compared to the parent molecule. However, in other embodiments, the characteristics of a molecule comprising a functional variant or functional fragment, e.g., the binding characteristic of a binding domain comprising a functional fragment or functional variant, may be enhanced compared to the parent molecule. In certain embodiments, a "functional variant" is a "functional fragment," e.g., an amino acid sequence that is truncated at the N-terminus and / or C-terminus compared to the parent molecule, but retains or essentially retains one or more or all of the functions of the parent molecule, as described above, and in particular is functionally equivalent to the parent molecule.

[0068] The term "functional variant" of an amino acid sequence includes "functional" fragments of said amino acid sequence. An amino acid sequence (peptide, protein, or polypeptide, e.g., VH, VL, CH1, CH2, or CH3) "derived from" a designated amino acid sequence (peptide, protein, or polypeptide, e.g., VH, VL, CH1, CH2, or CH3) refers to the origin of the first amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof, preferably a functional variant thereof as disclosed herein, including a functional fragment. For example, one of skill in the art will understand that amino acid sequences suitable for use herein can be modified to alter the sequence, including amino acid insertions, deletions, additions, and / or substitutions, from the naturally occurring or native sequence from which they are derived while retaining or essentially retaining the desired activity of the native sequence. For example, the amino acid sequences of the VH, VL, CH1, CH2, and / or CH3 domains on the peptide chains of the binding agents disclosed herein are derived from the amino acid sequences of the VH, VL, CH1, CH2, and / or CH3 domains of an immunoglobulin, but may be altered compared to the domains from which they are derived. For example, in the present invention, a VH or VL derived from an immunoglobulin may comprise an amino acid sequence that may be identical to the amino acid sequence of the respective VH or VL from which it is derived, or may differ at one or more amino acid positions compared to the sequence of the respective parent VH or VL. For example, the VH domain of a binding agent disclosed herein may comprise an amino acid sequence that comprises one or more amino acid insertions, additions, deletions, and / or substitutions compared to the amino acid sequence of the VH domain from which it is derived. For example, the VL domain of a binding agent disclosed herein may comprise an amino acid sequence that comprises one or more amino acid insertions, additions, deletions, and / or substitutions compared to the amino acid sequence of the VL domain from which it is derived. Preferably, a VH or VL having an amino acid sequence that is a functional variant of the amino acid sequence of a parent VH or VL provides the same or essentially the same function as the amino acid sequence of the parent VH or VL, for example, in terms of binding specificity, binding strength, etc.However, as will be appreciated by those skilled in the art, in certain embodiments it may be preferable to provide functional variants of, for example, the VH or VL amino acid sequences, which have altered properties compared to the amino acid sequence of the parent molecule. The same considerations apply, for example, to the amino acid sequences of the CDRs, and other amino acid sequences, for example, the amino acid sequences of the CH1, CH2, CH3 and / or CL domains.

[0069] When a bispecific binding agent is described as comprising a VH "derived from" an immunoglobulin and a VL "derived from" the same or a different immunoglobulin, the term "derived from" indicates that the bispecific binding agent was produced by recombining the VH and VL from the immunoglobulin into the resulting bispecific binding agent by any known method. "Recombinant" in this context is not intended to be limited by any particular recombinant method, and thus includes all methods for producing bispecific binding agents disclosed herein or known in the art, including, for example, recombination at the nucleic acid level and / or via co-expression of different molecules in the same cell.

[0070] 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 that has two different antigen-binding domains defined by different amino acid sequences. In some embodiments, the different antigen-binding domains bind to different epitopes on the same antigen. However, in preferred embodiments, the different antigen-binding domains bind to different target antigens. A binding agent can bind different antigens or different epitopes, respectively, with one, two, or more binding domains, i.e., it can bind different antigens or different epitopes, respectively, monovalently, bivalently (or bivalently), trivalently, tetravalently, and even higher valencies.

[0071] CLDN18.2 is not substantially expressed in cells when the expression level is low compared to its expression in gastric cells or gastric tissue. Preferably, the expression level is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05%, or even lower, than that in gastric cells or gastric tissue. Preferably, CLDN18.2 is not substantially expressed in cells when the expression level exceeds the expression level in non-cancerous tissue other than the stomach by no more than 2-fold, preferably no more than 1.5-fold, and preferably does not exceed the expression level in the non-cancerous tissue. Preferably, CLDN18.2 is not substantially expressed in cells when the expression level is below the detection limit and / or when the expression level is too low to bind to a CLDN18.2-specific antibody added to the cells. CLDN18.2 is expressed in a cell when the expression level is preferably more than 2-fold, preferably 10-fold, 100-fold, 1000-fold, or 10,000-fold higher than the expression level in non-cancerous tissues other than the stomach. Preferably, CLDN18.2 is expressed in a cell when the expression level exceeds the detection limit and / or when the expression level is high enough to be able to bind to a CLDN18.2-specific antibody added to the cell. Preferably, CLDN18.2 expressed in a cell is expressed or exposed on the surface of the cell.

[0072] The term "disease" according to the present invention refers to any pathological condition, including cancer, particularly the forms of cancer described herein. Any reference herein to cancer or a particular form of cancer also includes cancer metastases thereof. In a preferred embodiment, the disease treated herein involves cells that express CLDN18.2. As used herein, a "disease involving cells expressing CLDN18.2," "disease associated with cells expressing CLDN18.2," or similar expressions means that CLDN18.2 is expressed in cells of a diseased tissue or organ. In one embodiment, CLDN18.2 expression in cells of a diseased tissue or organ is higher than in a healthy tissue or organ. An increase refers to an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10,000% or more. In one embodiment, expression is found only in diseased tissue, while expression in healthy tissue is suppressed. In the present invention, diseases associated with cells expressing CLDN18.2 include cancer diseases. In the present invention, cancer diseases are preferably characterized by CLDN18.2 expressed in cancer cells. As used herein, the term "cancer disease" or "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. "Cancer cells" refer to abnormal cells that grow by rapid, uncontrolled cell proliferation and continue to grow after the stimulus that initiated the new growth has ceased. Preferably, "cancer diseases" are characterized by cells that express CLDN18.2, and the cancer cells express CLDN18.2. The CLDN18.2-expressing cells are preferably cancer cells, preferably cancer cells of a cancer disclosed herein. The term "cancer" according to the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, rectal cancer, large intestine cancer, stomach cancer, intestinal cancer, head and neck cancer, bile duct cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, cancer of the gastroesophageal junction (GEJ), colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, and lung cancer, as well as metastases thereof. Examples are lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer, or metastases of the above cancer types or tumors. The term "cancer" according to the present invention also includes cancer metastases. "Carcinomas" according to the present invention are malignant tumors derived from epithelial cells. This group represents the most common cancers, including common forms of breast, prostate, lung and colon cancer. "Adenocarcinoma" is a cancer originating from glandular tissue, which is also part of a larger tissue classification known as epithelial tissue. Epithelial tissue includes skin, glands, and various other tissues that support body cavities and organs. Epithelium is embryologically derived from the ectoderm, endoderm, and mesoderm. To be classified as adenocarcinoma, cells need not necessarily be part of a gland, as long as they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while less differentiated adenocarcinomas may not. By staining cells from a biopsy, a pathologist determines whether the tumor is adenocarcinoma or another cancer. Adenocarcinoma can arise in many tissues throughout the body due to the ubiquitous nature of glands in the body. Although each gland may not secrete the same substances, as long as the cells have exocrine function, it is considered a gland, and therefore its malignant form is called adenocarcinoma. Malignant adenocarcinomas often invade other tissues and, given enough time, metastasize. Ovarian adenocarcinoma is the most common type of ovarian cancer. It includes serous and mucinous adenocarcinoma, clear cell adenocarcinoma and endometrioid adenocarcinoma.

[0073] "Metastasis" refers to the spread of cancer cells from their original site to other parts of the body. The formation of metastases is an extremely complex process, involving the detachment of malignant cells from the primary tumor, the invasion of the extracellular matrix, the penetration of the endothelial basement membrane to enter body cavities and blood vessels, and then the invasion of target organs after transport by the blood. Finally, the growth of new tumors at the target site is via angiogenesis. Tumor metastases may recur even after removal of the primary tumor, as tumor cells or components may remain and metastasize. In one embodiment, the term "metastasis" according to the present invention refers to "distant metastasis," which refers to metastasis away from the primary tumor and the regional lymph node system. In one embodiment, the term "metastasis" according to the present invention refers to lymph node metastasis. One particular form of metastasis treatable using the treatment or method of the present invention is metastasis originating from gastric cancer as a primary site. In a preferred embodiment, the gastric cancer metastasis is a Krukenberg tumor, peritoneal metastasis, and / or lymph node metastasis.

[0074] Krukenberg tumor is a rare metastatic tumor of the ovary, accounting for 1% to 2% of all ovarian tumors. The prognosis for Krukenberg tumor remains extremely poor, and there is no established treatment for Krukenberg tumor. Krukenberg tumor is a metastatic signet-ring cell carcinoma of the ovary. The stomach is the primary site for most Krukenberg tumor cases (70%). Carcinomas of the colon, appendix, and breast (mainly invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg tumor originating from carcinomas of the gallbladder, bile duct, pancreas, small intestine, ampulla of Vater, cervix, and bladder / urachus have been reported.

[0075] "Treating" means administering a compound or composition, or a combination of compounds or compositions, to a subject to prevent, ameliorate, or eliminate disease, including reducing tumor size or the number of tumors in the subject; halting or slowing disease in a subject; inhibiting or slowing the onset of new disease in a subject; reducing the frequency or severity of symptoms and / or recurrences in a subject currently suffering from or previously suffering from disease; and / or extending, i.e., prolonging, the lifespan of a subject. In particular, the term "treatment of a disease" includes curing, shortening the duration, ameliorating, preventing, slowing or inhibiting the progression or worsening of a disease or a symptom thereof, or preventing or delaying its onset. The terms "protect", "prevent", "prophylactic", "preventive", or "protective", etc., in the context of the present invention, relate to the prevention or treatment, or both, of the occurrence and / or spread of a disease in a subject, in particular to minimizing the likelihood that a subject will develop a disease or delaying the onset of a disease. For example, a person at risk of cancer is a candidate for treatment to prevent cancer. "At risk" refers to a subject who has been identified as having a higher than normal likelihood of developing a disease, particularly cancer, compared to the general population. Furthermore, a subject who has had or currently has a disease, particularly cancer, is at high risk of developing the disease, and the subject may continue to develop the disease. Subjects who currently have or have had cancer also have a high risk of cancer metastasis.

[0076] According to the present invention, the terms "individual" and "subject" are used interchangeably herein. They refer to a human or other mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may be affected by or susceptible to a disease or disorder (e.g., cancer), but may or may not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and thus encompass adults, elderly people, children, and newborns. In an embodiment of the present disclosure, an "individual" or "subject" is a "patient." The term "patient" according to the present invention refers to a subject for treatment, in particular an affected subject, including humans, non-human primates or other animals, in particular mammals such as cows, horses, pigs, sheep, goats, dogs, cats, or rodents such as mice and rats. In a particularly preferred embodiment, the patient is a human.

[0077] "Target cell" means any unwanted cell, such as a cancer cell, etc. In a preferred embodiment, the target cell expresses CLDN18.2.

[0078] As used herein, the terms "activated" or "stimulated" refer to the state of an immune effector cell, such as a T cell, that has been stimulated sufficiently to induce detectable cell proliferation. Activation can also be associated with the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector function. The term "activated immune effector cell" refers, inter alia, to an immune effector cell that is undergoing cell division. According to the present invention, the term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen and differentiates into an effector cell, such as an effector T cell. The term "clonal expansion" or "expansion" refers to the process by which 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, causing proliferation and amplification of specific immune effector cells that recognize that antigen. Preferably, clonal expansion causes differentiation of immune effector cells.

[0079] As used herein, the term "interact" means that two molecular species, e.g., two polypeptide chains or portions thereof, are physically associated with one another. Associations characterized as interactions include non-covalent and / or covalent, preferably non-covalent, interactions such as charge-charge interactions, charge-dipole interactions, dipole-dipole interactions, van der Waals forces, hydrogen bonding, and / or hydrophobic forces. The terms "bind" or "binding" refer to non-covalent interactions with a target. In certain embodiments, the terms "bind" or "binding" refer to specific binding. As used herein, the terms "specific binding" or "specifically bind" refer to a molecule, such as an antibody, that recognizes a specific target molecule but does not substantially recognize or bind other molecules in a sample or subject. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more other species. However, such cross-species reactivity does not in itself alter the specific classification of the antibody. 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 in itself alter the specific classification of the antibody.

[0080] In certain instances, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, antibodies recognize and bind to specific protein structures rather than proteins in general. If an antibody is specific for epitope "A," then 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.

[0081] Disclosed herein are binding moieties and agents that bind to CLDN18.2 and / or CD3, which can form complexes with CLDN18.2 and / or CD3. As used herein, the term "binding agent" refers to any agent capable of binding to a desired antigen. Binding agents can also include synthetic, modified, or non-naturally occurring moieties, which can link a desired antigen-binding functional group or region, such as, for example, an antibody or antibody fragment. In certain embodiments, binding agents are synthetic constructs comprising antigen-binding CDRs or variable regions. In certain embodiments, binding agents disclosed herein include bispecific or multispecific binding agents, such as bispecific antibody-derived binding agents comprising a first and 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.

[0082] As used herein, the term "binding domain" or "antigen-binding domain" refers to any region, moiety, group, or domain that interacts with an antigen. In certain embodiments, the term "binding domain" or "antigen-binding domain" refers to the portion of a binding agent disclosed herein that binds to an antigen and comprises the antigen-binding portion of the binding agent. In certain embodiments, the binding domain is or comprises an antibody, an antibody fragment, or any other binding protein, or any combination thereof. A binding domain can be composed of heavy and light chain variable domains (VH and VL), each of which contains four conserved framework regions (FR) and three CDRs. The CDRs differ in sequence and determine specificity for a particular antigen. The VH and VL domains together can form a site that binds, e.g., specifically binds, a particular antigen. A "binding domain specific" for an antigen refers, for example, to a binding domain of a binding agent disclosed herein that binds, preferably specifically binds, to the antigen.

[0083] An agent, such as an antibody according to the present invention, is capable of binding to a predetermined target if it has significant affinity for the predetermined target and binds to the predetermined target in a standard assay. "Affinity" or "binding affinity" is defined as the equilibrium dissociation constant (K D ) Preferably, the term "significant affinity" is used in -5 M or less, 10 -6 M or less, 10 -7M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 This refers to the case where the value is M or less. An agent is (substantially) incapable of binding to a target if it has no significant affinity for the target and does not bind significantly, particularly not detectably, to the target in a standard assay. Preferably, the agent does not detectably bind to the target when present at concentrations up to 2 μg / ml, preferably up to 10 μg / ml, more preferably up to 20 μg / ml, particularly 50 μg / ml or 100 μg / ml or higher. Preferably, the K for binding to a given target to which the agent is capable of binding is D At least 10 times, 100 times, or 10 times 3 double, 10 4 double, 10 5 double, or 10 6 Twice as high as K D If a drug binds to a target at a K D is 10 -7 M, the K for binding to a target for which the drug has no significant affinity D is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 I am M.

[0084] A binding agent such as an antibody is specific for a given target if it can bind to the given target but cannot bind to other targets, i.e., it has no significant affinity for the other targets and does not significantly bind to the other targets in standard assays. In the present invention, a binding agent is specific for CLDN18.2 if it can bind to CLDN18.2 but cannot (substantially) bind to other targets. Preferably, a binding agent is specific for CLDN18.2 if its affinity and binding to the other targets do not significantly exceed its affinity or binding to non-CLDN18.2-related proteins such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or non-claudin transmembrane proteins such as MHC molecules or transferrin receptors, or any other specific polypeptide. Preferably, a binding agent has a K for binding to a target for which it is not specific. D At least 10 times, 100 times, or 10 times 3 double, 10 4 double, 10 5 double, or 10 6 Twice as high as K D For example, a binding agent is specific for a target if it binds to the target at a K D is 10 -7 M, it is the K for binding to a non-specific target D is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 I am M.

[0085] As used herein, the term "k d " (sec -1 ) refers to the dissociation rate constant of a particular antibody-antigen interaction. off Also called value. As used herein, the term "K D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. The binding of a binder to a target can be experimentally determined using any suitable method, such as Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, WE, Ed., Raven Press, New York, NY (1984); Kuby, Janis, Immunology, WH Freeman and Company, New York, NY (1992), and the methods described herein. Affinity can be readily determined using conventional techniques, such as equilibrium dialysis using a BIAcore 2000 instrument using the general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by other methods known to those skilled in the art. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann NY Acad. ScL, 51:660 (1949). The measured affinity of a particular interaction between a binder and an antigen can vary when measured under different conditions, such as salt concentration and pH. Therefore, affinity and other antigen binding parameters (e.g., K D , IC50) measurements are preferably performed using standardized solutions of binding agent and antigen and standardized buffers.

[0086] The term "compete" refers to the competition between two binding agents, such as antibodies, for binding to a target antigen. If two binding agents do not inhibit each other from binding to a target antigen, the binding agents are non-competitive, which indicates that the binding agents do not bind to the same part of the target antigen, i.e., epitope. Methods for testing the competition between binding agents for binding to a target antigen are well known to those skilled in the art. An example of such a method is the so-called cross-competition assay, which can be carried out, for example, as an ELISA or by flow cytometry. Two binding agents, such as antibodies, have the "same specificity" if they bind to the same antigen and the same epitope. The binding agents compete for binding in a competitive binding assay. In some embodiments, binding agents that bind to the same epitope are considered to bind to the same amino acid on the target molecule. Binding of antibodies to the same epitope on a target antigen can be determined by standard alanine scanning or antibody-antigen crystallization experiments known to those skilled in the art.

[0087] The ability of a binding agent to compete for binding to an antigen indicates whether the binding agent can bind to the same epitope region of the antigen or whether binding to another epitope sterically interferes with the binding of the binding agent to that particular epitope region. Competitive binding agents can be easily 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, competition between binding agents can be detected by cross-blocking assays. For example, a competitive ELISA assay can be performed by coating the target antigen on the wells of a microtiter plate and adding an antigen-binding agent and a candidate competitive test binding agent. The amount of antigen-binding agent bound to the antigen in the well indirectly correlates with the binding function of a candidate competitive test binding agent that competes for binding to the same epitope, for example. Specifically, the greater the affinity of the candidate competitive test binding agent for the same epitope, the less antigen-binding agent will bind to the antigen-coated well. The amount of antigen-binding substance bound to the well can be measured by labeling the binding substance with a detectable or measurable label. As described in WO 2013 / 173223 and known in the art, overlapping regions for different epitope regions recognized by binding agents can be identified using surface plasmon resonance analysis, for example, using a Biacore instrument. Alternatively, competition can be determined using biolayer interferometry.

[0088] A binding agent that competes with another binding agent for binding to an antigen, e.g., a binding agent comprising the heavy and light chain variable regions disclosed herein, or a binding agent specific for the antigen of another binding agent, e.g., a binding agent comprising the heavy and light chain variable regions disclosed herein, such as an antibody, can be a variant of the heavy and / or light chain variable regions disclosed herein, e.g., a binding agent that comprises modifications and / or a degree of identity in the CDRs disclosed herein.

[0089] The term "antigen" preferably relates to a molecule, such as a protein or peptide, containing an epitope against which an agent is and / or should be directed to induce an immune response. In one embodiment, the antigen or its processing product, e.g., a T cell epitope, is bound by a T cell receptor or an immunoglobulin molecule, e.g., an antibody. Thus, the antigen or its processing product can specifically react with an antibody or a T lymphocyte (T cell). In a preferred embodiment, the antigen is a tumor-associated antigen, such as CLDN18.2, i.e., a component of cancer cells that can originate from the cytoplasm, cell surface, and cell nucleus, particularly an antigen that is preferably produced in large amounts intracellularly or as a surface antigen on cancer cells.

[0090] The term "tumor-associated antigen" or "cancer-associated antigen" in the context of the present invention relates to a protein that is preferably specifically expressed under normal conditions in a limited number of tissues and / or organs or at a particular stage of development and that is expressed or aberrantly expressed in one or more tumor or cancer tissues. In the context of the present invention, tumor-associated antigens are preferably associated with the cell surface of cancer cells and are preferably not expressed or only rarely expressed in normal tissues.

[0091] The term "epitope" refers to an antigenic determinant in a molecule, e.g., a portion of a molecule that is recognized by the immune system, e.g., by an antibody. For example, an epitope is a distinct three-dimensional site on an antigen that is recognized by the immune system. Epitopes usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents. An epitope of a protein preferably comprises a continuous or discontinuous portion of the protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids in length; for example, the epitope may be preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0092] The term "immunoglobulin" refers to proteins of the immunoglobulin superfamily, preferably antibodies or antigen receptors such as B-cell receptors (BCRs). Immunoglobulins are characterized by structural domains with a characteristic immunoglobulin (Ig) fold, i.e., immunoglobulin domains. The term includes membrane-bound immunoglobulins as well as soluble immunoglobulins, which are commonly referred to as antibodies. Immunoglobulins generally comprise a chain, usually two identical heavy chains and two identical light chains linked via disulfide bonds. The chains are primarily composed of immunoglobulin domains, such as a VL (variable light chain) domain, a CL (constant light chain) domain, a VH (variable heavy chain), and CH (constant heavy chain) domains CH1, CH2, CH3, and CH4. There are five types of mammalian immunoglobulin heavy chains, i.e., α, β, d, ε, and g, which correspond to the different immunoglobulin classes, i.e., IgA, IgD, IgE, IgG, and IgM. Immunoglobulin class is also referred to as "isotype" (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM), which refers to the immunoglobulin class encoded by heavy chain constant region genes. When a particular isotype, e.g., IgG1, is referred to herein, the term is not limited to a particular isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g., IgG1, than to other isotypes. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at the carboxy terminus. In mammals, there are two types of light chains: lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved among different immunoglobulin isotypes, while the variable portion is highly diverse and is responsible for antigen recognition.

[0093] The term "antibody" refers to an immunoglobulin or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and chimeric antibodies. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (amino acid residues 118-447 of human IgG1 according to EU numbering) comprising CH1, CH2, and CH3 domains, with CH1 typically connected to CH2-CH3 by a peptide linker (also referred to as a "hinge"). Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The terms "region" and "domain" are used interchangeably herein. The VH and VL domains can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino 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 specified or contradicted by the context, CDR sequences herein are identified by the Kabat numbering system, and references to amino acid positions in the constant region herein are according to EU numbering (Edelman et al., (1969) Proc. Natl. Acad. Sci. USA 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition. 1991 NIH Publication No. 91-3242). The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0094] As used herein, the term "amino acid corresponding to a position ..." refers to the amino acid position number in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence that "corresponds" to an amino acid or segment in another sequence will have at least 50%, at least 80%, at least 90%, or at least 95% identity to the human IgG1 heavy chain when aligned with the other amino acid or segment using ALIGN, ClustalW, or similar standard sequence alignment programs, usually with default settings. Methods for aligning sequences or segments in sequences to determine positions in sequences that correspond to amino acid positions according to the invention are well known in the art.

[0095] As used herein, the term "IgG Fc ligand" refers to a molecule, preferably a polypeptide, that binds to the Fc region of an IgG immunoglobulin to form an Fc / Fc ligand complex. Fc ligands include, but are not limited to, FcγRI, FcγRII, FcγRII, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRHs), a family of Fc receptors homologous to FcγR (Davis et al., (2002) Immunol. Rev. 190:123-136). Specific IgG Fc ligands are FcRn and Fc gamma receptors. As used herein, "Fc ligands" can be derived from any organism, including mice, humans, and cynomolgus monkeys.

[0096] "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to any member of a family of proteins that bind to the IgG Fc region and are encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., (2002) Immunol. Lett. 82:57-65). FcγRs can be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. Murine FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2).

[0097] As used herein, "FcRn" or "neonatal Fc receptor" refers to a protein that binds to the IgG Fc region and is at least partially encoded by the FcRn gene. FcRn can be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. Functional FcRn proteins often contain two polypeptides: a heavy chain (encoded by the FcRn gene) and a light chain (β-2-microglobulin). Unless otherwise specified, "FcRn" or "FcRn protein" refers to the complex of the FcRn heavy chain and β-2-microglobulin. As used herein, "FcRn variants" may have enhanced binding to the FcRn receptor and may also have enhanced serum half-life.

[0098] As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising the CH2 and CH3 domains of an IgG molecule and, optionally, a peptide linker such as a hinge. The CH2-CH3 domain of human IgG1 comprises amino acid positions 231-447, and the hinge comprises amino acid positions 216-230, according to EU numbering. Thus, the term "Fc domain" as used herein with respect to IgG includes amino acid positions 231-447 (CH2-CH3) and 216-447 (hinge-CH2-CH3) according to EU numbering, as well as functional variants thereof, including functional fragments thereof. "Fc fragments" are functional variants that may contain fewer amino acids, e.g., N- or C-terminal truncation variants, but still retain the ability to form dimers with other Fc domains or Fc fragments, as can be detected, for example, using standard size-based methods (e.g., non-denaturing chromatography, size exclusion chromatography, etc.). In the present invention, the IgG Fc domain is preferably a human IgG Fc domain comprising an Fc domain derived from human IgG1, IgG2, or IgG4.

[0099] As used herein, the terms "hinge," "hinge region," "antibody hinge region," or "hinge domain" refer to a peptide linker comprising the amino acids between CH1 and CH2 of an immunoglobulin, e.g., an IgG. Structurally, in a naturally occurring IgG, e.g., an IgG1 molecule, CH1 ends at amino acid position 215 according to EU numbering, and CH2 begins at amino acid position 231 according to EU numbering. Thus, the hinge for an IgG comprises amino acid positions 216-230 according to EU numbering.

[0100] A "variant Fc domain" comprises amino acid modifications compared to a parent Fc domain. Thus, a "variant IgG1 Fc domain," e.g., a variant human IgG1 Fc domain, comprises amino acid modifications (e.g., amino acid substitutions and / or deletions) at positions corresponding to positions in an IgG1 Fc domain, e.g., a human IgG1 Fc domain, and is preferably a functional variant of the parent Fc domain. Such variant IgG Fc domains retain at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding parent human IgG Fc domain. In some cases, the variant Fc domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the parent Fc domain. Optionally, the variant Fc domain may have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the parent Fc domain. Preferably, the variant Fc domain retains the ability to form dimers with other Fc domains, as determined using techniques disclosed herein or known in the art, such as non-denaturing gel electrophoresis.

[0101] Fc modification The binding agents described herein comprise three distinct polypeptide chains, and two of the polypeptide chains, e.g., the first and second polypeptide chains, preferably comprise CH2-CH3 regions derived from an IgG, such as IgG1, particularly human IgG1. Preferably, the first and second polypeptide chains comprising the CH2-CH3 regions are capable of interacting, e.g., dimerizing, thereby forming a heterodimer comprising the first and second polypeptide chains of the binding agents disclosed herein. Preferably, the CH2-CH3 regions of the binding agents are derived from IgG1, more preferably human IgG1, although CH2s and CH3s derived from other serotypes may be used as described herein. Furthermore, as discussed herein, the binding agents self-assemble, e.g., within a production host cell. For example, it is envisioned 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. The Fc fragment may comprise one or more amino acid modifications, or "Fc modifications," as described herein for human IgG1, to promote interaction between the CH2s on the first and second polypeptide chains and / or the CH3s on the first and second polypeptide chains, and / or to facilitate purification of heteromultimers, such as heterodimers, containing first and second polypeptide chains that interact with each other, relative to homomultimers containing only one type of polypeptide chain, and / or to confer additional beneficial functionality, as described herein. The amino acid modifications described herein may also be contained in the CH1 domain, e.g., within the first and / or second polypeptide chains of the binding agents disclosed herein. Additionally, peptide linkers within the scFv portion or peptide linkers connecting additional domains of the binding agent, e.g., between CH1 and scFv, or CH2 and scFv, or CH1 and CH2, may comprise one or more amino acid modifications disclosed herein.For example, a peptide linker connecting a CH2-CH3 region to another region or domain, such as VH(CD3) or CH1, may have a serine at the amino acid position corresponding to position 220 according to EU numbering in naturally occurring IgG1 (where cysteine ​​would normally be found). Using a peptide linker containing a serine at this position corresponding to position 220 in human IgG1 reduces disulfide formation between the two chains comprising the CH2-CH3 region. Thus, the formation of the binders described herein preferably utilizes distinct monomers (e.g., first and second polypeptide chains described herein) comprising CH1, CH2, and / or CH3 domains containing amino acid substitutions, such as those that "skew" the formation of heterodimers formed by the monomers relative to the homodimers described herein, in combination with "pI modifications" that may facilitate the purification of heterodimers from homodimers, and optionally in combination with "ablation modifications" and additional Fc modifications described herein. One of skill in the art will appreciate that any of the amino acid modifications described herein with respect to the CH1, CH2, and / or CH3 domains and peptide linkers, e.g., hinge mutants, can be combined with additional amino acid modifications described herein or known in the art. For example, any of the skewing and pI modifications can be independently combined with ablation modifications and other Fc modifications. Fc modifications according to the present invention can be amino acid insertions, additions, deletions, or substitutions. The Fc modifications described herein are defined according to the amino acid modifications that comprise them. For example, N434S is an Fc modification in which serine at position 434 (EU numbering) is substituted with asparagine relative to the parent human IgG1 Fc polypeptide. The identity of the parent amino acid may not be specified, in which case the variant is referred to as 434S, i.e., the CH2-CH3 region containing the Fc modification contains serine at the amino acid position corresponding to EU numbering 434 in human IgG1.

[0102] pI modification pI modifications can increase the isoelectric point (pI) difference between monomers, allowing homo- and heteromultimeric proteins to be purified isoelectrically. Generally, pI modifications increase the pI of the polypeptide chain (a basic change) or decrease the pI of the polypeptide chain (an acidic change). As described herein, a pI difference of at least 0.1, e.g., 0.2, 0.3, 0.4, or 0.5, between two polypeptide chains allows for separation by ion exchange chromatography or isoelectric focusing, or other methods known in the art that are sensitive to isoelectric point. That is, the inclusion of a pI modification that changes the pI of each of interacting polypeptide chains, e.g., a first and second polypeptide chain described herein, such that the pIs of the polypeptide chains are different and the pIs of heteromultimers formed by the polypeptide chains are also different, facilitates isoelectric purification of binding agents that include the heteromultimers. Such substitutions can also aid in the determination and monitoring of any contaminating, undesired homo- or heteromultimer formation.

[0103] pI modifications can be contained in one or both chains of a heteromultimer comprising a heavy chain constant region, e.g., in the first and second polypeptide chains of the binding agents disclosed herein, and in the CH1, CH2, and / or CH3 domains, and / or in a peptide linker, such as the linker connecting the VH and VL domains of an scFv portion. For example, pI variants can be contained in the first and / or second polypeptide chains of the binding agents disclosed herein to reduce or prevent homomultimer formation. Typically, when contained in both the first and second polypeptide chains, pI variants are used to increase the pI of one polypeptide chain and decrease the pI of the second polypeptide chain. This can be done, for example, by substituting a neutral amino acid residue with a positively or negatively charged amino acid residue, or vice versa, or by changing a charged amino acid residue from a positive charge to a negative charge, or vice versa, as discussed herein. Thus, in certain embodiments of the present invention, the pI of at least one of the polypeptide chains of a binding agent disclosed herein can be altered sufficiently to purify, for example, heteromultimers of a first and second polypeptide chain from homomultimers. In certain embodiments, pI differences of 0.1, 0.2, 0.3, 0.4, or 0.5 pH units or greater are used in the present invention. The amount of pI modification to be included on one or more polypeptide chains of a binding agent disclosed herein to achieve good separation will depend, in part, on the starting pI of the polypeptide chain, the pI of the CH region, the Fv scaffold region, etc., as will be understood by those of skill in the art. The pI change can be measured by any method known in the art, e.g., based on the CH region, e.g., by the method of Sillero and Maldonado (Sillero, Maldonado, (2006) Comput. Biol. Med. 36(2):157-166). Alternatively, the pI of each polypeptide chain can be compared. Additionally, heteromultimers can be separated by their size. In certain embodiments, modifications such as pI modifications, skewing modifications, additional or deleting Fc modifications, etc. are not included in the variable regions of the binding agents disclosed herein.

[0104] In one embodiment, the pI modifications are derived from different IgG isotypes so that the pI of each polypeptide chain is varied without introducing immunogenicity (see U.S. Patent Application Publication No. 2014 / 0370013). Preferably, the pI modifications are derived from human IgG isotypes to reduce the risk of introducing immunogenicity. While the modifications described herein are described with respect to human IgG1, all IgG isotypes may be altered, as may isotype hybrids. When the heavy chain constant domain is derived from IgG2-4, R133E and R133Q can also be used.

[0105] IgG1 is a common isotype for therapeutic antibodies for various reasons, including its high effector function. However, the pI of the CH region of IgG1 is higher than that of IgG2. Introducing IgG2-derived residues into the IgG1 backbone at specific positions can lower or increase the pI of the resulting monomer and, further, increase its serum half-life. For example, human IgG1 has a glycine residue (pI approximately 5.97) at position 137 (EU numbering), while human IgG2 has a glutamic acid residue (pI approximately 3.22) at the corresponding position. Substituting the glycine residue with a glutamic acid residue affects the pI of the resulting polypeptide. Lowering the pI of the antibody constant region can also increase serum half-life in vivo (USSN 13 / 194,904; Ghetie and Ward, 1997, Immunol Today. 18(12): 592-598). Additionally, variable regions with lower pI may further extend serum half-life (Igawa et al., (2010) PEDS 23(5): 385-392). In a preferred combination of pI modifications, one polypeptide chain (e.g., a first polypeptide chain of a binding agent disclosed herein, e.g., VH(CLDN18.2), CH1, CH2, and CH3) comprises an aspartic acid residue at position 208, a glutamic acid residue at position 295, an aspartic acid residue at position 384, a glutamic acid residue at position 418, and an aspartic acid residue at position 421; and the other polypeptide chain (e.g., a second polypeptide chain of the binding agent, e.g., comprising VH(CLDN18.2), CH1, VL(CD3), VH(CD3), CH2, and CH3) comprises a positively charged peptide linker (an "scFv linker") connecting VH(CD3) and VL(CD3), such as a polypeptide linker comprising or consisting of the amino acid sequence (GKPGS)4 or a functional variant thereof.

[0106] In polypeptide chains that do not contain CH1 and do not contain the amino acid position corresponding to position 208 according to EU numbering, the following negative pI modifications can be used: glutamic acid residue at position 295, aspartic acid residue at position 384, glutamic acid residue at position 418, and aspartic acid residue at position 421 according to EU numbering (human IgG1: Q295E / N384D / Q418E / N421D). In certain embodiments, one polypeptide chain, e.g., a first polypeptide chain, contains a set of mutants as discussed herein, and the polypeptide chain that interacts with it, e.g., a second polypeptide chain, comprises a charged scFv linker, such as the positively charged scFv linker of SEQ ID NO: 2 or a functional variant thereof, or a negatively charged scFv linker.

[0107] Skew Modification A "skew modification" is a steric modification that promotes interaction between polypeptide chains containing the modification. One strategy utilizing steric modifications is known in the art as "knobs and holes," which refers to amino acid engineering, in which a protuberance is introduced into a first heavy chain polypeptide, usually in the Fc region (CH2-CH3), and a cavity corresponding to the Fc region (CH2-CH3) is introduced into a second heavy chain polypeptide, such that the protuberance is located in the cavity at the interface between the two heavy chains, promoting heterodimer formation and preventing homodimer formation (USSN 61 / 596,846, Ridgway et al., (1996) Protein Engineering 9(7):617; Atwell et al., (1997) J. Mol. Biol. 270:26; U.S. Patent No. 8,216,805). The "protuberance" is constructed by replacing a small amino acid side chain from the interface of the first heavy chain polypeptide with a larger side chain. Compensatory "cavities" of identical or similar size to the protrusions are created at the interface of the second heavy chain polypeptide by replacing large amino acid side chains with smaller ones (U.S. Patent No. 5,731,168). The "knob and hole" modification can be combined with disulfide bonds to skew heteromultimerization, e.g., heterodimerization of the first and second heavy chain polypeptides (Merchant et al., (1998) Nature Biotech. 16:677).

[0108] Useful skewed modifications include, but are not limited to, the following pairs of double modifications: 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, for human IgG1, according to EU numbering. and T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C, where one moiety of each pair of dual modifications is present in one polypeptide chain of a binding agent described herein (e.g., a first polypeptide chain described herein) and the second moiety is present in the other polypeptide chain of a binding agent described herein (e.g., a second polypeptide chain described herein). Preferably, L368D / K370S:S364K / E357Q is used in the binding agents disclosed herein. The skew modifications disclosed herein can also have an effect on pI (Gunasekaran et al., (2010) J. Biol. Chem. 285(25):19637) and therefore can also be considered pI variants for purification purposes.

[0109] removal modifier As used herein, "ablation" refers to the reduction or elimination of activity. "Ablation of FcγR binding" means that an Fc region comprising one or more ablation modifications loses more than 50% of its FcγR binding activity compared to an Fc region that does not contain the specified modifications. Preferably, an Fc region comprising one or more ablation modifications loses more than 70%, 80%, 90%, 95%, 98% or more of its FcγR binding activity. Preferably, the FcγR binding activity of an Fc region comprising one or more ablation modifications is below the level of detectable binding in a Biacore, SPR, or BLI assay compared to an Fc region that does not contain the specified modifications.

[0110] As is known, the Fc domain of human IgG1 has the highest binding affinity to Fcγ receptors; therefore, when the constant domain of a binding agent is derived from IgG1, a deletion modification can be used. Alternatively, or by a deletion modification and a mutation at glycosylation position 297 (typically to A or S), binding to FcγRIIIa can be significantly eliminated, for example. Human IgG2 and IgG4 naturally bind less well to Fcγ receptors (Parren et al., 1992, J. Clin Invest. 90: 1537-1546; Bruhns et al., 2009, Blood 113: 3716-3725); therefore, the CH1, CH2, and CH3 domains derived from IgG2 or IgG4 can be used in the binding agents disclosed herein, with or without a deletion modification. For amino acid modifications that eliminate FcγR binding, see, e.g., Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.; 75(24):12161-12168 (2001).

[0111] Thus, the Fc portion of the binding agents disclosed herein can comprise one or more "FcγR-deleting modifications" or "Fc knockout (FcKO or KO) modifications." In certain embodiments, it is desirable to reduce or eliminate binding of the Fc domain to one or more or all of the Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, etc.). In certain embodiments, it is desirable to eliminate FcγRIIIa binding of binding agents that bind monovalently to CD3, such as the binding agents disclosed herein, to eliminate or significantly reduce ADCC activity. Thus, in the binding agents described herein, one or more of the polypeptide chains of the binding agents described herein, e.g., the first polypeptide chain and the second polypeptide chain, comprise one or more FcγR-deleting variants. In a preferred embodiment, the one or more deletion mutants 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 / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del, S239K / S267K, 267K / P329K, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G and E233P / L234V / L235A / G236del, where "del" refers to the deletion of an amino acid at the indicated position. Preferably, the modifications E233P / L234V / L235A / G236del / S267K, according to EU numbering and with respect to human IgG1, are used in both the first and second polypeptide chains of the binding agents described herein. Note that the deleting modifications disclosed herein eliminate FcγR binding, but generally do not eliminate FcRn binding. However, techniques for reducing or increasing FcRn binding are known and can be used to reduce or increase the serum half-life of a binding agent (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). For example, the first polypeptide chain of a binding agent described herein comprises the amino acid sequence set forth in SEQ ID NO:7, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:8, and the first and second polypeptide chains comprise a skew modification of the set L368D / K370S:S364K / E357Q, and the first polypeptide chain further comprises a pI modification of the set N208D / Q295E / N384D / Q418E / N421D, and both the first and second polypeptide chains further comprise a deletion modification of the set E233P / L234V / L235A / G236del / S267K, e.g., the first polypeptide chain comprises a VH(CLDN18.2) and a CH1, and the second polypeptide chain comprises an scFv(CD3), a VH(CLDN18.2) and a CH1. Of course, further modifications may be included in the amino acid sequence of each binding agent, for example, a binding agent may include additional amino acid modifications, such as substitutions, in addition to the modifications described above.

[0112] Further Fc modifications In addition to other modifications disclosed herein, such as pI, skew, and deletion modifications, certain useful modifications that alter binding of one or more FcγR receptors, such as altered binding to the FcRn receptor, can be used. Thus, there are certain useful amino acid substitutions that can be made to alter the binding of the binding agents disclosed herein to one or more FcγR receptors. Substitutions that result in increased binding as well as decreased binding can be useful. For example, increased binding to FcγRIIIa is known to increase ADCC. Similarly, decreased binding to FcγRIIb can be beneficial. Amino acid substitutions that can be used in the present invention include those listed in U.S. Ser. Nos. 11 / 124,620, 11 / 174,287, 11 / 396,495, and 11 / 538,406, all of which are incorporated herein by reference in their entireties. Particular useful 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. Additionally, as disclosed in U.S. Ser. No. 12 / 341,769 (incorporated herein by reference in its entirety), there are additional modifications useful for enhancing binding to FcRn and increasing serum half-life, including, but not limited to, 434S, 434A, 428L, 308F, 259I, 428L / 434S, 259I / 308F, 436I / 428L, 436I / 434S, 436V / 434S, 436V / 428L, and 259I / 308F / 428L. Additionally, the CH3 on one or both polypeptide chains, preferably on both polypeptide chains, forming the Fc heterodimer may contain the modifications M428L / N434S, which confers a longer serum half-life.

[0113] As one of skill in the art will appreciate, the modifications described herein can be independently combined with other modifications. In some embodiments, one polypeptide chain (e.g., a first polypeptide chain) of a binding agent described herein comprises N208D, Q295E, N384D, Q418E, and N481D according to EU numbering, and the other polypeptide chain (e.g., a second polypeptide chain) of the binding agent comprises a positively charged scFv linker described herein. Preferably, the first polypeptide chain of the binding agent further comprises K370S and L368D according to EU numbering, and the second polypeptide chain of the binding agent further comprises E357Q and S364K. Additionally, 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 the 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 ​​typically paired with the light chain.

[0114] The term "monoclonal binding agent" as used herein includes "monoclonal antibodies" and refers to a preparation of binding agent molecules of single molecular composition. A "monoclonal antibody" displays a single binding specificity and affinity for a particular epitope. As used herein, the term "recombinant binding agent" includes "recombinant antibody" and includes all binding agents that are prepared, expressed, produced or isolated by recombinant techniques.

[0115] As used herein, the term "human binding agent" includes "human antibodies," and is intended to include binding agents with 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). As used herein, the term "humanized binding agent," including "humanized antibodies," refers to a molecule in which the antigen-binding site is substantially derived from an immunoglobulin derived from a non-human species, with the remaining immunoglobulin structure of the molecule being based on the structure and / or sequence of a human immunoglobulin. This can be achieved, for example, by grafting the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen-binding site, onto homologous human acceptor framework regions (FRs) (see WO 92 / 22653 and EP 0 629 240). The antigen-binding site can comprise either a complete variable domain fused to a constant domain or only the complementarity-determining regions (CDRs) grafted into appropriate framework regions in the variable domain. Substitution of framework residues (backmutations) from the parent binding agent (i.e., a non-human binding agent, e.g., a murine antibody) with human framework regions may be necessary to fully reconstitute the binding affinity and specificity of the parent binding agent. Structural homology modeling can be advantageous in identifying amino acid residues in the framework regions that are important for the binding properties of the binding agent. The antigen-binding site may be wild-type or may be modified with one or more amino acid substitutions, e.g., modified to more closely resemble human immunoglobulins. Some forms of humanized binding agents preserve all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from the mouse antibody). Other forms have one or more CDRs altered relative to the original binding agent, e.g., antibody. Thus, a humanized binding agent may contain non-human CDR sequences, primarily human framework regions, and fully human constant regions, optionally containing one or more amino acid backmutations to non-human amino acid sequences.

[0116] As used herein, the term "chimeric binding agent" includes "chimeric antibodies" and refers to binding agents in which portions of the amino acid sequences of the heavy and light chains are homologous to corresponding sequences in binding agents, e.g., antibodies, derived from a particular species or belonging to a particular class, while the remaining segments of the chains are homologous to corresponding sequences in the other. Typically, the variable regions of both the light and heavy chains mimic the variable regions of antibodies derived from one mammalian species, while the constant portions are homologous to antibody sequences derived from another species. One obvious advantage of such chimeric forms is that the variable regions can be easily derived from currently known sources, for example, using readily available B cells or hybridomas derived from non-human host organisms in combination with constant regions derived from human cell preparations. While variable regions have the advantage of being easily prepared and specificity is not affected by the source, human constant regions are less likely to elicit an immune response from a human subject when the binding agent is injected than constant regions derived from non-human sources. However, the definition is not limited to this specific example. The binding agents or fragments thereof, such as the variable and / or constant regions, can be derived from different species, including, but not limited to, mouse, rat, rabbit, guinea pig, and human.

[0117] Immunoglobulins disclosed herein include IgA, such as IgA1 or IgA2, IgG1 (including allotypes with polymorphisms at amino acid positions 356 (D or E) and 358 (L or M) according to EU numbering), IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the immunoglobulin is an IgG1 antibody, more specifically an IgG1 kappa or IgG1 lambda isotype (i.e., IgG1, κ, λ), an 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). The amino acid sequence disclosed herein for the IgG1 allotype 356D / 358M also includes the allotype 356E / 358L.

[0118] The term "IgG subclass modification" or "isotype modification" refers to an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid in a different, aligned IgG isotype. For example, IgG1 contains a tyrosine at amino acid position 296 according to EU numbering, and IgG2 contains a phenylalanine, so an F296Y substitution in IgG2 is considered an IgG subclass modification.

[0119] As used herein, the term "heteroligand" includes "heteroligand antibodies" and is defined in relation to the transgenic organism producing the binding agent. The term refers to a binding agent that has an amino acid sequence or encoding nucleic acid sequence that corresponds to an amino acid sequence or encoding nucleic acid sequence found in an organism other than the transgenic organism, and that is generally derived from a species other than the transgenic organism. As used herein, the term "heterohybrid binding agent" includes "heterohybrid antibodies" and refers to binding agents in which the light and heavy chains are of different biological origins. For example, an antibody in which the human heavy chain is associated with a murine light chain is a heterohybrid antibody. Binding agents, including antibodies, disclosed herein are preferably isolated. As used herein, the term "isolated" refers to a binding agent that is substantially free of other agents with different antigen specificities (e.g., an isolated binding agent that specifically binds to CLDN18.2 and CD3 is substantially free of binding agents that specifically bind to antigens other than CLDN18.2 and CD3). However, an isolated binding agent that specifically binds to an epitope, isoform, or variant of human CLDN18.2 may be cross-reactive with other related antigens, such as related antigens from other species (e.g., CLDN18.2 species homologs). Furthermore, an isolated binding agent may be substantially free of other cellular material and / or chemicals.

[0120] The terms "antigen-binding portion" (or simply "binding portion") of a binding agent such as an antibody or "antigen-binding fragment" (or simply "binding fragment") of a binding agent such as an antibody, or like terms, refer to one or more fragments of the binding agent that retain the function of specifically binding 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 by the term "antigen-binding portion" of a binding agent such as an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) ab' fragments derived from the F(ab')2 fragment and containing free sulfhydryl groups that can be alkylated or used for conjugation with enzymes, toxins, or other proteins of interest, and which may contain a small portion of the Fc; (iv) a Fd fragment consisting of the VH and CH domains; (v) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); (vii) an isolated complementarity-determining region (CDR); and (viii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be recombinantly engineered into a single protein chain in which the VL and VH domains pair to form a monovalent molecule (known as a single-chain Fv (scFv); e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-58833). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of a binding agent such as an antibody. A further example is a binding domain immunoglobulin fusion protein comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are further disclosed in US 2003 / 0118592 and US 2003 / 0133939. Such antibody fragments are obtained using conventional techniques known to those of skill in the art, and are screened for utility in the same manner as are intact antibodies.

[0121] Single-chain variable fragments (scFvs) are fusion proteins of the variable regions of immunoglobulin heavy (VH) and light (VL) chains, connected by a short linker peptide, typically 10 to about 30 amino acids, such as an scFv linker, as shown in SEQ ID NO: 2. The linker is typically rich in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Bivalent (or divalent) single-chain variable fragments (di-scFvs, bi-scFvs) can be engineered by linking two scFvs. This can be done by creating a single peptide chain with two VH and two VL domains, resulting in tandem scFvs. The present invention also encompasses multispecific molecules comprising one or more scFv binding domains. One common flexible linking peptide is (G4S)x, where x can be 2, 3, 4, 5, or 6. Preferably, the linker connecting the VH and VL domains of the scFv comprises, and preferably consists of, the amino acid sequence (GKPGS)x or a functional variant thereof, where x can be 2, 3, 4, 5, or 6. In some cases, the association of VH and VL can be stabilized by one or more intermolecular disulfide bonds.

[0122] A Fab (Fragment Antigen Binding) antibody fragment is an immunoreactive polypeptide comprising a monovalent antigen-binding domain of an antibody composed of a polypeptide consisting of a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1) portion, and a polypeptide consisting of a light chain variable region (VL) and a light chain constant region (the CL and CH1 portions are both linked, for example, by disulfide bonds between Cys residues). Preferably, the CH1 and CL of the Fab fragments described herein are of human origin. In one embodiment, the CL is a kappa-type CL. In one embodiment, the CH1 is derived from IgG1, preferably human IgG1.

[0123] For purposes of the present invention, all antibodies and antibody derivatives, e.g., antibody fragments, as disclosed herein are encompassed by the term "antibody." The term "antibody derivative" refers to any modified form of an antibody, e.g., a conjugate of an antibody with another agent or antibody, or an antibody fragment. Furthermore, the antibodies and antibody derivatives disclosed herein are useful for generating the binding agents disclosed herein. Naturally occurring antibodies are generally monospecific, binding to a single antigen. The bispecific binding agents disclosed herein bind to cytotoxic cells, such as T cells, by binding to the CD3 receptor and to target cells, such as cancer cells, by binding to CLDN18.2. The binding agents disclosed herein bind to at least two different types of antigens and are at least bispecific or multispecific, e.g., trispecific, tetraspecific, etc.

[0124] In some embodiments, the binding agents described herein are at least trivalent. As used herein, the terms "valent," "multivalent," or other grammatical variations refer to the number of antigen-binding sites or binding domains in a binding agent. Antigen-binding sites that bind to the same antigen may recognize different epitopes or, preferably, the same epitope. The binding agents described herein may also have a valency of four or more. The binding agents described herein are preferably engineered proteins (including protein complexes) that may be composed of fragments of at least two different antibodies (wherein the fragments of the at least two different antibodies form at least two different binding domains) and thus bind to at least two different types of antigens. The binding agents disclosed herein are engineered to simultaneously bind to immune cells, such as immune effector cells, particularly T cells, such as cytotoxic cells (e.g., by binding to CD3), and to target cells, such as cancer cells, to be destroyed (e.g., by binding to the tumor-associated antigen CLDN18.2).

[0125] In one embodiment, the binding agents described herein are in the form of a Fab2-scFv construct, i.e., a construct comprising two Fab fragments (each comprising a VH and CH1 domain on one polypeptide chain and corresponding VL and CL domains on the other polypeptide chain, wherein the antigen-binding domain is formed by the interaction of each of said sets of polypeptide chains) and an scFv portion (comprising VH and VL domains connected to each other by a polypeptide linker on the same polypeptide chain, wherein the VH and VL interact to form the antigen-binding domain). In certain embodiments, a binding agent described herein is a tetramer composed of four polypeptide chains, wherein the first polypeptide chain comprises a VH from an immunoglobulin, e.g., an immunoglobulin of a first specificity; the second polypeptide chain comprises a VH from an immunoglobulin, e.g., an immunoglobulin of the first specificity, and an scFv portion comprising a VH from an immunoglobulin, e.g., an immunoglobulin of a second specificity, and a VL from an immunoglobulin, e.g., an immunoglobulin of the second specificity; the third polypeptide chain comprises a VL from an immunoglobulin, e.g., an immunoglobulin of the first specificity; and the fourth polypeptide chain is identical to the third polypeptide chain. In certain embodiments, the first and second polypeptide chains further comprise a CH1 from an immunoglobulin, e.g., the C-terminus of the VH from the immunoglobulin of the first specificity, and the third and fourth polypeptide chains further comprise a CL 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., at the C-terminus of the Fab fragment and scFv portion, respectively. Thus, in some embodiments, a binding agent described herein comprises a first polypeptide chain comprising a VH-CH1 linked to CH2-CH3, a second polypeptide chain comprising a VH-CH1 linked to an scFv portion linked to CH2-CH3, and third and fourth polypeptide chains each comprising a VL-CL. In some embodiments, the first polypeptide chain interacts with the second polypeptide chain. In some embodiments, the first polypeptide chain interacts with the third polypeptide chain.In some embodiments, the second polypeptide chain interacts with a fourth polypeptide chain. In some embodiments, the first and second polypeptide chains interact, the first polypeptide chain further interacts with a third polypeptide chain, and the second polypeptide chain further interacts with a fourth polypeptide chain. In 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 CH1 on the first polypeptide chain interacts with the CL on the third polypeptide chain. In some embodiments, the VH on the second polypeptide chain (not part of the scFv portion) interacts with the VL on the fourth polypeptide chain to form a binding domain, and / or the CH1 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 CL and a cysteine ​​residue in CH1. One or both of the polypeptide chains comprising CH2-CH3 may comprise one or more amino acid modifications, e.g., Fc modifications disclosed herein, e.g., pI, skew, additional Fc, and removal modifications, e.g., to facilitate polypeptide chain interaction. According to the present invention, the VH and VL of the scFv portion are preferably connected by a peptide linker ("scFv linker"). In some embodiments, CH1 on the first and / or second polypeptide chain is connected to CH2 on the same polypeptide chain by a peptide linker. In some embodiments, the scFv is linked to CH2 by a peptide linker. In one embodiment, a VH on a first polypeptide chain interacts with a VL on a third polypeptide chain to form a binding domain specific for CLDN18.2, an additional VH on a second polypeptide chain that is not part of an scFv interacts with a VL on a fourth polypeptide chain to form a binding domain specific for CLDN18.2, and the VH and VL of the scFv on the second polypeptide chain interact to form a binding domain specific for CD3.

[0126] The term "linker" refers to any means that functions to connect two different functional units (e.g., domains or regions on a polypeptide chain). Types of linkers include, but are not limited to, chemical linkers, peptide linkers, and polypeptide linkers. The sequences of peptide linkers and polypeptide linkers are not limited. Peptide linkers are preferably non-immunogenic and flexible (e.g., containing serine and glycine sequences). Depending on the construct, linkers can be long or short. In a preferred embodiment, the scFv linker, i.e., the linker connecting the 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)x or a functional variant thereof, where 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 linked to 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.

[0127] According to the present invention, the linker connecting the scFv and CH1, preferably at the C-terminus of CH1, preferably comprises, preferably consists of, the amino acid sequence (G4S)x 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 present invention, the linker connecting the CH2 and scFv, preferably at the N-terminus of CH2, preferably comprises, preferably consists of, the amino acid sequence (G4S)2KTHTCPPC (SEQ ID NO: 4) or a variant thereof. According to the present invention, the linker connecting the CH1 and CH2 preferably comprises, preferably consists of, the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 6) or a functional variant thereof. According to the present invention, for example, the CH1 and scFv on the second polypeptide chain are connected by a peptide linker that preferably comprises, preferably consists of, the amino acid sequence (G4S)2 (SEQ ID NO: 5) or a functional variant thereof. However, other linkers may be used as known in the art.

[0128] In certain embodiments, a binding agent described herein comprises a first, second, third, and 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) a third polypeptide chain comprising, 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 Here, VH(CLDN18.2) on the first polypeptide chain interacts with VL(CLDN18.2) on the third polypeptide chain to form a binding domain specific for CLDN18.2, VH(CLDN18.2) on the second polypeptide chain interacts with VL(CLDN18.2) on the fourth polypeptide chain to form a binding domain specific for CLDN18.2, and VH(CD3) and VL(CD3) interact to form a binding domain specific for CD3, and the domains on the polypeptide chains are preferably connected to each other by a peptide linker disclosed herein.

[0129] In certain embodiments, a binding agent described herein comprises: a) a first polypeptide chain comprising a VH(CLDN18.2) and 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, and 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 b) a VH(CLDN18.2) and a CH1 described herein. a second polypeptide chain in which VH(CD3) and VL(CD3) are linked to each other via a charged scFv linker having the amino acid sequence (GKPGS)4 (SEQ ID NO:2), and CH2 and CH3 contain 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.

[0130] In certain embodiments, a binding agent described herein comprises a first, second, third, and fourth polypeptide chain, wherein i) the first polypeptide chain is, from the N-terminus to the C-terminus, VH(CLDN18.2)-CH1-linker-CH2-CH3 ii) the second polypeptide chain is N-terminally to C-terminally VH(CLDN18.2)-CH1-linker-VL(CD3)-linker-VH(CD3)-linker-CH2-CH3 iii) a third polypeptide chain comprising, 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 Here, VH(CLDN18.2) on the first polypeptide chain interacts with VL(CLDN18.2) on the third polypeptide chain to form a binding domain specific for CLDN18.2, VH(CLDN18.2) on the second polypeptide chain interacts with VL(CLDN18.2) on the fourth polypeptide chain to form a binding domain specific for CLDN18.2, and VH(CD3) and VL(CD3) interact to form a binding domain specific for CD3.

[0131] In certain embodiments, a binding agent described herein comprises a first, second, third, and fourth polypeptide chain, wherein i) the first polypeptide chain is, from the N-terminus to the C-terminus, VH(CLDN18.2)-CH 1-linker 1-CH 2-CH 3 ii) the second polypeptide chain is N-terminally to C-terminally VH(CLDN18.2)-CH1-linker2-VL(CD3)-linker3-VH(CD3)-linker4-CH2-CH3 iii) a third polypeptide chain consisting of, from the N-terminus to the 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; Linker 2 comprises the amino acid sequence (G4S)x or a functional variant thereof, where x is 2, 3, 4, 5 or 6, preferably x is 2; Linker 3 comprises the amino acid sequence (GKPGS)x or a functional variant thereof, where x is 2, 3, 4, 5 or 6, preferably x is 4; Linker 4 comprises the amino acid sequence (G4S)2KTHTCPPCP or a functional variant thereof; and Here, VH(CLDN18.2) on the first polypeptide chain interacts with VL(CLDN18.2) on the third polypeptide chain to form a binding domain specific for CLDN18.2, VH(CLDN18.2) on the second polypeptide chain interacts with VL(CLDN18.2) on the fourth polypeptide chain to form a binding domain specific for CLDN18.2, and VH(CD3) and VL(CD3) interact to form a binding domain specific for CD3.

[0132] The binding agents described herein, and / or the first, second, third, and fourth polypeptide chains of the binding agents described herein, may also comprise an amino acid sequence to facilitate secretion of the binding agent or polypeptide chain, such as an N-terminal secretory signal, and / or one or more epitope tags to facilitate binding, purification, or detection of the molecule. Preferably, the secretory signal is a signal sequence that is sufficient to allow passage through the secretory pathway and / or secretion of the binding agent or its polypeptide chain into the extracellular environment. Preferably, the secretory signal sequence may be cleaved and removed from the mature binding agent or polypeptide chain. The secretory signal sequence is preferably selected with respect to the cell or organism in which the binding agent or polypeptide chain is produced.

[0133] The amino acid sequence of the epitope tag may be introduced at any position within the amino acid sequence of the binding agent or polypeptide chain, may be in the form of a loop within the encoded protein structure, or may be fused N- or C-terminally to the binding agent or polypeptide chain. Preferably, the epitope tag is fused C-terminally to the binding agent or polypeptide chain. The epitope tag may include a cleavage site that allows removal of the tag from the binding agent or polypeptide chain. The epitope tag may be any type of epitope tag that is functional under native and / or denaturing conditions, preferably a histidine tag, most preferably a tag containing six histidines. In addition to the first, second, and third binding domains, the binding agents described herein may comprise one or more further binding domains, which serve, for example, to enhance selectivity for tumor cells. This can be achieved, for example, by providing binding domains that bind to other antigens expressed on tumor cells.

[0134] The term "post-translational modification" or like terms refers to modifications of proteins, such as covalent and enzymatic modifications, that occur after protein biosynthesis. As is generally known in the art, binding agents, such as antibodies, expressed in cells are often post-translationally modified. For example, post-translational modifications of binding agents, such as antibodies, can occur, for example, on amino acid side chains of the heavy or light chains or at the N- or C-terminus of the first and / or second polypeptide chains disclosed herein. Examples of post-translational modifications that may occur in the binding agents disclosed herein include, but are not limited to, cleavage of the C-terminal lysine of the heavy chain, e.g., of the first and / or second polypeptide chain, by, e.g., carboxypeptidase; modification of the N-terminal glutamine or glutamic acid of the heavy chain, e.g., of the first and / or second polypeptide chain, to pyroglutamic acid by pyroglutamylation; modification of the N-terminal glutamine or glutamic acid of the light chain, e.g., of the third and / or fourth polypeptide chain, to pyroglutamic acid by pyroglutamylation; glycosylation; oxidation; deamidation; and glycation. Such post-translational modifications are known to occur in various binding agents (Liu et al., 2008, J. Pharmacol. Sci. 97(7):2426-2447). Post-translational modifications by pyroglutamylation at the N-terminus and deletion of lysine at the C-terminus generally do not have any effect on the activity of the binders (Lyubuskaya et al., 2006, Analyt. Biochem. 348(1):24-39).

[0135] Thus, in certain embodiments, the binding agents disclosed herein may comprise one or more post-translational modifications. In certain embodiments, the one or more post-translational modifications comprise pyroglutamylation of the N-terminus of one or more polypeptide chains of the binding agent. In certain embodiments, the one or more post-translational modifications comprise pyroglutamylation of the N-terminus of one or more VH(CLDN18.2). In certain embodiments, the one or more post-translational modifications comprise deletion of a lysine at the C-terminus of a first polypeptide chain. In certain embodiments, the one or more post-translational modifications comprise deletion of a lysine at the C-terminus of a second polypeptide chain.

[0136] The binding agents disclosed herein in the context of the present invention are preferably capable of eliciting one or more immune effector functions as disclosed herein, preferably directed against cells bearing the cancer-associated antigen CLDN18.2 on their surface. The term "immune effector function" in the context of the present invention includes any function mediated by a component of the immune system that results in the inhibition of cancer growth and / or the inhibition of cancer development, including, for example, the inhibition of cancer dissemination and metastasis. Preferably, the immune effector function results in the killing of cancer cells. Immune effector functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis in cells bearing cancer-associated antigens, cytolysis of cells bearing cancer-associated antigens, and / or inhibition of proliferation of cells bearing cancer-associated antigens. The binding agents disclosed herein preferably recruit and redirect T cells, such as CD4 and / or CD8 T cells, particularly CD107a+ T cells, to disease-associated cells such as cancer cells, and thus act via redirected T cell cytotoxicity (RTCC), i.e., the redirected T cells preferably kill disease-associated cells, e.g., cancer cells. CD107a expression is known to be associated with the cytolytic potential of CD4 and CD8 T cells. Preferably, the CD107a+ T cells are capable of degranulation, i.e., they can release cytotoxic molecules such as perforin, granzymes, and the like, and can release one or more cytokines, such as tumor necrosis factor alpha (TNFα), interleukin-2 (IL2), interferon gamma (IFNγ), and the like, thereby inducing the death of target cells, e.g., cancer cells, to which the T cells are redirected by the binding agents described herein. Binding agents can also exert their effects simply by binding to cancer-associated antigens on the surface of cancer cells. For example, binding agents can block the function of cancer-associated antigens or induce apoptosis by binding to cancer-associated antigens on the surface of cancer cells.

[0137] The term "immune effector cell" or "effector cell" in the context of the present invention relates to a cell that exerts an effector function during an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells. The term "MHC-dependent T cell" or similar terms refers to a T cell that recognizes an antigen when presented in the context of an MHC and preferably exerts a T cell effector function, e.g., killing target cells expressing the antigen.

[0138] 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 the T cell receptor (TCR). The thymus is the primary organ responsible for the maturation of T cells. Different subsets of T cells have been discovered, each with distinct functions. T helper cells assist other white blood cells in immunological processes, including, among other functions, the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. These cells express the CD4 glycoprotein on their surface and are therefore also known as CD4+ T cells. Helper T cells are activated when presented with peptide antigens, usually by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or support active immune responses.

[0139] Cytotoxic T cells destroy virus-infected and cancer cells and are involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells normally recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of almost all cells in the body.

[0140] All T cells have a T cell receptor (TCR), which exists as a complex of proteins. The TCR on a T cell binds to major histocompatibility complex (MHC) molecules and can interact with immunogenic peptides (epitopes) displayed on the surface of target cells. Specific binding of the TCR triggers a signaling cascade within the T cell, leading to proliferation and differentiation into mature effector T cells. In the majority of T cells, the actual T cell receptor is produced by independent T cell receptor alpha and beta (TCRα and TCRβ) genes and consists of two distinct peptide chains, the α- and β-TCR chains. A very rare group of T cells (2% of all T cells), γδ T cells (gamma delta T cells), consists of one γ chain and one δ chain and has a different T cell receptor (TCR) on its surface. All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells populate the thymus, where they proliferate by cell division to generate a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8 and are therefore classified as double-negative (CD4-CD8-) cells. As they progress through development, they become double-positive thymocytes (CD4+CD8+) and ultimately mature into single-positive (CD4+CD8- or CD4-CD8+) thymocytes before being released from the thymus into peripheral tissues.

[0141] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of T cell receptors. Human MHC molecules are commonly referred to as HLA (human leukocyte antigen) molecules. There are two major classes of MHC molecules: class I and class II. MHC class I antigens are found on almost all nucleated cells in the body. The primary function of this class of MHC molecule is to present peptide fragments of intracellular proteins to CTLs. This presentation allows CTLs to attack those presenting MHC-bound peptides, including disease-associated peptides (antigens) such as cancer antigens. CD8+ T cells are typically cytotoxic (hence the name cytotoxic T cells, or CTLs) and recognize 9-10 amino acid peptides that are processed intracellularly from any intracellular protein and presented on the cell surface by MHC class I molecules. Therefore, surface expression of MHC class I molecules plays an important role in determining the susceptibility of target cells to CTLs.

[0142] The binding agents disclosed herein can 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 cells, and in particular, kills cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Suitable therapeutic agents for forming conjugates include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decylcarbazine), alkylating agents (e.g., mechlorethamine, thioepa), These include, but are not limited to, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibodies (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and antimitotic agents (e.g., vincristine and vinplastine). In preferred embodiments, the therapeutic agent is a cytotoxic or radiotoxic agent. In other embodiments, the therapeutic agent is an immunosuppressant. In yet other embodiments, the therapeutic agent is GM-CSF. In preferred embodiments, the therapeutic agent is doxorubicin, cisplatin, bleomycin sulfate, carmustine, chlorambucil, cyclophosphamide, or ricin A. The binding agents may also be coupled to radioisotopes, such as iodine-131, yttrium-90, or indium-111, to create cytotoxic radiopharmaceuticals.

[0143] Techniques for conjugating such therapeutic moieties to binding agents are well known and are described, for example, in: Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al. (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: See 119-58 (1982).

[0144] As used herein, the term "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes. As used herein, the term "isotype switching" refers to the phenomenon in which the class or isotype of an antibody changes from one Ig class to one of the other Ig classes. As used herein, the term "naturally-occurring" as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by humans in the laboratory is naturally-occurring.

[0145] As used herein, the term "rearranged" refers to the configuration of a heavy or light chain immunoglobulin locus in which a V segment is positioned immediately adjacent to a DJ or J segment in a conformation that essentially encodes a complete VH or VL domain, respectively. Rearranged immunoglobulin (antibody) loci can be identified by comparison with germline DNA, where the rearranged locus has at least one recombined heptamer / nonamer homology element. The term "unrearranged" or "germline configuration" as used herein with respect to a V segment refers to a configuration in which the V segment has not recombined directly adjacent to a D or J segment.

[0146] In certain embodiments, the binding agents described herein are capable of binding to, or preferably bind to, CLDN18.2, i.e., to an epitope present on CLDN18.2, preferably an epitope located in the extracellular domain of CLDN18.2, particularly the first extracellular loop, preferably within amino acid positions 29 to 78 of CLDN18.2. In certain embodiments, an agent capable of binding to CLDN18.2 binds to an epitope on CLDN18.1 that is not present on CLDN18.2. The agent capable of binding to CLDN18.2 preferably binds to human, mouse, and / or cynomolgus monkey CLDN18.2, but preferably does not bind to human, mouse, and / or cynomolgus monkey CLDN18.1. Preferably, the agent binding to CLDN18.2 preferably does not bind to human, mouse, and / or cynomolgus monkey CLDN9. Preferably, the agent capable of binding to CLDN18.2 is specific for CLDN18.2. Preferably, the agent capable of binding to CLDN18.2 binds to CLDN18.2 expressed on the cell surface. In a particularly preferred embodiment, the agent capable of binding to CLDN18.2 binds to a natural epitope of CLDN18.2 present on the surface of living cells.

[0147] In one embodiment, the binding domain comprises an antibody fragment. The term "fragment" particularly refers to one or more complementarity determining regions (CDRs) of the heavy chain variable region (VH) and / or light chain variable region (VL), preferably at least the CDR3 variable region. In one embodiment, the one or more complementarity determining regions (CDRs) are selected from the set of complementarity determining regions CDR1, CDR2, and CDR3. In a particularly preferred embodiment, the term "fragment" refers to the complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL). In one embodiment, a binding domain comprising one or more CDRs, sets of CDRs, or combinations of sets of CDRs disclosed herein comprises the CDRs together with their intervening framework regions. Preferably, this portion also comprises at least about 50% of either or both of the first and fourth framework regions, the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of binding agents produced by recombinant DNA techniques may also introduce residues C-terminal to the encoded variable regions by linkers introduced to facilitate cloning or other engineering steps, including the introduction of linkers for linking the variable regions to additional protein sequences, including immunoglobulin heavy chains, other variable regions, or protein tags. In one embodiment, a binding domain comprising one or more CDRs, a set of CDRs or a combination of a set of CDRs disclosed herein comprises the CDRs in a human antibody framework.

[0148] The precise identification of CDR regions depends on computational methods used to determine the amino acid residues involved. For example, according to Kabat et al., supra, the variable region generally encompasses amino acid residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in the VL and approximately 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) in the VH; the variable region may also include residues forming hypervariable loops (e.g., residues 26-32 (CDR1), 50-52 (CDR2), and 91-96 (CDR3) in the VL and residues 26-32 (CDR1), 53-55 (CDR2), and 96-101 (CDR3) in the VH (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917)). Those skilled in the art will understand that the exact identification of the CDR positions within the sequences disclosed herein may vary slightly depending on the numbering system used, as shown in Table 1 below (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)):

[0149] [Table 2] Thus, the CDR sequences disclosed herein include variants derived from different numbering systems, and thus, disclosure of each VH is a disclosure of the CDRs (e.g., CDR1, CDR2, and CDR3) derivable therefrom, and disclosure of each VL is a disclosure of the CDRs (e.g., CDR1, CDR2, and CDR3) derivable therefrom.

[0150] Throughout this specification, when referring to residues in the variable regions of the binding domains with specificity for CD3 or CLDN18.2 as disclosed herein (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is used, and for the CH1 and CH2-CH3 (optionally including the hinge) regions, the EU numbering system is used.

[0151] In one embodiment, the binding domain of a binding agent described herein that is specific for CLDN18.2 comprises a VH that comprises the complementarity determining regions CDR1, CDR2 and / or CDR3 identified within the amino acid sequence of SEQ ID NO: 16. In one embodiment, the binding domain of the binding agent described herein that is specific for CLDN18.2 comprises a VL that includes the complementarity determining regions CDR1, CDR2 and / or CDR3 identified within the amino acid sequence set forth in SEQ ID NO: 17.

[0152] In a preferred embodiment, the binding domain with specificity for CLDN18.2 of the binding agents described herein comprises the following set of CDRs: 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. In one embodiment, the VH further comprises a CDR1 or a functional variant thereof comprising the sequence set forth in SEQ ID NO: 10, and / or a CDR2 or a functional variant thereof comprising the sequence set forth in SEQ ID NO: 11, and / or the VL further comprises a CDR1 or a functional variant thereof comprising the sequence set forth in SEQ ID NO: 13, and / or a CDR2 or a functional variant thereof comprising the sequence set forth in SEQ ID NO: 14.

[0153] In a preferred embodiment, the binding domain with specificity for CLDN18.2 of the binding agents described herein comprises the following set of CDRs: VH comprises CDR1 comprising the sequence set forth in SEQ ID NO: 10 or a functional variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 11 or a functional variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 12 or a functional variant thereof, and VL comprises CDR1 comprising the sequence set forth in SEQ ID NO: 13 or a functional variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 14 or a functional variant thereof, and the sequence set forth in SEQ ID NO: 15 or a functional variant thereof. Preferably, VH comprises CDR1, 2 and 3 of SEQ ID NOs: 10, 11 and 12, and VL comprises CDR1, 2 and 3 of SEQ ID NOs: 13, 14 and 15.

[0154] In some embodiments, the heavy and light chain variable regions comprise the 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, the complementarity determining regions and framework regions are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0155] In a preferred embodiment, the binding domain of the binding agent described herein that is specific for CLDN18.2 comprises the amino acid sequence set forth in VH(CLDN18.2) SEQ ID NO: 16 over the entire length of the CLDN161 domain of CLDN0161, or a functional variant thereof. In a preferred embodiment, the binding domain of the binding agent described herein that has specificity for CLDN18.2 comprises VL(CLDN18.2) comprising the amino acid sequence set forth in SEQ ID NO: 17 or a functional variant thereof. In particularly preferred embodiments, the binding domain with specificity for CLDN18.2 of the binding agents described herein comprises the following combination of VH(CLDN18.2) and VL(CLDN18.2): VH(CLDN18.2) comprises the amino acid sequence shown in SEQ ID NO: 16 or a functional variant thereof, and VL(CLDN18.2) comprises the amino acid sequence shown in SEQ ID NO: 17 or a functional variant thereof.

[0156] In preferred embodiments, the framework regions of the VH and VL domains present in the binding agents disclosed herein may contain amino acid changes but retain at least 80%, 85%, or 90% identity to human germline sequences. In a further embodiment, the binding domain of the binding agent described herein that is specific for CLDN18.2 comprises the heavy and light chain variable regions of an antibody that (i) competes with an antibody comprising the heavy and light chain variable regions described above for CLDN18.2 binding, and / or (ii) is specific for CLDN18.2 of an antibody comprising the heavy and light chain variable regions described above. In one embodiment, the binding domain specific for CLDN18.2 of the binding agents described herein is in the format of a Fab molecule described herein, where VH(CLDN18.2) is a portion of a first and second polypeptide chain described herein, and VL(CLDN18.2) is a portion of a third polypeptide chain and a fourth polypeptide chain identical to the third polypeptide chain of the binding agents described herein. It should be understood that the binding domains that bind to CLDN18.2 of the binding agents described herein in Fab2-scFv format can be identical or essentially identical and therefore can bind to the same or essentially the same epitope on CLDN18.2. Thus, both binding domains that bind to CLDN18.2 of the binding agents described herein in Fab2-scFv format can correspond to, or essentially correspond to, one of the binding domains that bind to CLDN18.2 described herein. Preferably, the binding domain with specificity for CD3 is capable of specifically recognizing human CD3 in the context of other TCR subunits present on activated primary human T cells expressing the TCR in its native configuration.

[0157] In certain embodiments, a binding domain specific 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. In one embodiment, the binding domain specific for CD3 of the binding agents described herein comprises a VH comprising the following set of CDR1, CDR2 and CDR3: CDR1: SEQ ID NO: 18 CDR2: SEQ ID NO: 23 CDR3: SEQ ID NO: 19 or is a functional variant thereof. In a preferred embodiment, the binding domain specific for CD3 of the binding agents described herein comprises a VL comprising the CDR1, CDR2 and / or CDR3 identified within the amino acid sequence set forth in SEQ ID NO:24. In a preferred embodiment, the binding domain with specificity for CD3 of the binding agents described herein comprises a VL comprising the following set of CDR1, CDR2 and CDR3: CDR1: SEQ ID NO: 20 CDR2: SEQ ID NO: 21 CDR3: SEQ ID NO: 22 or is a functional variant thereof. In preferred embodiments, the binding domains with specificity for CD3 of the binding agents described herein comprise the following combinations of VH and VL, each comprising a set of CDR1, CDR2 and CDR3: 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.

[0158] In a preferred embodiment, the binding domain with specificity for CD3 of the binding agents described herein comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 25 or a functional variant thereof. In a preferred embodiment, the binding domain with specificity for CD3 of the binding agents disclosed herein comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 24 or a functional variant thereof. In a preferred embodiment, the binding domain with specificity for CD3 of the binding agents described herein comprises the following VH and VL: VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25 or a functional variant thereof; and VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or a functional variant thereof. In one embodiment, the binding domain with specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 26 or a functional variant thereof. In certain embodiments, the CH1 of the first and / or second polypeptide chain of a binding agent disclosed herein is derived from IgG, preferably IgG1, more preferably human IgG1. In certain 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 IgG1, more preferably human IgG1. In certain embodiments, the CL of the third and / or fourth polypeptide chain of a binding agent disclosed herein is derived from Igκ or Igλ, preferably from Igκ, more preferably from human Igκ.

[0159] In certain embodiments, the first polypeptide chain of a binding agent disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 7 or a functional variant thereof. In certain embodiments, the second polypeptide chain of a binding agent disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 8 or a functional variant thereof. In certain embodiments, the third and / or fourth polypeptide chain of a binding agent disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 9 or a functional variant thereof. In a preferred embodiment, the binding agents described herein comprise 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, the third polypeptide chain comprises SEQ ID NO: 9 or a functional variant thereof, and the fourth polypeptide chain is identical to the third polypeptide chain.

[0160] In some embodiments, a binding agent described herein comprises at least two binding domains specific for CLDN18.2 and at least one binding domain specific for CD3. In some embodiments, a 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, a 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, a third polypeptide chain of a binding agent disclosed 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 that bind to CLDN18.2 and at least one binding domain that binds to CD3 comprises a set of first, second, third, and fourth polypeptide chains according to SEQ ID NOs: 27, 28, 29, and 29.

[0161] It should be understood that the binding agents described herein can be delivered to a patient by administering a nucleic acid, such as RNA, encoding the agent and / or by administering a host cell containing a nucleic acid, such as RNA, encoding the agent. When 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, the administered nucleic acid may be a mixture of different nucleic acid molecules, such as a set of nucleic acids. When administered to a subject, e.g., a patient, the nucleic acid or set of nucleic acids encoding the binding agent may be present in naked form, in a suitable delivery vehicle, such as a liposome, nanoparticle, or viral particle, or within a host cell. The provided nucleic acid or set of nucleic acids may produce the agent over an extended period of time in a sustained manner, which at least partially alleviates the instability observed with therapeutic antibodies. The nucleic acid or set of nucleic acids delivered to a patient may be produced by recombinant means. When the nucleic acid or set of nucleic acids is administered to a patient without being present in the host cell, it is preferably taken up by the patient's cells for expression of the binding agent encoded by the nucleic acid. If the nucleic acid or set of nucleic acids is administered to a patient while present within host cells, it is preferably expressed by the host cells within the patient to produce the binding agent encoded by the nucleic acid.

[0162] The term "recombinant" in the context of the present invention means "produced by genetic recombination." Preferably, "recombinant matter," such as a recombinant nucleic acid, in the context of the present invention is not naturally occurring. As used herein, the term "naturally occurring" refers to the fact that an entity can be found in nature. For example, a peptide or nucleic acid is naturally occurring if it is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in the laboratory. As used herein, the term "nucleic acid" is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0163] A nucleic acid or a set of nucleic acids can be contained in a vector. A set of nucleic acids can also be contained in a set of vectors, such that each nucleic acid in the set of nucleic acids is contained in a vector. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a specific desired DNA fragment and may lack functional sequences necessary for expression of the desired DNA fragment.

[0164] In the context of the present invention, the term "RNA" includes ribonucleotide residues, and preferably relates to molecules composed entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such modifications can include the addition of non-nucleotide material to the end of the RNA or internally, for example, at one or more nucleotides of the RNA. Nucleotides in an RNA molecule can also include non-standard nucleotides, such as unnatural nucleotides or chemically synthesized nucleotides or deoxynucleotides. Such modified RNAs may be referred to as analogs or analogs of naturally occurring RNA. The term "RNA" according to the present invention includes and preferably relates to "mRNA," which means "messenger RNA," and relates to a "transcript" that can be produced using DNA as a template and encodes a peptide or protein. mRNA typically comprises a 5'-untranslated region (5'-UTR), a protein or peptide coding region, and a 3'-untranslated region (3'-UTR). mRNA has a limited half-life both intracellularly and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In certain embodiments of the present invention, RNA is obtained by in vitro transcription or chemical synthesis. Methodologies for in vitro transcription are known to those skilled in the art. For example, various in vitro transcription kits are commercially available. In some embodiments of the invention, the RNA is a self-replicating RNA, such as a single-stranded self-replicating RNA. In some embodiments, the self-replicating RNA is a positive-sense single-stranded RNA. In some embodiments, the self-replicating RNA is viral RNA or RNA derived from viral RNA. In some embodiments, the self-replicating RNA is or is derived from an alphavirus genomic RNA. Alphavirus RNA can 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 which encodes a binding agent disclosed herein. In some embodiments, when the RNA is or is derived from viral RNA, the transgene can partially or completely replace viral sequences, such as viral sequences encoding structural proteins. In some embodiments, the self-replicating RNA is an in vitro transcribed RNA.

[0165] To increase the expression and / or stability of the RNA used according to the invention, it can be modified, preferably without changing the sequence of the expressed peptide or protein. As used herein, the term "modification" in the context of RNA includes any modification of RNA that does not naturally occur in that RNA. In one embodiment of the invention, the RNA used in accordance with the invention is free of uncapped 5'-triphosphates, which can be achieved by treating the RNA with a phosphatase.

[0166] The RNA of the present invention may contain modified naturally occurring or synthetic ribonucleotides to increase its stability and / or reduce its cytotoxicity and / or immunogenicity. For example, in some embodiments, 5-methylcytidine is partially or completely, preferably completely, substituted for cytidine in the RNA used in the present invention. Alternatively or additionally, in some embodiments, pseudouridine is partially or completely, preferably completely, substituted for uridine in the RNA used in the present invention.

[0167] In one embodiment, the term "modified" refers to the provision of RNA with a 5'-cap or 5'-cap analog. The term "5'-cap" refers to a cap structure found at the 5' end of an mRNA molecule, generally consisting of a guanosine nucleotide attached to the mRNA via an unusual 5'-5'-triphosphate linkage. In one embodiment, this guanosine is methylated at position 7. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably the 7-methylguanosine cap (m7G). The term "5'-cap" in the context of the present invention includes 5'-cap (capping) analogs that have been modified to resemble the RNA cap structure and function to stabilize the RNA when bound to it, preferably in vivo and / or in cells. Providing RNA with a 5' cap or 5' cap analog can be achieved by in vitro transcription of a DNA template in the presence of the 5' cap or 5' cap analog, and the 5' cap is co-transcriptionally incorporated into the generated RNA strand, or the RNA can be generated, for example, by in vitro transcription, and the 5' cap can be attached to the RNA post-transcriptionally using a capping enzyme, for example, vaccinia virus capping enzyme.

[0168] The RNA may comprise further modifications. For example, further modifications of the RNA used in the present invention may be extension or truncation of the naturally occurring poly(A) tail, or alteration of the 5'- or 3'-untranslated region (UTR), for example, introduction of a UTR not associated with the coding region of the RNA, for example, insertion of one or more, preferably two copies of a 3'-UTR from a globin gene, for example, alpha2-globin, alpha1-globin, beta-globin, preferably beta-globin, more preferably human beta-globin. Therefore, to enhance the stability and / or expression of the RNA used according to the present invention, it may be modified to include a poly(A) sequence having a length of preferably 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, and particularly preferably 100 to 150 adenosine residues. In a particularly preferred embodiment, the length of the poly(A) sequence is approximately 120 adenosine residues. Furthermore, incorporating two or more 3' untranslated regions (UTRs) into the 3' untranslated region of the RNA molecule can enhance translation efficiency. In one specific embodiment, the 3'-UTR is derived from the human β-globin gene. Preferably, the RNA, when delivered to, ie, transfected into, a cell, particularly a cell present in vivo, expresses the protein, peptide or antigen that it encodes.

[0169] The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of the present invention, the term "transfection" also includes the introduction of nucleic acids into or the uptake of nucleic acids by cells, where the cells may be present in a subject (e.g., a patient). Thus, according to the present invention, cells for transfection of nucleic acids as described herein can be present in vitro or in vivo; for example, the cells can form part of an organ, tissue, and / or organism of a patient. Transfection according to the present invention can be transient or stable. For transfection applications, it is sufficient if the transfected genetic material is only transiently expressed. Because nucleic acids introduced during the transfection process are not usually integrated into the nuclear genome, the foreign nucleic acid is diluted or degraded through mitosis. Cells capable of amplifying nucleic acid episomes greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remain in the genome of the cell and its daughter cells, stable transfection must occur. The RNA can be transfected into cells to transiently express the encoded protein.

[0170] The term "stability" of RNA relates to the "half-life" of the RNA. "Half-life" relates to the period of time required to eliminate half of the activity, amount or number of a molecule. The half-life of an RNA in the context of the present invention indicates the stability of said RNA. The half-life of an RNA may affect the "duration of expression" of the RNA. RNA with a long half-life can be expected to be expressed for a long period of time.

[0171] The term "transcription" in the context of the present invention relates to the process by which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription", which relates to a process by which RNA, in particular mRNA, is synthesized outside the body in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is applied for the production of the transcript. Such a cloning vector is generally referred to as a transcription vector and is included in the term "vector" according to the present invention. The term "translation" according to the present invention relates to the process in the ribosomes of a cell in which a chain of messenger RNA directs the assembly of a sequence of amino acids to make a peptide or protein.

[0172] The term "expression" is used according to the present invention in its most general sense and includes, for example, the production of RNA and / or peptides or proteins by transcription and / or translation. With respect to RNA, the terms "expression" or "translation" particularly relate to the production of peptides or proteins. It also includes partial expression of nucleic acids. Furthermore, expression can be transient or stable. According to the present invention, the term "expression" also includes "aberrant expression" or "aberrant expression". "Aberrant expression" or "aberrant expression" according to the present invention means that expression is altered, preferably increased, compared to a reference, e.g., the state of a subject not suffering from a disease associated with abnormal or aberrant expression of a particular protein, e.g., a tumor antigen. Increased expression refers to an increase of at least 10%, particularly at least 20%, at least 50%, or at least 100%, or more. In one embodiment, expression is found only in diseased tissue, while expression in healthy tissue is suppressed. The term "specifically expressed" means that a protein is expressed essentially only in a particular tissue or organ. For example, a tumor antigen specifically expressed in the gastric mucosa means that the protein is expressed primarily in the gastric mucosa and not in other tissues or not expressed to a significant extent in other tissues or organ types. Thus, a protein that is expressed exclusively in cells of the gastric mucosa and to a significantly lesser extent in any other tissue (e.g., testis) is specifically expressed in cells of the gastric mucosa. In certain embodiments, a tumor antigen may also be specifically expressed in two or more tissue types or organs, e.g., two or three tissue types or organs, but preferably no more than three different tissue types or organs, under normal conditions. In this case, the tumor antigen is specifically expressed in the organ. For example, if a tumor antigen is expressed to approximately equal extent in the lung and stomach under normal conditions, the tumor antigen is specifically expressed in the lung and stomach.

[0173] According to the present invention, the term "RNA encoding" means that the RNA can be expressed to produce the protein or peptide that it encodes when present in the appropriate environment, preferably within a cell. One aspect of the invention relies on adoptive transfer of host cells that are transfected ex vivo with nucleic acid, such as RNA, encoding a binding agent described herein, and, preferably after ex vivo expansion from low precursor frequencies to clinically relevant cell numbers, transferred into a recipient, such as a patient. Host cells used for therapy according to the invention may be autologous, allogeneic, or syngeneic to the recipient being treated. The term "autologous" is used to describe something that comes from the same subject. For example, the term "autologous transplant" refers to the transplantation of tissue or organs that come from the same subject. This procedure is advantageous because it overcomes the immunological barriers that result in rejection. The term "allogeneic" is used to describe something that is derived from different individuals of the same species. Two or more individuals are allogeneic to one another if the genes at one or more loci are not identical. The term "syngeneic" is used to describe individuals or tissues that are genotypically identical, i.e., derived from identical twins or the same inbred strain of animals, or tissues thereof. The term "xenogeneic" is used to describe something that is made up of multiple dissimilar elements. As an example, transplanting one individual's bone marrow into a different individual constitutes a xenogeneic transplant. A xenogeneic gene is a gene that originates from a source other than the subject.

[0174] The term "peptide" according to the present invention includes oligo- and polypeptides and refers to a substance comprising two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably nine or more, preferably ten or more, preferably thirteen or more, preferably sixteen or more, preferably twenty one or more, and preferably up to 8, 10, 20, 30, 40 or 50, especially 100 amino acids covalently linked by peptide bonds. The term "protein" refers to large peptides, preferably peptides with more than 100 amino acid residues, although in general the terms "peptide" and "protein" are synonymous and are used interchangeably herein. Any teachings given herein regarding specific amino acid sequences, e.g., those set forth in the Sequence Listing, should also be construed to relate to sequences functionally equivalent to the specific sequence, e.g., variants of the specific sequence that result in an amino acid sequence exhibiting the same or similar properties as the specific amino acid sequence. One important property is retaining target binding or effector function. Preferably, a sequence that is variant with respect to a specific sequence, when replacing a specific sequence in an antibody, retains the binding of the antibody to CLDN18.2 and / or CD3 and, preferably, the function of the antibody disclosed herein. Furthermore, preferably, a sequence that is variant with respect to a specific sequence, when replacing a specific sequence in a binding agent, retains the binding of the binding agent to CLDN18.2 and / or CD3 and, preferably, the function of the binding agent disclosed herein, e.g., cytotoxic T cell-mediated lysis. For example, the sequences shown in the sequence listing can be modified to remove one or more, preferably all, free cysteine ​​residues, in particular by substituting cysteine ​​residues with amino acids other than cysteine, preferably serine, alanine, threonine, glycine, tyrosine, tryptophan, leucine or methionine.

[0175] In particular, those skilled in the art will understand that the sequences of the CDRs, hypervariable regions, and variable regions can be modified without losing the ability to bind to CLDN18.2 and / or CD3. For example, the CDR regions can be identical to or highly homologous to the regions identified herein. By "highly homologous," it is contemplated that the CDRs can have 1 to 5, preferably 1 to 4, e.g., 1 to 3, or 1 or 2 substitutions. Furthermore, the hypervariable and variable regions can be modified to exhibit substantial homology with the regions specifically disclosed herein. In certain embodiments, the variable region sequences only deviate in the framework sequences from the variable region sequences specifically disclosed herein.

[0176] The binding agents described herein can be produced intracellularly (e.g., in the cytosol, periplasm, or in inclusion bodies) and then isolated from the host cells and optionally further purified, or 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 recombinant production of polypeptides (e.g., particular appropriate expression vectors, transformation or transfection methods, selectable markers, methods for inducing protein expression, culture conditions, etc.) are known in the art. Similarly, protein isolation and purification techniques are well known to those of skill in the art.

[0177] The term "cell" or "host cell" preferably relates to a whole cell, i.e. a cell with an intact membrane that has not released its normal intracellular components such as enzymes, organelles, or genetic material. A whole, intact cell is preferably a viable cell, i.e. a living cell that is able to carry out its normal metabolic functions. Preferably, the term relates in the present invention to any cell that can be transfected with an exogenous nucleic acid. Preferably, the cell is transfected with an exogenous nucleic acid and is able to express the nucleic acid in the recipient when transferred to the recipient. The term "cell" includes bacterial cells; other useful cells are yeast cells, fungal cells, or mammalian cells. Suitable bacterial cells include cells from gram-negative bacterial strains (e.g., strains of Escherichia coli, Proteus, and Pseudomonas), and gram-positive bacterial strains (e.g., strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus). Suitable fungal cells include cells from the genera Trichoderma, Neurospora, and Aspergillus. Suitable yeast cells include cells from the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and the methylotrophic yeast 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 expression of heterologous proteins can also be used.Mammalian cells, such as cells from humans, mice, hamsters, pigs, goats, and primates, are particularly preferred for adoptive transfer. Cells can be derived from numerous tissue types and include primary cells and cell lines, such as cells of the immune system, particularly 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. Antigen-presenting cells are cells that present antigens in the context of major histocompatibility complexes on their surface. T cells can recognize this complex using their T cell receptors (TCRs).

[0178] The ability of antibodies and other binding agents to bind to antigens can be determined using standard binding assays (e.g., ELISA, Western blot, immunofluorescence and flow cytometry analysis). The binding agents disclosed herein can also be tested in in vivo models (e.g., in immunodeficient mice bearing xenograft tumors inoculated with cell lines expressing CLDN18.2) to determine their effectiveness in controlling the growth of CLDN18.2-expressing tumor cells.

[0179] As used herein, "reduce," "decrease," or "inhibit" refers to an overall decrease or function that causes an overall decrease, e.g., in the level of expression or in the level of cell proliferation, preferably by 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more. The term "increase" or "enhance" and the like preferably relates to an increase or enhancement of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or more.

[0180] Antibody-dependent cell-mediated cytotoxicity ADCC refers to the cell killing ability of effector cells, particularly lymphocytes, disclosed herein, which preferably requires that the target cell be marked by an antibody. ADCC preferably occurs when an antibody binds to an antigen on a tumor cell and the antibody Fc domain binds to an Fc receptor (FcR) on the surface of an immune effector cell. Certain families of Fc receptors have been identified, and specific cell populations characteristically express defined Fc receptors. ADCC can be viewed as a mechanism that directly induces varying degrees of immediate tumor destruction, resulting in antigen presentation and the induction of tumor-directed T cell responses. Preferably, in vivo induction of ADCC results in tumor-directed T cell responses and host-derived antibody responses.

[0181] Antibody-dependent cell-mediated phagocytosis Antibody-mediated phagocytosis (ADCP) is one of the mechanisms of action of many antibody therapeutics. It is defined as a highly regulated process in which antibodies eliminate bound targets by connecting their Fc domain to specific receptors on phagocytes and inducing phagocytosis. ADCP can be mediated by monocytes, macrophages, neutrophils, and dendritic cells via FcγRIIa, FcγRI, and FcγRIIIa, of which FcγRIIa (CD32a) on macrophages represents the major pathway. ADCP preferably occurs when nonspecific phagocytes expressing FcγR recognize antibodies bound to target cells, such as diseased cells, including tumor cells, and subsequently trigger phagocytosis of the target cells, such as diseased cells, including tumor cells. ADCP also stimulates downstream adaptive immune responses by promoting antigen presentation or stimulating the secretion of inflammatory mediators. ADCP can be improved in vivo by co-treatment with immunomodulatory drugs. The Fc receptor-dependent function of ADCP provides a mechanism for the clearance of viruses and virus-infected cells and for the stimulation of downstream adaptive immune responses by promoting antigen presentation or stimulating the secretion of inflammatory mediators.

[0182] Complement-dependent cytotoxicity CDC is yet another method of cell killing that can be directed by antibodies. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also both highly effective at directing CDC via the classical complement activation pathway. Preferably, in this cascade, formation of an antigen-antibody complex exposes multiple closely spaced C1q binding sites on the CH2 domain of participating antibody molecules, such as IgG molecules (C1q is one of the three subcomponents of complement C1). Preferably, the unshielded C1q binding sites convert the previously low-affinity C1q-IgG interaction to a high-avidity one, triggering a cascade of events involving a series of other complement proteins, resulting in the proteolytic release of the effector cell chemotactic / activating factors C3a and C5a. Preferably, the complement cascade culminates in the formation of a membrane attack complex, which forms pores in the cell membrane, facilitating the entry and exit of water and solutes into and out of the cell.

[0183] Chimerization Repeated application of unlabeled mouse antibodies can be highly immunogenic in humans, reducing their therapeutic efficacy. The primary immunogenicity is mediated by the heavy chain constant region. The immunogenicity of mouse antibody-derived binding agents in humans can be reduced or completely avoided if the binding agents are chimerized or humanized, respectively. Chimeric binding agents are binding agents in which different portions are derived from different animal species, e.g., a variable region derived from a mouse antibody and a human immunoglobulin constant region. Chimerization of binding agents can be achieved by linking the variable regions of mouse antibody heavy and light chains with human heavy and light chain constant regions (e.g., as described in Kraus et al., "Methods in Molecular Biology," Recombinant antibodies for cancer therapy, ISBN-0-89603-918-8). In a preferred embodiment, chimeric binding agents are generated by linking a human κ light chain constant region to a mouse light chain variable region. In another preferred embodiment, chimeric binding agents can be generated by linking a human λ light chain constant region to a mouse light chain variable region. Preferred heavy chain constant regions for the generation of chimeric binding agents are IgG1, IgG3, and IgG4. Other preferred heavy chain constant regions for the generation of chimeric binding agents are IgG2, IgA, IgD, and IgM.

[0184] Humanization Binding agents, such as antibodies, interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity-determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse among individual antibodies than sequences outside the CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, recombinant binding agents, e.g., antibodies, that mimic the properties of a particular naturally occurring antibody can be expressed by constructing an expression vector containing the CDR sequences from a particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (e.g., Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-1003). The framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. The germline sequences differ from mature antibody gene sequences because they do not contain fully assembled variable genes formed by V(D)J joining during B-cell maturation. Germline gene sequences also differ from the sequences of high affinity secondary repertoire antibodies individually and uniformly across the variable regions.

[0185] Immune checkpoint inhibitors As used herein, the term "immune checkpoint" refers to regulators of the immune system, particularly costimulatory and inhibitory signals that modulate the amplitude and quality of T cell receptor recognition of antigen. In one embodiment, an immune checkpoint is an inhibitory signal. In one embodiment, an inhibitory signal is the interaction between PD-1 and PD-L1 and / or PD-L2. In one embodiment, an inhibitory signal is the interaction between CTLA-4 and CD80 or CD86 to displace CD28 binding. In one embodiment, an inhibitory signal is the interaction of LAG-3 with an MHC class II molecule. In one embodiment, an inhibitory signal is the interaction of TIM-3 with one or more of its ligands, such as galectin-9, PtdSer, HMGB1, and CEACAM1. In one embodiment, an inhibitory signal is the interaction between one or more KIRs and their ligands. In one embodiment, an inhibitory signal is the interaction between TIGIT and one or more of its ligands, PVR, PVRL2, and PVRL3. In some embodiments, the inhibitory signal is the interaction between CD94 / NKG2A and HLA-E. In some embodiments, the inhibitory signal is the interaction between VISTA and its binding partner. In some embodiments, the inhibitory signal is the interaction between one or more Siglecs and their ligands. In some embodiments, the inhibitory signal is the interaction between GARP and one or more of its ligands. In some embodiments, the inhibitory signal is the interaction between CD47 and SIRPα. In some embodiments, the inhibitory signal is the interaction between PVRIG and PVRL2. In some embodiments, the inhibitory signal is the interaction between CSF1R and CSF1. In some embodiments, the inhibitory signal is the interaction between BTLA and HVEM. In some embodiments, the inhibitory signal is the interaction between A2AR and / or A2BR and adenosine, which are produced by portions of the adenosinergic pathway, e.g., CD39 and CD73. In some embodiments, the inhibitory signal is the interaction between B7-H3 and its receptor and / or between B7-H4 and its receptor. In certain embodiments, the inhibitory signal is mediated by IDO, CD20, NOX, or TDO.

[0186] The "programmed cell death-1 (PD-1)" receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed primarily 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). As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs that share at least one epitope with hPD-1. "Programmed cell death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs that share at least one common epitope with hPD-L1. As used herein, the term "PD-L2" includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, and analogs that share at least one common epitope with hPD-L2. PD-1 ligands (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 downregulates T cell activation. Cancer cells expressing PD-L1 and / or PD-L2 can switch off PD-1-expressing T cells, thereby suppressing anti-cancer immune responses. The interaction of PD-1 with its ligands results in a reduction in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion by cancerous cells. Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and the effect is additive when the interaction between PD-1 and PD-L2 is similarly blocked.

[0187] Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) (also known as CD152) is a T-cell surface molecule and a member of the immunoglobulin superfamily. This protein binds to CD80 (B7-1) and CD86 (B7-2) to downregulate the immune system. As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, as well as analogs that share at least one epitope with hCTLA-4. CTLA-4 is a homolog of the stimulatory checkpoint protein CD28, which has much higher binding affinity for CD80 and CD86. CTLA-4 is expressed on the surface of activated T cells, and its ligand is expressed on the surface of professional antigen-presenting cells. Binding of CTLA-4 to its ligand prevents the costimulatory signal of CD28 and generates an inhibitory signal. Thus, CTLA-4 downregulates T-cell activation.

[0188] The "T cell immunoreceptor with Ig and ITIM domains" (also known as TIGIT, WUCAM, or Vstm3) is an immunoreceptor on T cells and natural killer (NK) cells that binds to PVR (CD155), PVRL2 (CD112; nectin-2), and PVRL3 (CD113; nectin-3) on DCs, macrophages, etc., and regulates T cell-mediated immunity. As used herein, the term "TIGIT" includes human TIGIT (hTIGIT), variants, isoforms, and species homologs of hTIGIT, as well as analogs that share at least one common epitope with hTIGIT. As used herein, the term "PVR" includes human PVR (hPVR), variants, isoforms, and species homologs of hPVR, as well as analogs that share at least one common epitope with hPVR. As used herein, the term "PVRL2" includes human PVRL2 (hPVRL2), variants, isoforms, and species homologs of hPVRL2, and analogs that share at least one epitope in common with hPVRL2. As used herein, the term "PVRL3" includes human PVRL3 (hPVRL3), variants, isoforms, and species homologs of hPVRL3, and analogs that share at least one epitope in common with hPVRL3. The term "B7 family" refers to inhibitory ligands with as yet undefined receptors. The B7 family includes B7-H3 and B7-H4, both of which are upregulated on tumor cells and tumor-infiltrating cells. As used herein, the terms "B7-H3" and "B7-H4" include human B7-H3 (hB7-H3) and human B7-H4 (hB7-H4), their variants, isoforms, and species homologs, as well as analogs that share at least one epitope with B7-H3 and B7-H4, respectively.

[0189] "B and T lymphocyte attenuator" (BTLA, also known as CD272) is a TNFR family member expressed on Th1 cells but not on Th2 cells. BTLA expression is induced during T cell activation, particularly on the surface of CD8+ T cells. As used herein, the term "BTLA" includes human BTLA (hBTLA), variants, isoforms, and species homologs of hBTLA, as well as analogs that share at least one epitope with hBTLA. BTLA expression is gradually downregulated during differentiation of human CD8+ T cells toward an 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. As used herein, the term "HVEM" includes human HVEM (hHVEM), variants, isoforms, and species homologs of hHVEM, as well as analogs that share at least one epitope with hHVEM. The BTLA-HVEM complex negatively regulates T cell immune responses.

[0190] "Killer cell immunoglobulin-like receptors" (KIRs) are receptors for MHC class I molecules on NK T cells and NK cells that are involved in the differentiation between healthy and diseased cells. KIRs bind to human leukocyte antigens (HLA) A, B, and C, which suppress normal immune cell activation. As used herein, the term "KIR" includes human KIRs (hKIRs), variants, isoforms, and species homologs of hKIRs, as well as analogs that share at least one epitope in common with hKIR. As used herein, the term "HLA" includes variants, isoforms, and species homologs of HLA, as well as analogs that share at least one epitope in common with HLA. As used herein, KIR refers, inter alia, to KIR2DL1, KIR2DL2, and / or KIR2DL3.

[0191] Lymphocyte-activation gene-3 (LAG-3), also known as CD223, is an inhibitory receptor associated with the inhibition of lymphocyte activity by binding to MHC class II molecules. This receptor enhances Treg cell function and inhibits CD8+ effector T cell function, resulting in a suppressed immune response. LAG-3 is expressed on activated T cells, NK cells, B cells, and DCs. As used herein, the term "LAG-3" includes human LAG-3 (hLAG-3), variants, isoforms, and species homologs of hLAG-3, as well as analogs that share at least one common epitope.

[0192] "T-cell membrane protein-3 (TIM-3)" (also known as HAVcr-2) is an inhibitory receptor involved in the inhibition of lymphocyte activity by inhibiting Th1 cell responses. Its ligand is galectin 9 (GAL9), which is upregulated in various types of cancer. Other TIM-3 ligands include phosphatidylserine (PtdSer), high mobility group protein 1 (HMGB1), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). As used herein, the term "TIM-3" includes human TIM3 (hTIM-3), variants, isoforms, and species homologs of hTIM-3, as well as analogs that share at least one common epitope. As used herein, the term "GAL9" includes human GAL9 (hGAL9), variants, isoforms, and species homologs of hGAL9, as well as analogs that share at least one common epitope. As used herein, the term "PdtSer" includes variants and analogs that share at least one common epitope. As used herein, the term "HMGB1" includes human HMGB1 (hHMGB1), variants, isoforms, and species homologs of hHMGB1, and analogs that share at least one common epitope. As used herein, the term "CEACAM1" includes human CEACAM1 (hCEACAM1), variants, isoforms, and species homologs of hCEACAM1, and analogs that share at least one common epitope.

[0193] The term "CD94 / NKG2A" refers to an inhibitory receptor primarily expressed on the surface of natural killer cells and CD8+ T cells. As used herein, the term "CD94 / NKG2A" includes human CD94 / NKG2A (hCD94 / NKG2A), hCD94 / NKG2A variants, isoforms, and species homologs, as well as analogs that share at least one common epitope. The CD94 / NKG2A receptor is a heterodimer containing CD94 and NKG2A. It inhibits NK cell activation and CD8+ T cell function, likely by binding to ligands such as HLA-E. CD94 / NKG2A limits cytokine release and cytotoxic responses 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 on tumor-infiltrating cells, while HLA-E is overexpressed in certain cancers.

[0194] The term "indoleamine 2,3-dioxygenase" (IDO) is a tryptophan catabolic enzyme with immunosuppressive properties. As used herein, the term "IDO" includes human IDO (hIDO), variants, isoforms, and species homologs of hIDO, as well as analogs that share at least one common epitope. IDO is the rate-limiting enzyme in tryptophan degradation, catalyzing the conversion of tryptophan to kynurenine. Thus, IDO is involved in the depletion of essential amino acids. It is known to be involved in the suppression of T cells and NK cells, the generation and activation of Tregs and myeloid-derived suppressor cells, and the promotion of tumor angiogenesis. IDO is overexpressed in many cancers, promoting immune system escape by tumor cells, and, when induced by local inflammation, promoting chronic tumor progression.

[0195] As used herein, in the "adenosinergic pathway" or "adenosine signaling pathway," ATP is converted to adenosine by the ectonucleotidases CD39 and CD73, resulting in inhibitory signaling via 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, present at high concentrations in the tumor microenvironment, limiting immune cell infiltration, cytotoxicity, and cytokine production. Thus, adenosine signaling is a strategy used by cancer cells to evade host immune system clearance. Adenosine signaling via A2AR and A2BR is a critical checkpoint in cancer therapy, activated by the high adenosine concentrations normally 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 via A2AR and A2BR counteracts T cell receptor-mediated activation of immune cells, increases the number of Tregs, and reduces the activation of DCs and effector T cells. As used herein, the term "CD39" includes human CD39 (hCD39), variants, isoforms, and species homologs of hCD39, as well as analogs that share at least one common epitope. As used herein, the term "CD73" includes human CD73 (hCD73), variants, isoforms, and species homologs of hCD73, as well as analogs that share at least one common epitope. As used herein, the term "A2AR" includes human A2AR (hA2AR), variants, isoforms, and species homologs of hA2AR, and analogs that share at least one common epitope. As used herein, the term "A2BR" includes human A2BR (hA2BR), variants, isoforms, and species homologs of hA2BR, and analogs that share at least one common epitope.

[0196] The term "V-domain Ig suppressor of T-cell activation" (VISTA, also known as C10orf54) shares homology with PD-L1 but displays a unique expression pattern restricted to the hematopoietic compartment. As used herein, the term "VISTA" includes human VISTA (hVISTA), variants, isoforms, and species homologs of hVISTA, as well as analogs that share at least one shared epitope. VISTA induces T-cell suppression and is expressed by leukocytes within tumors.

[0197] The term "sialic acid-binding immunoglobulin-type lectin" (Siglec) family members recognize sialic acid and are involved in distinguishing between "self" and "non-self." As used herein, the term "hSiglecs" includes human Siglecs (hSiglecs), variants, isoforms, and species homologs of hSiglecs, as well as analogs that share at least one epitope with one or more Siglecs. The human genome contains 14 Siglecs, some of which are involved in immunosuppression, including but not limited to Siglec-2, Siglec-3, Siglec-7, and Siglec-9. Siglec receptors bind to sialic acid-containing glycans but differ in their recognition of the binding site chemistry and the spatial distribution of sialic acid residues. Members of this family also have different expression patterns. A wide range of malignancies overexpress one or more Siglecs.

[0198] The term "CD20" refers to an antigen expressed on the surface of B cells and T cells. CD20 can be highly expressed in cancers such as B cell lymphoma, hairy cell leukemia, B cell chronic lymphocytic leukemia, and melanoma cancer stem cells. As used herein, the term "CD20" includes human CD20 (hCD20), variants, isoforms, and species homologs of hCD20, and analogs that share at least one common epitope.

[0199] The term "glycoprotein A repeat dominant" (GARP) plays a role in immune tolerance and tumor evasion by a patient's immune system. As used herein, the term "GARP" includes human GARP (hGARP), hGARP variants, isoforms, and species homologs, as well as analogs that share 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 likely binds to latent "transforming growth factor beta" (TGF-β). Disruption of GARP signaling in Tregs reduces tolerance and inhibits Treg migration to the intestine and increased proliferation of cytotoxic T cells.

[0200] The term "CD47" refers to a transmembrane protein that binds to the ligand "signal regulatory protein alpha" (SIRPα). As used herein, the term "CD47" includes human CD47 (hCD47), variants, isoforms, and species homologs of hCD47, as well as analogs that share at least one epitope with hCD47. As used herein, the term "SIRPα" includes human SIRPα (hSIRPα), variants, isoforms, and species homologs of hSIRPα, as well as analogs that share at least one epitope with hSIRPα. 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 a "no-take" signal to macrophages. Blocking CD47 signaling via inhibitory anti-CD47 or anti-SIRPα antibodies allows macrophage phagocytosis of cancer cells and promotes the activation of cancer-specific T lymphocytes.

[0201] The term "poliovirus receptor-related immunoglobulin domain containing" (PVRIG, also known as CD112R) binds to "poliovirus receptor-related 2" (PVRL2). PVRIG and PVRL2 are overexpressed in certain cancers. PVRIG expression also induces TIGIT and PD-1 expression, and PVRL2 and PVR (TIGIT ligand) are co-overexpressed in certain cancers. Blockade of the PVRIG signaling pathway results in increased T cell function and CD8+ T cell responses, thus reducing immunosuppression and enhancing interferon responses. As used herein, the term "PVRIG" includes human PVRIG (hPVRIG), variants, isoforms, and species homologs of hPVRIG, as well as analogs that share at least one shared epitope with hPVRIG. As used herein, "PVRL2" includes hPVRL2 as defined above.

[0202] The "colony-stimulating factor 1" pathway is another checkpoint that can be targeted by the present disclosure. CSF1R is a myeloid growth factor receptor that binds to CSF1. Blockade of CSF1R signaling can functionally reprogram macrophage responses to enhance antigen presentation and anti-tumor T cell responses. As used herein, "CSF1R" includes human CSF1R (hCSF1R), variants, isoforms, and species homologs of hCSF1R, and analogs that share at least one epitope in common with hCSF1R. As used herein, "CSF1" includes human CSF1 (hCSF1), variants, isoforms, and species homologs of hCSF1, and analogs that share at least one epitope in common with hCSF1.

[0203] "Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase" refers to an enzyme in the NOX family of enzymes in myeloid cells that generates immunosuppressive reactive oxygen species (ROS). Five NOX enzymes (NOX1-NOX5) have been found to be involved in cancer development and immunosuppression. Elevated ROS levels are detected in almost all cancers and promote many aspects of tumor development and progression. NOX-generated ROS impair NK and T cell function, and inhibiting NOX in myeloid cells improves the antitumor function of neighboring NK and T cells. As used herein, the term "NOX" includes human NOX (hNOX), hNOX variants, isoforms, and species homologs, as well as analogs that share at least one epitope with hNOX.

[0204] Another immune checkpoint that can be targeted according to the present disclosure is signaling mediated by "tryptophan-2,3-dioxygenase" (TDO). TDO represents an alternative pathway to IDO in tryptophan degradation and is involved in immunosuppression. Because tumor cells can catabolize tryptophan via TDO instead of IDO, TDO may represent an additional target for checkpoint blockade. Indeed, certain cancer cell lines have been found to upregulate TDO, and TDO may complement IDO inhibition. As used herein, the term "TDO" includes human TDO (hTDO), variants, isoforms, and species homologs of hTDO, as well as analogs that share at least one epitope with hTDO.

[0205] Many immune checkpoints are regulated by the interaction between specific receptor-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 utilize such checkpoint pathways to protect themselves from attack by the immune system. Therefore, the function of the checkpoint proteins regulated by the present disclosure is typically to regulate T cell activation, T cell proliferation, and / or T cell function. Thus, immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Many immune checkpoint proteins belong to the B7:CD28 family or the tumor necrosis factor receptor (TNFR) superfamily, and upon binding to specific ligands, activate signaling molecules recruited to their cytoplasmic domains (Suzuki et al., 2016, Jap J Clin Onc, 46:191-203).

[0206] As used herein, the term "immune checkpoint modulator" or "checkpoint modulator" refers to a molecule or compound that modulates the function of one or more checkpoint proteins. Immune checkpoint modulators are typically capable of modulating self-tolerance and / or the amplitude and / or duration of an immune response. Preferably, immune checkpoint modulators used in accordance with the present disclosure are "human checkpoint modulators" because they modulate the function of one or more human checkpoint proteins. In a preferred embodiment, a human checkpoint modulator used herein is an immune checkpoint inhibitor.

[0207] As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or negatively regulates one or more checkpoint proteins, or completely or partially reduces, inhibits, interferes with, or negatively regulates the expression of one or more checkpoint proteins. In some embodiments, an immune checkpoint inhibitor binds to one or more checkpoint proteins. In some embodiments, an immune checkpoint inhibitor binds to one or more molecules that regulate checkpoint proteins. In some embodiments, an immune checkpoint inhibitor binds to precursors of one or more checkpoint proteins, e.g., at the DNA or RNA level. Any agent that functions as a checkpoint inhibitor according to the present disclosure can be used.

[0208] As used herein, the term "partial" 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% of the level, e.g., of inhibition of a checkpoint protein.

[0209] In certain embodiments, immune checkpoint inhibitors suitable for use herein are antagonists of inhibitory signals, such as antibodies targeting PD-1, PD-L1, CTLA-4, LAG-3, B7-H3, B7-H4, or TIM-3. Such ligands and receptors are reviewed in Pardoll, D., Nature. 12:252-264, 2012. Additional immune checkpoint proteins that can be targeted by the present disclosure are disclosed herein. In some embodiments, the immune checkpoint inhibitor prevents inhibitory signaling associated with an immune checkpoint. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that disrupts inhibitory signaling associated with an immune checkpoint. In some embodiments, the immune checkpoint inhibitor is a small molecule inhibitor that disrupts inhibitory signaling. In some embodiments, the immune checkpoint inhibitor is a peptide-based inhibitor that disrupts inhibitory signaling. In some embodiments, the immune checkpoint inhibitor is an inhibitory nucleic acid molecule that disrupts inhibitory signaling.

[0210] In some embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that prevents an 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 some embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that prevents the interaction between CTLA-4 and CD80 or CD86. In some embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that prevents the interaction between LAG-3 and its ligand, or between TIM-3 and its ligand. In some embodiments, the immune checkpoint inhibitor prevents inhibitory signaling via CD39 and / or CD73 and / or the interaction of A2AR and / or A2BR with adenosine. In some embodiments, the immune checkpoint inhibitor prevents the interaction of B7-H3 with its receptor and / or the interaction of B7-H4 with its receptor. In some embodiments, the immune checkpoint inhibitor prevents the interaction of BTLA with its ligand HVEM. In some embodiments, the immune checkpoint inhibitor prevents the interaction of one or more KIRs with their respective ligands. In some embodiments, the immune checkpoint inhibitor prevents the interaction of LAG-3 with one or more of its ligands. In some 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 some embodiments, the immune checkpoint inhibitor prevents the interaction of TIGIT with one or more of its ligands: PVR, PVRL2, and PVRL3. In some embodiments, the immune checkpoint inhibitor prevents the interaction of CD94 / NKG2A with HLA-E. In some embodiments, the immune checkpoint inhibitor prevents the interaction of VISTA with one or more of its binding partners. In some embodiments, the immune checkpoint inhibitor prevents the interaction of one or more Siglecs with their respective ligands. In some embodiments, the immune checkpoint inhibitor prevents CD20 signaling.In some embodiments, the immune checkpoint inhibitor prevents the interaction of GARP with one or more of its ligands. In some embodiments, the immune checkpoint inhibitor prevents the interaction of CD47 with SIRPα. In some embodiments, the immune checkpoint inhibitor prevents the interaction of PVRIG with PVRL2. In some embodiments, the immune checkpoint inhibitor prevents the interaction of CSF1R with CSF1. In some embodiments, the immune checkpoint inhibitor prevents NOX signaling. In some embodiments, the immune checkpoint inhibitor prevents IDO and / or TDO signaling. As disclosed herein, inhibiting or blocking inhibitory immune checkpoint signaling prevents or reverses immune suppression and establishes or enhances T cell immunity against cancer cells. In one embodiment, inhibiting immune checkpoint signaling as disclosed herein reduces or inhibits immune system dysfunction. In one embodiment, inhibiting immune checkpoint signaling as disclosed herein alleviates dysfunction of dysfunctional immune cells. In one embodiment, inhibiting immune checkpoint signaling as disclosed herein alleviates dysfunction of dysfunctional T cells. As used herein, the term "dysfunction" refers to a state of decreased 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 subsequent immune response is ineffective in controlling infection or tumor growth. Dysfunction also includes states in which antigen recognition is delayed due to dysfunctional immune cells. As used herein, the term "dysfunction" refers to immune cells in a state of reduced immune responsiveness to antigenic stimulation. Dysfunction includes unresponsiveness to antigen recognition and impaired translation of antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2), and / or target cell killing. As used herein, the term "anergy" refers to a state of unresponsiveness to antigenic stimulation resulting from defective or insufficient signals delivered via the T cell receptor (TCR). T cell anergy can also occur upon stimulation with an antigen in the absence of costimulation, resulting in cells becoming refractory to subsequent activation by antigen, even in the context of costimulation. The unresponsive state can often be abrogated by the presence of IL-2. Anergic T cells do not undergo clonal expansion and / or acquire effector function. As used herein, the term "exhaustion" refers to immune cell exhaustion, such as T cell exhaustion, a state of T cell dysfunction resulting from persistent TCR signaling, which occurs during many chronic infections and cancers. It is distinct from anergy in that it results from persistent signaling, rather than incomplete or absent signaling. Exhaustion is defined by insufficient effector function, persistent expression of inhibitory receptors, and a transcriptional state that differs from that of functional effector or memory T cells. Exhaustion prevents optimal control of disease (e.g., infection and tumors). Exhaustion can result from both extrinsic negative regulatory pathways (e.g., immunomodulatory cytokines) and cell-intrinsic negative regulatory pathways (e.g., inhibitory immune checkpoint pathways, as disclosed herein).

[0211] "Enhancing T cell function" means inducing, causing, or stimulating T cells so that they have sustained or amplified biological function, or to regenerate or reactivate exhausted or inactive T cells. Examples of enhanced T cell function include increased secretion of gamma interferon from CD8+ T cells, increased proliferation, and increased antigen responsiveness (e.g., tumor clearance) compared to pre-intervention levels. In one embodiment, the level of enhancement is at 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. Methods for measuring this enhancement are known to those skilled in the art.

[0212] An immune checkpoint inhibitor may be an inhibitory nucleic acid molecule. As used herein, the term "inhibitory nucleic acid" or "inhibitory nucleic acid molecule" refers to a nucleic acid molecule, e.g., DNA or RNA, that completely or partially reduces, inhibits, interferes with, or negatively regulates one or more checkpoint proteins. Inhibitory nucleic acid molecules include, but are not limited to, oligonucleotides, siRNAs, shRNAs, antisense DNA or RNA molecules, and aptamers (e.g., DNA or RNA aptamers).

[0213] As used herein, the term "oligonucleotide" refers to a nucleic acid molecule capable of reducing protein expression, particularly the expression of a checkpoint protein, such as the checkpoint proteins disclosed herein. Oligonucleotides are short DNA or RNA molecules typically containing 2 to 50 nucleotides. Oligonucleotides can be single-stranded or double-stranded. Checkpoint inhibitor oligonucleotides can be antisense oligonucleotides. Antisense oligonucleotides are single-stranded DNA or RNA molecules complementary to a predetermined sequence, particularly the nucleic acid sequence (or a fragment thereof) of a checkpoint protein. Antisense RNA is typically used to bind to and prevent protein translation of mRNA, such as mRNA encoding a checkpoint protein. Antisense DNA is typically used to target a specific complementary (coding or non-coding) RNA. Upon binding, the DNA / RNA hybrid can be degraded by the enzyme RNase H. Furthermore, morpholino antisense oligonucleotides can be used for gene knockdown in vertebrates. For example, Kryczek et al., 2006 (J Exp Med, 203:871-81) designed a B7-H4-specific morpholino that specifically blocked B7-H4 expression in macrophages, resulting in increased T cell proliferation and reduced tumor volume in mice with tumor-associated antigen (TAA)-specific T cells.

[0214] The terms "siRNA," "small interfering RNA," or "small inhibitory RNA" are used interchangeably herein to refer to double-stranded RNA molecules, typically 20-25 base pairs in length, that interfere with the expression of a specific gene, such as a gene encoding a checkpoint protein, to which they have a complementary nucleotide sequence. In one embodiment, the siRNA interferes with mRNA, thus blocking translation, e.g., of an immune checkpoint protein. Transfection of exogenous siRNA can be used for gene knockdown, but the effect may be only transient, especially in rapidly dividing cells. Stable transfection can be achieved, for example, by RNA modification or using an expression vector. Useful modifications and vectors for stable transfection of cells with siRNA are known in the art. siRNA sequences can also be modified to introduce a short loop between the two strands, resulting in "small hairpin RNA" or "shRNA." shRNA can be processed into functional siRNA by Dicer. shRNA degradation and turnover rates are relatively slow. Thus, immune checkpoint inhibitors can be shRNAs.

[0215] As used herein, the term "aptamer" refers to a single-stranded nucleic acid molecule, such as DNA or RNA, typically 25-70 nucleotides in length, that can bind to a target molecule, such as a polypeptide. In one embodiment, an aptamer binds to an immune checkpoint protein, such as the immune checkpoint proteins disclosed herein. For example, an aptamer according to the present disclosure can specifically bind to an immune checkpoint protein or polypeptide, or a molecule in a signaling pathway that regulates 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., U.S. Pat. No. 5,475,096).

[0216] The term "small molecule inhibitor" or "small molecule" is used interchangeably herein and refers to a low molecular weight organic compound, usually up to 1000 daltons, that completely or partially reduces, inhibits, interferes with or negatively regulates one or more checkpoint proteins.Such small molecule inhibitors are usually synthesized by organic chemistry, but can also be isolated from natural sources such as plants, fungi and microorganisms.Because of their small molecular weight, small molecule inhibitors can rapidly diffuse across cell membranes.For example, various A2AR antagonists known in the art are organic compounds with molecular weights of less than 500 daltons.

[0217] The immune checkpoint inhibitor can be an antibody, an antigen-binding fragment thereof, an antibody mimetic, or a fusion protein comprising an antibody portion with an antigen-binding fragment of the required specificity. The antibody or antigen-binding fragment thereof is as disclosed 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. The antibody or antigen-binding fragment may also be conjugated to a further moiety, as disclosed herein. In particular, the antibody or antigen-binding fragment thereof is a chimerized, humanized, or human antibody. Preferably, the immune checkpoint inhibitor antibody or antigen-binding fragment thereof is an antagonist of the immune checkpoint receptor or immune checkpoint receptor ligand. In a preferred embodiment, the antibody that is an immune checkpoint inhibitor is an isolated antibody. An antibody or antigen-binding fragment thereof that is an immune checkpoint inhibitor according to the present disclosure may be an antibody that cross-competes with a known immune checkpoint inhibitor antibody for antigen binding. In certain embodiments, the immune checkpoint inhibitor antibody cross-competes with one or more of the immune checkpoint inhibitor antibodies disclosed herein. The function of a cross-competing antibody for antigen binding is indicated when the antibody can bind to the same epitope region of the antigen or when binding to another epitope sterically interferes with the binding of a known immune checkpoint inhibitor antibody to that particular epitope region. Such a cross-competing antibody may have functional properties very similar to those of its cross-competing counterpart, as it is expected to block immune checkpoint binding to its ligand by binding to the same epitope or by sterically interfering with ligand binding. Cross-competing antibodies can be readily identified based on their ability to cross-compete with one or more known antibodies in standard binding assays, such as surface plasmon resonance analysis, ELISA assays, or flow cytometry (see, e.g., WO 2013 / 173223).

[0218] In one embodiment, an antibody or antigen-binding fragment thereof that cross-competes with one or more known antibodies for binding to a given antigen or that binds to the same epitope region of a given antigen is a monoclonal antibody. For administration to human patients, the cross-competing antibody can be a chimeric antibody, or a humanized or human antibody. The chimeric, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art. The checkpoint inhibitor may also be in the form of a soluble form of the molecule (or a variant thereof) itself, for example, a soluble PD-L1 or a PD-L1 fusion.

[0219] In the context of the present disclosure, more than one checkpoint inhibitor can be used, where the more than one checkpoint inhibitor targets different checkpoint pathways or the same checkpoint pathway. Preferably, the more than one checkpoint inhibitor is a separate checkpoint inhibitor. Preferably, when more than one separate checkpoint inhibitor is used, particularly at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 separate checkpoint inhibitors are used, preferably 2, 3, 4 or 5 separate checkpoint inhibitors are used, more preferably 2, 3 or 4 separate checkpoint inhibitors are used, even more preferably 2 or 3 separate checkpoint inhibitors are used, and most preferably 2 separate checkpoint inhibitors are used. Preferred examples of combinations of different checkpoint inhibitors include 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, and an inhibitor of PD-1 signaling and an inhibitor of IDO signaling. and combinations of 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.

[0220] In one embodiment, the inhibitory immunomodulator (immune checkpoint blocker) is a component of the PD-1 / PD-L1 or PD-1 / PD-L2 signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering to a subject a checkpoint inhibitor of the PD-1 signaling pathway. In one embodiment, the checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 inhibitor. In some 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 antigen-binding portion thereof that interferes with the interaction between the PD-1 receptor and one or more of its ligands, PD-L1 and / or PD-L2. Antibodies that bind to PD-1 and disrupt the interaction between PD-1 and one or more of its ligands are known in the art. In one embodiment, the antibody or antigen-binding portion thereof specifically binds to PD-1. In some embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby enhancing immune activity. In one embodiment, the antibody, or antigen-binding portion thereof, specifically binds to PD-L2 and inhibits its interaction with PD-1, thereby enhancing immune activity.

[0221] In some embodiments, the inhibitory immunomodulator is a component of the CTLA-4 signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering to a subject a checkpoint inhibitor of the CTLA-4 signaling pathway. In some embodiments, the checkpoint inhibitor of the CTLA-4 signaling pathway is a CTLA-4 inhibitor. In some embodiments, the checkpoint inhibitor of the CTLA-4 signaling pathway is a CTLA-4 ligand inhibitor.

[0222] In some embodiments, the inhibitory immunomodulator is a component of the TIGIT signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering a checkpoint inhibitor of the TIGIT signaling pathway to a subject. In some embodiments, the checkpoint inhibitor of the TIGIT signaling pathway is a TIGIT inhibitor. In some embodiments, the checkpoint inhibitor of the TIGIT signaling pathway is a TIGIT ligand inhibitor.

[0223] In some embodiments, the inhibitory immunomodulator is a component of the B7 family signaling pathway. In some embodiments, the B7 family members are B7-H3 and B7-H4. Some embodiments of the present disclosure provide for administering a B7-H3 and / or B7-4 checkpoint inhibitor to a subject. Accordingly, some embodiments of the present disclosure provide for administering an antibody or antigen-binding portion thereof that targets B7-H3 or B7-H4 to a subject. The B7 family does not have any defined receptors, but their ligands are upregulated on tumor cells or tumor-infiltrating cells. Preclinical mouse models have shown that blocking these ligands can enhance anti-tumor immunity. In some embodiments, the inhibitory immunomodulator is a component of the BTLA signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering a checkpoint inhibitor of the BTLA signaling pathway to a subject. In some embodiments, the checkpoint inhibitor of the BTLA signaling pathway is a BTLA inhibitor. In some embodiments, the checkpoint inhibitor of the BTLA signaling pathway is an HVEM inhibitor.

[0224] In some embodiments, the inhibitory immunomodulator is a component of one or more KIR signaling pathways. Accordingly, some embodiments of the present disclosure provide for administering to a subject a checkpoint inhibitor of one or more KIR signaling pathways. In some embodiments, the checkpoint inhibitor of one or more KIR signaling pathways is a KIR inhibitor. In some embodiments, the checkpoint inhibitor of one or more KIR signaling pathways is a KIR ligand inhibitor. For example, a KIR inhibitor according to the present disclosure may be an anti-KIR antibody that binds to KIR2DL1, KIR2DL2, and / or KIR2DL3.

[0225] In some embodiments, the inhibitory immunomodulator is a component of the LAG-3 signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering to a subject a checkpoint inhibitor of LAG-3 signaling. In some embodiments, the checkpoint inhibitor of the LAG-3 signaling pathway is a LAG-3 inhibitor. In some embodiments, the checkpoint inhibitor of the LAG-3 signaling pathway is a LAG-3 ligand inhibitor.

[0226] In some embodiments, the inhibitory immunomodulator is a component of the TIM-3 signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering a checkpoint inhibitor of the TIM-3 signaling pathway to a subject. In some embodiments, the checkpoint inhibitor of the TIM-3 signaling pathway is a TIM-3 inhibitor. In some embodiments, the checkpoint inhibitor of the TIM-3 signaling pathway is a TIM-3 ligand inhibitor.

[0227] In certain embodiments, the inhibitory immunomodulator is a component of the CD94 / NKG2A signaling pathway. Accordingly, certain embodiments of the present 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 CD94 / NKG2A ligand inhibitor. In some embodiments, the inhibitory immunomodulator is a component of the IDO signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering to a subject a checkpoint inhibitor of the IDO signaling pathway, e.g., an IDO inhibitor.

[0228] In some embodiments, the inhibitory immunomodulator is a component of the adenosine signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering to a subject a checkpoint inhibitor of the adenosine signaling pathway. In some embodiments, the checkpoint inhibitor of the adenosine signaling pathway is a CD39 inhibitor. In some embodiments, the checkpoint inhibitor of the adenosine signaling pathway is a CD73 inhibitor. In some embodiments, the checkpoint inhibitor of the adenosine signaling pathway is an A2AR inhibitor. In some embodiments, the checkpoint inhibitor of the adenosine signaling pathway is an A2BR inhibitor.

[0229] In some embodiments, the inhibitory immunomodulator is a component of the VISTA signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering a checkpoint inhibitor of the VISTA signaling pathway to a subject. In some embodiments, the checkpoint inhibitor of the VISTA signaling pathway is a VISTA inhibitor.

[0230] In some embodiments, the inhibitory immunomodulator is a component of one or more Siglec signaling pathways. Accordingly, some embodiments of the present disclosure provide for administering to a subject a checkpoint inhibitor of one or more Siglec signaling pathways. In some embodiments, the checkpoint inhibitor of one or more Siglec signaling pathways is a Siglec inhibitor. In some embodiments, the checkpoint inhibitor of one or more Siglec signaling pathways is a Siglec ligand inhibitor.

[0231] In some embodiments, the inhibitory immunomodulator is a component of the CD20 signaling pathway. Thus, some embodiments of the present disclosure provide for administering a checkpoint inhibitor of the CD20 signaling pathway to a subject. In some embodiments, the checkpoint inhibitor of the CD20 signaling pathway is a CD20 inhibitor.

[0232] In some embodiments, the inhibitory immunomodulator is a component of the GARP signaling pathway. Thus, some embodiments of the present disclosure provide for administering a checkpoint inhibitor of the GARP signaling pathway to a subject. In some embodiments, the checkpoint inhibitor of the GARP signaling pathway is a GARP inhibitor.

[0233] In some embodiments, the inhibitory immunomodulator is a component of the CD47 signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering to a subject a checkpoint inhibitor of the CD47 signaling pathway. In some embodiments, the checkpoint inhibitor of the CD47 signaling pathway is a CD47 inhibitor. In some embodiments, the checkpoint inhibitor of the CD47 signaling pathway is a SIRPα inhibitor. In some embodiments, the inhibitory immunomodulator is a component of the PVRIG signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering a checkpoint inhibitor of the PVRIG signaling pathway to a subject. In some embodiments, the checkpoint inhibitor of the PVRIG signaling pathway is a PVRIG inhibitor. In some embodiments, the checkpoint inhibitor of the PVRIG signaling pathway is a PVRIG ligand inhibitor.

[0234] In some embodiments, the inhibitory immunomodulator is a component of the CSF1R signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering a checkpoint inhibitor of the CSF1R signaling pathway to a subject. In some embodiments, the checkpoint inhibitor of the CSF1R signaling pathway is a CSF1R inhibitor. In some embodiments, the checkpoint inhibitor of the CSF1R signaling pathway is a CSF1 inhibitor. In some embodiments, the inhibitory immunomodulator is a component of the NOX signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering to a subject a checkpoint inhibitor of the NOX signaling pathway, e.g., a NOX inhibitor. In some embodiments, the inhibitory immunomodulator is a component of the TDO signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering to a subject a checkpoint inhibitor of the TDO signaling pathway, e.g., a TDO inhibitor.

[0235] Exemplary PD-1 inhibitors include, but are not limited to, BGB-A317 (BeiGene; see US 8,735,553, WO 2015 / 35606 and US 2015 / 0079109), cemiplimab (Regeneron; see WO 2015 / 112800), and lambrolizumab (disclosed, for example, as hPD109A and its humanized derivatives h409A1, h409A16, and h409A17 in WO 2008 / 156712), AB137132 (Abcam), EH12.2H7 and RMP1-14 (#BE0146; Bioxcell Lifesciences Pvt. LTD.), MIH4 (Affymetrix eBioscience), nivolumab (OPDIVO, BMS-936558; Bristol Myers Squibb), and NIH4 (BMS-936558; Bristol Myers Squibb). 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; 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), karelizumab (SHR-1210) (see WO2015 / 085847), and antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, INCSHR1210 (Jiangsu Hengrui), described in WO 2006 / 121168. Medicine; also known as karelizumab (SHR-1210); see WO2015 / 085847), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; 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), e.g., US 7488802, US 8008449, US 8168757, WO 03 / 042402, WO 2010 / 089411 (further disclosing anti-PD-L1 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-L1 and anti-TIGIT antibodies), US 8,008,449, Anti-PD-1 antibodies such as those disclosed in US 8,779,105, US 6,808,710, US 8,168,757, US 2016 / 0272708, and US 8,354,509; small molecule antagonists against the PD-1 signaling pathway such as those disclosed in, for example, Shaabani et al., 2018, Expert Op Ther Pat., 28(9):665-678 and Sasikumar and Ramachandra, 2018, BioDrugs, 32(5):481-497; siRNA against PD-1 such as those disclosed in, for example, WO 2019 / 000146 and WO 2018 / 103501; soluble PD-1 proteins such as those disclosed in WO 2018 / 222711; and soluble PD-1 proteins such as those disclosed in, for example, WO 2018 / 022831. Examples include oncolytic viruses containing a soluble form of PD-1, such as those described in [1].

[0236] In some embodiments, 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 karelizumab (SHR-1210).

[0237] Exemplary PD-1 ligand inhibitors include PD-L1 inhibitors and PD-L2 inhibitors, including, but not limited to, anti-PD-L1 antibodies, such as MEDI4736 (durvalumab; AstraZeneca; see WO 2011 / 066389), MSB-0010718C (see US 2014 / 0341917), YW243.55.S70 (see SEQ ID NO: 20 in WO 2010 / 077634 and US 8,217,149), MIH1 (Affymetrix eBioscience; see EP 3 230 319), MDX-1105 (Roche / Genentech; see WO 2013019906 and US 8,217,149), STI-1014 (Sorrento; WO2013 / 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, see WO 2013 / 173223), avelumab (bavencio; see US 2014 / 0341917), LY3300054 (Eli Lilly Co.), CX-072 (Proclaim-CX-072; also known as CytomX; see WO2016 / 149201), FAZ053, KN035 (see WO2017020801 and WO2017020802), sugemalimab (CS1001), MDX-1105 (see US 2015 / 0320859), anti-PD-L1 antibodies disclosed in US 7,943,743 (including 3G10, 12A4 (also known as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4), WO 2010 / 077634, US 8,217,149, WO 2010 / 036959, WO 2010 / 077634, WO 2011 / 066342, US 8,217,149, US 7,943,743, WO 2010 / 089411, US 7,635,757, US 8,217,149, US 2009 / 0317368, W.O. 2011 / 066389, WO2017 / 034916, WO2017 / 020291, WO2017 / 020858, WO2017 / 020801, W O2016 / 111645, WO2016 / 197367, WO2016 / 061142, WO2016 / 149201, WO2016 / 000619, WO2016 / 160792, WO2016 / 022630, WO2016 / 007235, WO2015 / 179654, WO2015 / 173267, WO2015 / 181342, WO2015 / 109124, WO Examples include the anti-PD-L1 antibodies described in WO2018 / 222711, WO2015 / 112805, WO2015 / 061668, WO2014 / 159562, WO2014 / 165082, and WO2014 / 100079.

[0238] Examples of CTLA-4 inhibitors include the monoclonal antibodies ipilimumab (Yervoy, Bristol Myers Squibb) and tremelimumab (Pfizer / MedImmune), trevilizumab, AGEN-1884 (Agenus) and ATOR-1015, WO 2001 / 014424, US 2005 / 0201994, EP 1212422, US 5,811,097, US 5,855,887, US 6,051,227, US 6,682,736, US 6,984,720, WO 01 / 14424, WO 00 / 37504, US 2002 / 0039581, US 2003 / 0039582, 2002 / 086014, WO 98 / 42752, US 6,207,156, US 5,977,318, US 7,109,003, and US 7,132,281, the dominant negative proteins abatacept (Orencia, see EP 2 855 533) and belatacept (Nulojix; see WO 2014 / 207748), which comprise the Fe region of IgG1 fused to the CTLA-4 ECD, second generation high affinity CTLA-4-Ig variants with two amino acid substitutions in the CTLA-4 ECD compared to abatacept, soluble CTLA-4 polypeptides, e.g., RG2077 and CTLA4-IgG4m (see US 6,750,334), anti-CTLA-4 aptamers and antibodies, e.g., those disclosed in US Examples of CTLA-4 ligand inhibitors include, but are not limited to, CTLA-4-directed siRNAs, such as those disclosed in US Pat. No. 2015 / 203848. Exemplary CTLA-4 ligand inhibitors are described in Pile et al., 2015 (Encyclopedia of Inflammatory Diseases, M. Parnham (ed.), doi:10.1007 / 978-3-0348-0620-6_20).

[0239] Exemplary checkpoint inhibitors of the TIGIT signaling pathway include, but are not limited to, anti-TIGIT antibodies, such as BMS-986207, COM902 (CGEN-15137; Compugen), AB154 (Arcus Biosciences), or etigilimab (OMP-313M32; OncoMed Pharmaceuticals), or the antibodies disclosed in WO2017 / 059095, particularly "MAB10," US 2018 / 0185482, WO 2015 / 009856, and US 2019 / 0077864.

[0240] Exemplary checkpoint inhibitors of B7-H3 include, but are not limited to, the Fc-optimized monoclonal antibody enoblituzumab (MGA271; Macrogenics; see US 2012 / 0294796) and the anti-B7-H3 antibodies MGD009 (Macrogenics) and pidilizumab (see US 7,332,582).

[0241] Exemplary B7-H4 inhibitors include, but are not limited to, antibodies such as those described in Dangaj et al., 2013 (Cancer Research 73:4820-9) and Smith et al., 2014 (Gynecol Oncol, 134:181-189), WO 2013 / 025779 (e.g., 2D1 encoded by SEQ ID NOs: 3 and 4, 2H9 encoded by SEQ ID NOs: 37 and 39, and 2E11 encoded by SEQ ID NOs: 41 and 43) and WO 2013 / 067492 (e.g., with amino acids selected from SEQ ID NOs: 1-8), morpholino antisense oligonucleotides such as those described by Kryczek et al., 2006 (J Exp Med, 203:871-81), or soluble recombinant forms of B7-H4 such as those disclosed in US 2012 / 0177645.

[0242] Exemplary BTLA inhibitors include, but are not limited to, anti-BTLA antibodies described in Crawford and Wherry, 2009 (J Leukocyte Biol 86:5-8), WO 2011 / 014438 (e.g., 4C7 or an antibody comprising a heavy chain and a light chain according to SEQ ID NOs: 8 and 15 and / or SEQ ID NOs: 11 and 18), WO 2014 / 183885 (e.g., the antibody deposited under number CNCM I-4752), and US 2018 / 155428.

[0243] Checkpoint inhibitors of KIR signaling include, but are not limited to, monoclonal antibodies lirilumab (1-7F9; IPH2102; see US 8,709,411), IPH4102 (Innate Pharma; see Marie-Cardine et al., 2014, Cancer 74(21): 6060-70), and the like, e.g., US 2018 / 208652, US 2018 / 117147, US 2015 / 344576, WO 2005 / 003168, WO 2005 / 009465, WO 2006 / 072625, WO 2006 / 072626, WO 2007 / 042573, WO 2008 / 084106 (e.g., an antibody comprising the heavy and light chains set forth in SEQ ID NOs: 2 and 3), WO 2010 / 065939, WO 2012 / 071411, WO 2012 / 160448, and WO 2014 / 055648.

[0244] LAG-3 inhibitors include anti-LAG-3 antibodies BMS-986016 (Bristol Myers Squibb; see WO 2014 / 008218 and WO 2015 / 116539), 25F7 (see US2011 / 0150892), IMP731 (see WO 2008 / 132601), H5L7BW (see WO2014140180), MK-4280 (28G-10; Merck; see WO 2016 / 028672), REGN3767 (Regneron / Sanofi), and BAP050 (WO 2017 / 019894), IMP-701 (LAG-525; Novartis), Sym022 (Symphogen), TSR-033 (Tesaro), MGD013 (a bispecific DART antibody targeting LAG-3 and PD-1 developed by MacroGenics), BI754111 (Boehringer Ingelheim), FS118 (a bispecific antibody targeting LAG-3 and PD-1 developed by F-star), GSK2831781 (GSK), and WO 2009 / 044273, WO 2008 / 132601, WO 2015 / 042246, EP 2 320 940, US 2019 / 169294, US 2019 / 169292, WO 2016 / 028672, WO 2016 / 126858, WO 2016 / 200782, WO 2015 / 200119, WO 2017 / 220569, WO 2017 / 087589, WO 2017 / 219995, WO 2017 / 019846, WO 2017 / 106129, WO 2017 / 062888, WO 2018 / 071500, WO 2017 / 087901, US 2017 / 0260271, WO 2017 / 198741, WO2017 / 220555, WO2017 / 015560, WO2017 / 025498, WO2017 / 149143, WO 2018 / 069500, WO2018 / 083087, WO2018 / 034227 and WO2014 / 140180, their LAG-3 antagonist protein AVA-017 (Avacta), soluble LAG-3 fusion protein IMP321 (eftilagimod alpha, Immutep, EP 2 205 257 and Brignone et al., 2007, J. Immunol., 179:4202-4211) and the soluble LAG-3 protein disclosed in WO 2018 / 222711.

[0245] Examples of TIM-3 inhibitors include, but are not limited to, antibodies that target TIM-3, such as F38-2E2 (BioLegend), covolimab (TSR-022; Tesaro), LY3321367 (Eli Lilly), MBG453 (Novartis), and antibodies disclosed in, for example, WO 2013 / 006490, WO 2018 / 085469 (e.g., antibodies comprising heavy and light chain sequences encoded by the nucleotide sequences represented by SEQ ID NOs: 3 and 4), WO 2018 / 106588, and WO 2018 / 106529 (e.g., antibodies comprising heavy and light chain sequences represented by SEQ ID NOs: 8 to 11).

[0246] Examples of TIM-3 ligand inhibitors include, but are not limited to, CEACAM1 inhibitors such as the anti-CEACAM1 antibody CM10 (cCAM Biotherapeutics; see WO 2013 / 054331 ), antibodies disclosed in WO 2015 / 075725 (e.g., CM-24, 26H7, 5F4, TEC-11, 12-140-4, 4 / 3 / 17, COL-4, F36-54, 34B1, YG-C28F2, D14HD11, M8.7.7, D11-AD11, HEA81, B 1.1, CLB-gran-10, F34-187, T84.1, B6.2, B 1.13, YG-C94G7, 12-140-5, scFv DIATHIS1, TET-2, cCAM Biotherapeutics), and antibodies disclosed in Watt et al. al., 2001 (Blood, 98: 1469-1479) and WO 2010 / 12557, as well as PtdSer inhibitors such as bavituximab (Peregrine).

[0247] CD94 / NKG2A inhibitors include, but are not limited to, monalizumab (IPH2201, Innate Pharma) and antibodies disclosed in US 9,422,368 (see, e.g., humanized Z199, EP 2 628 753), EP 3 193 929 and WO2016 / 032334 (see, e.g., humanized Z270, EP 2 628 753) and methods for their production.

[0248] IDO inhibitors include, but are not limited to, exiguamine A, epacadostat (INCB024360, InCyte, see US 9,624,185), indoximod (Newlink Genetics, CAS No. 110117-83-4), NLG919 (Newlink Genetics / Genentech, CAS No. 1402836-58-1), GDC-0919 (Newlink Genetics / Genentech, CAS No. 1402836-58-1), F001287 (Flexus Biosciences / BMS, CAS No. 2221034-29-1), KHK2455 (Cheong et al., 2018, Expert Opin Ther Pat. 28(4):317-330), PF-06840003 (WO 2016 / 181348), navoximod (RG6078, GDC-0919, NLG919; CAS No. 1402837-78-8), linrodostat (BMS-986205; Bristol-Myers Suibb; CAS No. 1923833-60-6), 1-methyl-tryptophan, pyrrolidine-2,5-dione derivatives (see WO 2015 / 173764), and the IDO inhibitors disclosed in Sheridan, 2015, Nat Biotechnol 33:321-322.

[0249] CD39 inhibitors include, but are not limited to, A001485 (Arcus Biosciences), PSB 069 (CAS number 78510-31-3), and the anti-CD39 monoclonal antibody IPH5201 (Innate Pharma; see Perrot et al., 2019, Cell Reports 8:2411-2425.E9).

[0250] CD73 inhibitors include, but are not limited to, CPI-006 (Corvus Pharmaceuticals), MEDI9447 (MedImmune; see WO2016075099), IPH5301 (Innate Pharma; see Perrot et al., 2019, Cell Reports 8:2411-2425.E9), anti-CD73 antibodies described in WO2018 / 110555, small molecule inhibitors PBS 12379 (Tocris Bioscience; CAS number 1802226-78-3), A000830, A001190, and A001421 (Arcus Biosciences; Becker et al., 2018, Cancer Research 78(13 Supplement):3691-3691, doi:10.1158 / 1538-7445.AM2018-3691), anti-CD73 antibodies such as CB-708 (Calithera Biosciences), and diphosphonates of the purine cytotoxic nucleoside analogue series as described in Allard et al., 2018 (Immunol Rev., 276(1):121-144).

[0251] A2AR inhibitors include, but are not limited to, isotoradefylline (KW-6002; CAS No. 155270-99-8), PBF-509 (Palobiopharma), ciforadenant (CPI-444: Corvus Pharma / Genentech; CAS No. 1202402-40-1), ST1535 ([2-butyl-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purin-6-xylamine]; CAS No. 496955-42-1), ST4206 (see Stasi et al., 2015, Europ J Pharm 761:353-361; CAS No. 1246018-36-9), tozadenant (SYN115; CAS No. 870070-55-6), V81444 (WO 2002 / 055082), preladenant (SCH420814; Merck; CAS no. 377727-87-2), bipadenant (BIIB014; CAS no. 442908-10-3), ST1535 (CAS no. 496955-42-1), SCH412348 (CAS no. 377727-26-9), SCH442416 (Axon 2283; Axon Medchem; CAS No. 316173-57-6), ZM241385 (4-(2-(7-amino-2-(2-furyl)-(1,2,4)triazolo(2,3-a)-(1,3,5)triazin-5-yl-amino)ethyl)phenol; CAS No. 139180-30-6), AZD4635 (AstraZeneca), AB928 (dual A2AR / A2BR small molecule inhibitor; Arcus Biosciences), and SCH58261 (see Popoli et al., 2000, Neuropsychopharm 22:522-529; CAS No. 160098-96-4).

[0252] A2BR inhibitors include, but are not limited to, AB928 (dual A2AR / A2BR small molecule inhibitor; Arcus Biosciences), MRS 1706 (CAS No. 264622-53-9), GS6201 (CAS No. 752222-83-6), and PBS 1115 (CAS No. 152529-79-8). VISTA inhibitors include, but are not limited to, anti-VISTA antibodies such as JNJ-61610588 (onvatilimab; Janssen Biotech) and the low molecular weight inhibitor CA-170 (anti-PD-L 1 / L 2 and anti-VISTA low molecular weight; CAS number 1673534-76-3).

[0253] Siglec inhibitors include, but are not limited to, the anti-Siglec-7 antibodies disclosed in US 2019 / 023786 and WO 2018 / 027203 (e.g., antibodies comprising a variable heavy chain region set forth in SEQ ID NO: 1 and a variable light chain region set forth in SEQ ID NO: 15), the anti-Siglec-2 antibody inotuzumab ozogamicin (Besponsa; see US 8,153,768 and US 9,642,918), the anti-Siglec-3 antibody gemtuzumab ozogamicin (Mylotarg; see US 9,359,442), or the antibodies disclosed in US 2019 / 062427, US 2019 / 023786, WO 2019 / 011855, WO Examples of such antibodies include the anti-Siglec-9 antibodies disclosed in US 2019 / 011852 (e.g., antibodies comprising CDRs set forth in SEQ ID NOs: 171 to 176, or 3 and 4, or 5 and 6, or 7 and 8, or 9 and 10, or 11 and 12, or 13 and 14, or 15 and 16, or 17 and 18, or 19 and 20, or 21 and 22, or 23 and 24, or 25 and 26), and the antibodies described in US 2017 / 306014 and EP 3146979.

[0254] CD20 inhibitors include, but are not limited to, anti-CD20 antibodies such as rituximab (RITUXAN; IDEC-102; IDEC-C2B8; see US 5,843,439), ABP 798 (rituximab biosimilar), ofatumumab (2F2; see WO 02004 / 035607), obinutuzumab, ocrelizumab (2h7; see WO 2004 / 056312), ibritumomab tiuxetan (Zevalin), tositumomab, ublituximab (LFB-R603; LFB Biotechnologies), and antibodies disclosed in US 2018 / 0036306 (e.g., antibodies comprising light chains and heavy chains according to SEQ ID NOS: 1-3 and 4-6, or 7 and 8, or 9 and 10).

[0255] GARP inhibitors include, but are not limited to, anti-GARP antibodies such as ARGX-115 (arGEN-X), as well as antibodies and methods of producing them disclosed in US 2019 / 127483, US 2019 / 016811, US 2018 / 327511, US 2016 / 251438, and EP 3 253 796. CD47 inhibitors include, but are not limited to, anti-CD47 antibodies such as HuF9-G4 (Stanford University / FourteSeven), CC-90002 / INBRX-103 (Celgene / Inhibrx), SRF231 (Surface Oncology), IBI188 (Innovent Biologics), and AO-176 (Arch Oncology), bispecific antibodies targeting CD47, including TG-1801 (NI-1701; a bispecific monoclonal antibody targeting CD47 and CD19; Novimmune / TG Therapeutics) and NI-1801 (a bispecific monoclonal antibody targeting CD47 and mesothelin; Novimmune), and ALX148 (ALX Oncology; Kauder et al., 2019, PLoS One, doi:10.1371 / journal.pone.0201832). SIRPα inhibitors include, but are not limited to, anti-SIRPα antibodies such as OSE-172 (Boehringer Ingelheim / OSE) and FSI-189 (FourteSeven), and anti-SIRPα fusion proteins such as TTI-621 and TTI-662 (Trillium Therapeutics; see WO 2014 / 094122).

[0256] PVRIG inhibitors include, but are not limited to, anti-PVRIG antibodies such as COM701 (CGEN-15029), as well as antibodies and methods for producing same disclosed in, for example, WO 2018 / 033798 (e.g., CHA.7.518.1H4(S241P), CHA.7.538.1.2.H4(S241P), CPA.9.086H4(S241P), CPA.9.083H4(S241P), CHA.9.547.7.H4(S241P), CHA.9.547.13.H4(S241P) of WO 2018 / 033798, or antibodies comprising a variable heavy chain domain according to SEQ ID NO: 5 and a variable light chain domain according to SEQ ID NO: 10, or antibodies comprising a heavy chain according to SEQ ID NO: 9 and a light chain according to SEQ ID NO: 14; WO WO 2018 / 033798 further discloses anti-TIGIT antibodies, and combination therapies with anti-TIGIT and anti-PVRIG antibodies), WO2016134333, WO2018017864 (e.g., antibodies comprising a heavy chain according to SEQ ID NOs: 5-7 that has at least 90% sequence identity to SEQ ID NO: 11 and / or a light chain according to SEQ ID NOs: 8-10 that has at least 90% sequence identity to SEQ ID NO: 12, or antibodies encoded by SEQ ID NOs: 13 and / or 14 or SEQ ID NOs: 24 and / or 29, or other antibodies disclosed in WO 2018 / 017864), and anti-PVRIG antibodies and fusion peptides disclosed in WO 2016 / 134335.

[0257] CSF1R inhibitors include anti-CSF1R antibodies such as cavalalizumab (FPA008; FivePrime; see WO 2011 / 140249, WO 2013 / 169264, and WO 2014 / 036357), IMC-CS4 (EiiLilly), emactuzumab (R05509554; Roche), and RG7155 (WO 2011 / 70024, WO 2011 / 107553, WO 2011 / 131407, WO 2013 / 87699, WO 2013 / 119716, WO 2013 / 132044) and the low molecular weight inhibitors BLZ945 (CAS number 953769-46-5) and pexidartinib (PLX3397; Selleckchem; CAS number 1029044-16-3). CSF1 inhibitors include, but are not limited to, anti-CSF1 antibodies disclosed in EP 1223980 and Weir et al., 1996 (J Bone Mineral Res 11:1474-1481), WO 2014 / 132072, and antisense DNA and RNA disclosed in WO 2001 / 030381.

[0258] Exemplary NOX inhibitors include, but are not limited to, the small molecule ML171 (Gianni et al., 2010, ACS Chem Biol 5(10):981-93), NOS31 (see Yamamoto et al., 2018, Biol Pharm Bull. 41(3):419-426), NOX2 inhibitors such as the low molecular weight Sepren (histamine dihydrochloride; CAS No. 56-92-8), BJ-1301 (Gautam et al., 2017, Mol Cancer Ther 16(10):2144-2156; CAS No. 1287234-48-3) and the inhibitors described in Lu et al., 2017, Biochem Pharmacol 143:25-38, NOX4 inhibitors such as the low molecular weight inhibitor VAS2870 (Altenhoefer et al., 2012, Cell Mol Life Sciences 69(14):2327-2343), diphenyleneiodonium (CAS No. 244-54-2), and GKT137831 (CAS No. 1218942-37-0; see Tang et al., 2018, 19(10):578-585).

[0259] TDO inhibitors include, but are not limited to, 4-(indol-3-yl)-pyrazole derivatives (see US 9,126,984 and US 2016 / 0263087), 3-indole substituted derivatives (see WO 2015 / 140717, WO 2017 / 025868, WO 2016 / 147144), 3-(indol-3-yl)-pyridine derivatives (see US 2015 / 0225367 and WO 2015 / 121812), WO 2015 / 150097, WO 2015 / 082499, WO 2016 / 026772, WO 2016 / 071283, WO 2016 / 071293, WO Dual IDO / TDO antagonists, such as the small molecule dual IDO / TDO inhibitor disclosed in 2017 / 007700, and the small molecule inhibitor CB548 (Kim, C, et al., 2018, Annals Oncol 29(suppl_8):viii400-viii441).

[0260] According to the present disclosure, immune checkpoint inhibitors are inhibitors of inhibitory checkpoint proteins, but preferably not stimulatory checkpoint proteins. As disclosed herein, certain CTLA-4, PD-1, TIGIT, B7-H3, B7-H4, BTLA, KIR, LAG-3, TIM-3, CD94 / NKG2A, IDO, A2AR, A2BR, VISTA, Siglec, CD20, CD39, CD73, GARP, CD47, PVRIG, CSF1R, NOX, and TDO inhibitors and their respective ligands are known, some of which are already in clinical trials or even approved. Based on these known immune checkpoint inhibitors, alternative immune checkpoint inhibitors may be developed. In particular, known inhibitors of preferred immune checkpoint proteins may be used as is, or analogs thereof, particularly chimeric, humanized, or human antibodies and antibodies that cross-compete with any of the antibodies described herein, may be used. It will be understood by those skilled in the art that other immune checkpoint targets can also be targeted by antagonists or antibodies, provided that targeting results in stimulation of an immune response, such as an anti-tumor immune response as reflected by increased T cell proliferation, enhanced T cell activation, and / or increased cytokine production (e.g., IFN-γ, IL2).

[0261] Checkpoint inhibitors can be administered by any method and by any route known in the art. The mode and route of administration will depend on the type of checkpoint inhibitor used. The checkpoint inhibitor may be administered in the form of any suitable pharmaceutical composition as disclosed herein. The checkpoint inhibitor can be administered in the form of a nucleic acid, e.g., a DNA or RNA molecule, encoding the immune checkpoint inhibitor, such as an inhibitory nucleic acid molecule or an antibody or fragment thereof. For example, an antibody can be delivered encoded in an expression vector, as disclosed herein. The nucleic acid molecule can be delivered by itself, e.g., in the form of a plasmid or mRNA molecule, or complexed with a delivery vehicle, e.g., a liposome, lipoplex, or nucleic acid-lipid particle. The checkpoint inhibitor can also be administered via an oncolytic virus containing an expression cassette encoding the checkpoint inhibitor. The checkpoint inhibitor can also be administered by administering endogeneic or allogeneic cells capable of expressing the checkpoint inhibitor, e.g., in the form of cell-based therapy.

[0262] The term "cell-based therapy" refers to the transplantation of cells (e.g., T lymphocytes, dendritic cells, or stem cells) expressing an immune checkpoint inhibitor into a subject for the purpose of treating a disease or disorder (e.g., a cancer disease). In one embodiment, the cell-based therapy comprises genetically engineered cells. In one embodiment, the genetically engineered cells express an immune checkpoint inhibitor as disclosed herein. In one embodiment, the genetically engineered cells express an immune checkpoint inhibitor that is an inhibitory nucleic acid molecule, such as an siRNA, shRNA, oligonucleotide, antisense DNA or RNA, aptamer, antibody or fragment thereof, or soluble immune checkpoint protein or fusion. The genetically engineered cells may also express additional agents that enhance T cell function. Such agents are known in the art. Cell-based therapies for use in inhibiting immune checkpoint signaling are disclosed, for example, in WO 2018 / 222711, which is incorporated herein by reference in its entirety.

[0263] As used herein, the term "oncolytic virus" refers to a virus that selectively replicates in cancerous or hyperproliferative cells, slowing their growth, or inducing their death, either in vitro or in vivo, while having no or minimal effect on normal cells. Oncolytic viruses for delivery of immune checkpoint inhibitors contain an expression cassette that can encode an immune checkpoint inhibitor, which is an inhibitory nucleic acid molecule such as siRNA, shRNA, oligonucleotide, antisense DNA or RNA, aptamer, antibody or fragment thereof, or soluble immune checkpoint protein or fusion. The oncolytic virus is preferably replication-competent, and the expression cassette is under the control of a viral promoter, such as a synthetic early / late poxvirus promoter. Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), rhabdoviruses (e.g., picornaviruses such as Seneca Valley virus; SVV-001), coxsackieviruses, parvoviruses, Newcastle disease virus (NDV), herpes simplex virus (HSV; OncoVEX GMCSF), retroviruses (e.g., influenza virus), measles virus, reovirus, Sindbis virus, vaccinia virus (exemplary of which are described in WO 2017 / 209053 (including the Copenhagen, Western Reserve, and Wyeth strains)), and adenoviruses (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAd1, H101, AD5 / 3-D24-GMCSF). The generation of recombinant oncolytic viruses comprising soluble forms of immune checkpoint inhibitors and methods for their use are disclosed in WO 2018 / 022831, which is incorporated herein by reference in its entirety. The oncolytic virus can be used as an attenuated virus. In some embodiments, the immune checkpoint inhibitor comprises an antibody selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, and a combination thereof. In certain embodiments, the immune checkpoint inhibitor comprises an anti-PD-1 antibody. In some embodiments, the anti-PD-1 is selected from the group consisting of cemiplimab (LIBTAYO, REGN2810), nivolumab (OPDIVO; BMS-936558), pembrolizumab (KEYTRUDA; MK-3475), pidilizumab (CT-011), spartalizumab (PDR001), MEDI0680 (AMP-514), dostarlimab (TSR-042), cetrelimab (JNJ 63723283), toripalimab (JS001), AMP-224 (GSK-2661380), PF-06801591, tislelizumab (BGB-A317), ABBV-181, BI 754091, sintilimab (IBI308), or karelizumab (SHR-1210).

[0264] In one embodiment, the immune checkpoint inhibitor comprises an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 agent comprises atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267), durvalumab (MEDI4736), BMS-936559, avelumab (BAVENCIO), lodapolimab (LY3300054), CX-072 (Proclaim-CX-072), FAZ053, KN035, sugemalimab (CS1001), or MDX-1105.

[0265] As disclosed herein, the bispecific binding agent is administered to a subject, e.g., a patient, along with a checkpoint inhibitor, i.e., co-administered. In some embodiments, the checkpoint inhibitor and bispecific binding agent are administered to a subject as a single composition. In some embodiments, the checkpoint inhibitor and bispecific binding agent are administered to a subject simultaneously (as separate compositions at the same time). In some embodiments, the checkpoint inhibitor and bispecific binding agent are administered separately to a subject. In some embodiments, the checkpoint inhibitor is administered to a subject before the bispecific binding agent. In some embodiments, the checkpoint inhibitor is administered to a subject after the bispecific binding agent. In some embodiments, the checkpoint inhibitor and bispecific binding agent are administered to a subject on the same day. In some embodiments, the checkpoint inhibitor and bispecific binding agent are administered to a subject on different days.

[0266] The term "pharmaceutical preparation" refers to any product intended for medical use. The term includes pharmaceutical compositions containing one or more active ingredients, as well as configurations (e.g., kits) of one or more active ingredients, which may be present together or separately (e.g., in separate vials), optionally together with informational material regarding, for example, their administration, effects, etc. The compounds and agents disclosed herein can be administered in the form of any suitable pharmaceutical composition. The pharmaceutical compositions described herein are preferably sterile and contain an effective amount of the compounds and agents described herein, and optionally additional agents described herein, to produce the desired response or desired effect. The pharmaceutical composition is usually provided in a uniform dosage form and can be prepared in a manner known per se. The pharmaceutical composition may, for example, be in the form of a solution or suspension. Pharmaceutical compositions may contain salts, buffering substances, preservatives, carriers, diluents and / or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to a non-toxic substance that does not interact with the action of the active ingredients of the pharmaceutical composition.

[0267] Pharmaceutically unacceptable salts may be used to prepare pharmaceutically acceptable salts and are included in the present invention. Pharmaceutically acceptable salts of this type include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts may also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, or calcium salts. Suitable buffering substances for use in pharmaceutical compositions include acetic acid in a salt form, citric acid in a salt form, boric acid in a salt form, and phosphoric acid in a salt form. Suitable preservatives for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens and thimerosal. Injectable formulations may include pharmaceutically acceptable excipients such as lactated Ringer's.

[0268] The term "carrier" refers to an organic or inorganic component, of natural or synthetic nature, with which the active ingredient is combined to facilitate, enhance, or enable application. According to the present invention, the term "carrier" includes one or more compatible solid or liquid fillers, diluents, or encapsulating substances that are suitable for administration to a patient. Possible carrier materials for parenteral administration are, for example, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes, and, particularly, biocompatible lactide polymer, lactide / glycolide copolymer, or polyoxyethylene / polyoxypropylene copolymer.

[0269] The term "excipient" as used herein is intended to refer to any substance that may be present in a pharmaceutical composition and that is not an active ingredient, such as, for example, a carrier, binder, lubricant, thickener, surfactant, preservative, emulsifier, buffer, flavoring agent, or coloring agent. The agents and compositions disclosed herein can be administered via any conventional route, such as parenteral administration, including injection or infusion. Administration is preferably parenteral, e.g., intravenous, intraarterial, subcutaneous, intradermal, or intramuscular. Compositions suitable for parenteral administration usually comprise a sterile aqueous or non-aqueous preparation of the active compound, which is preferably isotonic with the blood of the recipient. Examples of suitable carriers and solvents include Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are usually used as a solution or suspension medium.

[0270] The agents and compositions disclosed herein are administered in an effective amount. An "effective amount" refers to an amount that alone or together with further doses achieves the desired response or desired effect. In the case of treatment of a specific disease or a specific condition, the desired response preferably relates to inhibiting the course of the disease. This includes slowing the progression of the disease, and in particular, halting or reversing the progression of the disease. The desired response in the treatment of a disease or condition may also be delaying or preventing the onset of the disease or condition.

[0271] The effective amount of the agents or compositions disclosed herein depends on the condition being treated, the severity of the disease, individual patient parameters including age, physiological condition, size and weight, duration of treatment, type of concomitant therapy (if any), the specific route of administration, and similar factors. Thus, the dose administered of the agents described herein may depend on various such parameters. If the patient's response is inadequate with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) can be used.

[0272] The agents and compositions described herein can be administered to a patient, for example, in vivo, to treat or prevent various disorders, such as those described herein. Preferred patients include human patients with disorders that can be corrected or ameliorated by administering the agents and compositions described herein. This includes disorders involving cells characterized by an altered expression pattern of CLDN18.2. For example, in certain embodiments, the agents described herein can be used to treat a patient with a cancer disease, e.g., a cancer disease as described herein that is characterized by the presence of cancer cells that express CLDN18.2. The pharmaceutical compositions and treatment methods described by the present invention may also be used for immunization or vaccination to prevent the diseases disclosed herein.

[0273] The pharmaceutical compositions described herein may be administered with supplementary immune-enhancing substances, such as one or more adjuvants, to further enhance their efficacy, preferably to achieve an additive immune-stimulating effect. The term "adjuvant" refers to a compound that prolongs, enhances, or accelerates the immune response. In this regard, various mechanisms are possible depending on the type of adjuvant. For example, compounds capable of maturing DCs, such as lipopolysaccharides or CD40 ligand, form a first class of suitable adjuvants. Generally, any agent that influences the immune system, such as "danger signals" (e.g., LPS, GP96, dsRNA, etc.) or cytokines, such as GM-CSF, can be used as an adjuvant that can enhance and / or influence the immune response in a controlled manner. CpG oligodeoxynucleotides can also be used in this context, although their potential side effects under certain circumstances should be considered, as explained above. Particularly preferred adjuvants are cytokines, such as monokines, lymphokines, interleukins, or chemokines, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFα, INF-γ, GM-CSF, LT-α, or growth factors, such as hGH. Further known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils, such as Montanide®, most preferably Montanide® ISA51. Lipopeptides, such as Pam3Cys, are also suitable for use as adjuvants in the pharmaceutical compositions of the invention.

[0274] The agents and compositions provided herein may be used alone or in combination with conventional treatment regimens such as surgery, radiation, chemotherapy and / or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated). Cancer treatment represents a particularly desirable area for combination strategies, as the combined action of two, three, four, or more cancer drugs / therapies often produces synergistic effects significantly stronger than those of monotherapy approaches. Thus, in other embodiments of the present invention, cancer treatments utilizing immunotherapy- or vaccination-based mechanisms, such as the methods and compositions of the present invention, can be effectively combined with a variety of other drugs and / or methods targeting similar or other specific mechanisms. These include, for example, combinations with conventional tumor therapy, multi-epitope strategies, additional immunotherapies, and therapeutic approaches targeting angiogenesis or apoptosis (for reviews, see, e.g., Andersen et al. 2008: Cancer treatment: the combination of vaccination with other therapies. Cancer Immunology Immunotherapy, 57(11):1735-1743). Sequential administration of different drugs may inhibit cancer cell proliferation at different checkpoints, while other drugs may inhibit, for example, neovascularization, malignant cell survival, or metastasis, potentially transforming cancer into a chronic disease. The following list provides some non-limiting examples of anti-cancer agents and therapies that may be used in combination with the present invention.

[0275] 1.Chemotherapy Chemotherapy is the standard treatment for multiple types of cancer. Most common chemotherapeutic agents act by killing rapidly dividing cells, which is one of the main characteristics of cancer cells. Therefore, the combination of conventional chemotherapeutic agents, such as alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents that affect either cell division or DNA synthesis, can significantly improve the therapeutic effect of the present invention by eliminating suppressor cells, restarting the immune system, making tumor cells more susceptible to immune-mediated killing, or further activating cells of the immune system. The additive anticancer effects of chemotherapy and vaccination-based immunotherapy have been demonstrated in several studies (see, e.g., Quoix et al. 2011: Therapeutic vaccination with TG4010 and first-line chemotherapy in advanced non-small-cell lung cancer: a controlled phase 2B trial. Lancet Oncol. 12(12): 1125-33; Liseth et al. 2010: Combination of intensive chemotherapy and anticancer vaccines in the treatment of human malignancies: the hematological experience. J Biomed Biotechnol. 2010: 6920979; Hirooka et al. 2009: A combination therapy of gemcitabine with immunotherapy for patients with inoperable locally advanced pancreatic cancer. Pancreas 38(3): e69-74). In principle, hundreds of chemotherapy drugs are available that are suitable for combination therapy.Some (non-limiting) examples of chemotherapeutic agents that may be used in combination with the present invention include carboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ), cyclophosphamide (Cytoxan, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (VePesid), gemcitabine (Gemzar), imatinib mesylate (Gleevec), irinotecan (Camptosar), methotrexate (Folex, Mexate, Amethoterin), paclitaxel (Taxol, Abraxane), sorafinib (Nexavar), sunitinib (Sutent), topotecan (Hycamtin), vincristine (Oncovin, Vincasar), and the like. PFS), and vinblastine (Velban).

[0276] 2.Surgery Cancer surgery—operation to remove tumors—remains the cornerstone of cancer treatment. Surgery may be combined with other cancer treatments to remove any remaining tumor cells. Combining surgical methods with subsequent immunotherapeutic treatments is a promising approach that has been extensively documented.

[0277] 3. Radiation Radiation therapy remains an important component of cancer treatment, with approximately 50% of all cancer patients receiving radiation therapy during the course of their disease. The primary goal of radiation therapy is to eliminate the ability of cancer cells to proliferate (divide). The types of radiation used to treat cancer are photon radiation (X-rays and gamma rays) and particle radiation (electron, proton, and neutron beams). There are two ways to deliver radiation to the location of the cancer: external beam radiation, in which high-energy rays (photon, proton, or particle radiation) are directed and delivered from outside the body to the tumor location. Internal radiation, or brachytherapy, is delivered directly to the tumor site from within the body by a radioactive source enclosed in a catheter or seed. Radiation therapy techniques that can be used in conjunction with the present invention include, for example, fractionation (radiation therapy delivered in a fractionated regimen, e.g., 1.5-3 Gy daily fractions given over several weeks), 3D conformal radiation therapy (3DCRT; delivering radiation to the entire tumor volume), intensity-modulate...

Claims

1. (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 bispecific 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 that 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 that 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 that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:29; and (iv) the fourth polypeptide chain comprises an amino acid sequence of SEQ ID NO:29, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:29; and (b) immune checkpoint inhibitor 10. A composition or pharmaceutical preparation comprising:

2. (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 bispecific binding agent is encoded by one or more nucleic acid molecules, wherein: (i) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence represented by SEQ ID NO: 27; (ii) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence represented by SEQ ID NO: 28; and (iii) a third nucleic acid sequence encoding a third polypeptide chain comprising the amino acid sequence represented by SEQ ID NO: 29; and (b) an immune checkpoint inhibitor; 1. A composition or pharmaceutical preparation comprising: Optionally, the composition or pharmaceutical preparation, wherein the one or more nucleic acid molecules is a set of nucleic acids.

3. the bispecific binding agent comprises four polypeptide chains, wherein: (a) a first polypeptide chain is encoded by a first nucleic acid sequence; (b) the second polypeptide chain is encoded by a second nucleic acid sequence; (c) the third polypeptide chain is encoded by a third nucleic acid sequence; and (d) the fourth polypeptide chain is encoded by a third nucleic acid sequence; In some cases, here, (i) the first polypeptide chain comprises the amino acid sequence set forth in 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 set forth in 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 set forth in SEQ ID NO: 29; and / or (iv) the fourth polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 29; 3. A composition or pharmaceutical preparation according to claim 2.

4. 4. The composition or pharmaceutical preparation of any one of claims 1 to 3, wherein (i) the first polypeptide chain interacts with the second polypeptide chain and the third polypeptide chain, and / or (ii) the second polypeptide chain interacts with the fourth polypeptide chain.

5. (a) the first polypeptide chain comprises, from N-terminus to C-terminus: (i) the variable region of the heavy chain (VH) derived from an immunoglobulin that binds to human CLDN18.2 (VH(CLDN18.2)); (ii) a heavy chain constant region 1 (CH1) derived from an immunoglobulin or a functional variant thereof; (iii) a heavy chain constant region 2 (CH2) derived from an immunoglobulin or a functional variant thereof; and (iv) a heavy chain constant region 3 (CH3) derived from an immunoglobulin or a functional variant thereof; Including, (b) the second polypeptide chain comprises, from N-terminus to C-terminus: (i) the variable region of the heavy chain (VH) derived from an immunoglobulin that binds to human CLDN18.2 (VH(CLDN18.2)); (ii) a heavy chain constant region 1 (CH1) 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 heavy chain constant region 2 (CH2) derived from an immunoglobulin or a functional variant thereof; and (vi) a heavy chain constant region 3 (CH3) derived from an immunoglobulin or a functional variant thereof; Including, (c) the third polypeptide chain comprises, from N-terminus to C-terminus: (i) the variable region of the light chain (VL) derived from an immunoglobulin that binds to human CLDN18.2 (VL (CLDN18.2)); and (ii) a light chain (CL) constant region derived from an immunoglobulin or a functional variant thereof; and / or (d) the fourth polypeptide chain comprises, from N-terminus to C-terminus: (i) the variable region of the light chain (VL) derived from an immunoglobulin that binds to human CLDN18.2 (VL (CLDN18.2)); and (ii) a light chain constant region (CL) derived from an immunoglobulin or a functional variant thereof; and optionally, where: (i) 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; (ii) 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; and / or (iii) the VH(CD3) and the VL(CD3) interact to form a binding domain that binds to human CD3; 5. A composition or pharmaceutical preparation according to any one of claims 1, 3 and 4.

6. (a) the VH (CLDN18.2) comprises a CDR1 comprising the amino acid SYWIN (SEQ ID NO: 10), a CDR2 comprising the amino acid NIYPSDSYTNYNQKFQG (SEQ ID NO: 11), and a CDR3 comprising the amino acid SWRGNSFDY (SEQ ID NO: 12); (b) the VL (CLDN18.2) comprises a CDR1 comprising the amino acid KSSQSLLNSGNQKNYLT (SEQ ID NO: 13), a CDR2 comprising the amino acid WASTRES (SEQ ID NO: 14), and a CDR3 comprising the amino acid QNDYSYPFT (SEQ ID NO: 15); (c) the VH(CD3) comprises a CDR1 comprising the amino acids TYAMN (SEQ ID NO: 18), a CDR2 comprising the amino acids RIRSKANNYATYYADSVKG (SEQ ID NO: 23), and a CDR3 comprising the amino acids HGNFGDSYVSWFAY (SEQ ID NO: 19); (d) the VL(CD3) comprises a CDR1 comprising the amino acid sequence GSSTGAVTSTNYAN (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); (e) CH2 on the first polypeptide chain interacts with CH2 on the second polypeptide chain, and / or CH3 on the first polypeptide chain interacts with CH3 on the second polypeptide chain; (f) a CH1 on the first polypeptide chain interacts with a CL on the third polypeptide chain; and / or 6. The composition or pharmaceutical preparation of claim 5, wherein (g) a CH1 on the second polypeptide chain interacts with a CL on the fourth polypeptide chain.

7. 7. The composition or pharmaceutical preparation of claim 5 or 6, wherein the immunoglobulin is IgG1 or human IgG1.

8. (a) the VH (CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 16; (b) the VL (CLDN18.2) comprises or consists of the amino acid sequence represented by SEQ ID NO: 17; (c) the VL(CD3) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24; and / or (d) the VH(CD3) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25; and / or (e) the VH (CLDN18.2), the VL (CLDN18.2), the VH (CD3) and / or the VL (CD3) are humanized; A composition or pharmaceutical preparation according to any one of claims 5 to 7.

9. (a) on the first polypeptide chain, the CH1 is connected to the CH2 by a peptide linker L1; Optionally, the peptide linker L1 comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 6) or a functional variant thereof; and / or (b) the VL(CD3) is connected to the CH1 by a peptide linker L2, optionally wherein the peptide linker L2 comprises the amino acid sequence (GS)x or a functional variant thereof, where x is 2, 3, 4, 5, or 6; the peptide linker L2 comprises the amino acid sequence (GS)2 (SEQ ID NO: 5) or a functional variant thereof; (c) the VL(CD3) and the VH(CD3) are linked to each other by a peptide linker L3, and optionally the peptide linker L3 comprises the amino acid sequence (GKPGS)x or a functional variant thereof, where x is 2, 3, 4, 5, or 6, or the amino acid sequence (GKPGS) ４ (SEQ ID NO:2) or a functional variant thereof; and / or (d) the VH(CD3) is connected to the CH2 by a peptide linker L4, optionally the peptide linker L4 comprises the amino acid sequence (G4S)x or a functional variant thereof, where x is 2, 3, 4, 5, or 6; or wherein the peptide linker L4 comprises the amino acid sequence (GS)z (SEQ ID NO: 5) or a functional variant thereof; A composition or pharmaceutical preparation according to any one of claims 5 to 8.

10. (a) the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:7; (b) the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 8; and / or (c) the third polypeptide chain and / or the fourth polypeptide chain comprises the amino acid sequence represented by SEQ ID NO: 9; A composition or pharmaceutical preparation according to any one of claims 1 to 9.

11. the immune checkpoint inhibitor comprises one or more immune checkpoint inhibitors, and optionally The immune checkpoint inhibitor comprises an antibody selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, and a combination thereof, and preferably is one of the following: and / or, including cemiplimab (LIBTAYO, REGN2810), nivolumab (Opdivo; BMS-936558), pembrolizumab (KEYTRUDA; MK-3475), pidilizumab (CT-011), spartalizumab (PDR001), MEDI0680 (AMP-514), dostarlimab (TSR-042), cetrelimab (JNJ 63723283), toripalimab (JSOO1), AMP-224 (GSK-2661380), PF-06801591, tislelizumab (BGB-A317), ABBV-181, Bl 754091, sintilimab (IBI308), or karelizumab (5HR-1210); 11. The composition or pharmaceutical preparation of any one of claims 1 to 10, comprising atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267), durvalumab (MEDI4736), BMS-936559, avelumab (BAVENCIO), lodapolimab (LY3300054), CX-072 (Proclaim-CX-072), FAZ053, KN035, sugemalimab (CS1001), or MDX-1105.

12. 12. The composition or pharmaceutical preparation according to any one of claims 1 to 11, which is a pharmaceutical composition or a pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers, diluents and / or excipients.

13. Kit, or 12. The composition or pharmaceutical preparation of any one of claims 1 to 11, wherein the bispecific binding agent and the immune checkpoint inhibitor are in separate vials, optionally further comprising instructions for using the bispecific binding agent and the immune checkpoint inhibitor to treat or prevent cancer.

14. A composition or pharmaceutical preparation according to any one of claims 1 to 13 for pharmaceutical use, comprising In some cases, the following: (a) said medical use includes the therapeutic or prophylactic treatment of a disease or disorder, and optionally (b) the medical use comprises treating or preventing cancer; more preferably, the cancer comprises cancer cells expressing CLDN18.2; more preferably, the cancer is selected from the group consisting of gastric cancer, particularly gastric adenocarcinoma, esophageal cancer, gastroesophageal junction (GEJ) cancer, GEJ adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, lung cancer, non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, rectal cancer, colorectal cancer, liver cancer, head and neck cancer, bile duct cancer, gallbladder cancer and metastasis thereof, Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis. Composition or pharmaceutical preparation.

15. A composition or pharmaceutical preparation according to any one of claims 1 to 14 for administration to a human.

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