TW202541837A - Combination therapy involving bispecific binding agents binding to cldn18.2 and cd3 and agents stabilizing or increasing expression of cldn18.2 - Google Patents

Abstract

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

Description

Combination therapy containing a bispecific binder that binds to CLDN18.2 and CD3 and a drug that stabilizes or increases CLDN18.2 expression.

This invention provides a combination therapy involving a bispecific binder that binds to two binding domains of CLDN18.2 and a binding domain of CD3, and an antibody that binds to VEGFR2. This combination therapy is effective in treating cancers involving cancer cells expressing CLDN18.2.

Cancer is the second leading cause of death worldwide, projected to cause approximately 9.6 million deaths in 2018 (Bray, F. et al., CA: A Cancer Journal for Clinicians, 68: 394-424, 2018). Generally, once a solid tumor metastasizes, with certain exceptions such as germ cell tumors and some carcinoids, the 5-year survival rate rarely exceeds 25%. The poor prognosis of some cancers underscores the need for additional treatment options.

The tight-linking molecule CLDN18 is an integrated membrane protein (tetraspanin) with four transmembrane hydrophobic regions and two extracellular loops (loop 1 is located between hydrophobic regions 1 and 2; loop 2 is located between hydrophobic regions 3 and 4). Two distinct splice variants of CLDN18 have been described in mice and humans (Niimi et al., Mol. Cell. Biol. 21:7380-90, 2001). These splice variants (Genbank access numbers: splice variant 1 (CLDN18.1): NP_057453, NM_016369, and splice variant 2 (CLDN18.2): NM_001002026, NP_001002026) have a molecular weight of approximately 27.9 / 27.72 kD. The splice variants of CLDN18.1 and CLDN18.2 differ in the N-terminal portion, which includes the first transmembrane (TM) region and loop 1, while the major protein sequence at the C-terminus is the same.

In normal tissues, CLDN18.2 is almost undetectable except in the stomach, where it is only present on short-lived differentiated gastric epithelial cells. CLDN18.2 is maintained during malignant transformation and is therefore frequently observed on the surface of human gastric cancer cells. Furthermore, this pantumor antigen is also ectopically activated at significant levels in adenocarcinomas of the esophagus, pancreas, and lung. The CLDN18.2 protein is also localized to lymph node metastases and distant metastases of gastric adenocarcinomas, particularly in tumors that metastasize to the ovary (known as Krukenberg tumor).

The differential expression of tight-linked proteins like CLDN18.2 between cancer cells and normal cells, their membrane localization, and their absence in most normal tissues associated with their toxicity make these molecules attractive targets for cancer immunotherapy. Antibody-based therapies targeting CLDN18.2 are expected to provide high therapeutic specificity in cancer treatment.

Developed by Ganymed Pharmaceuticals, the chimeric IgG1 antibody IMAB362 (Zolbetuximab, formerly known as Claudiximab) exhibits high affinity and specificity for CLDN18.2, recognizing its first outer domain (ECD1). IMAB362 does not bind to other members of the tight junction protein family, including its closely associated splice variant 1 (CLDN18.1) of tight junction protein. IMAB362 demonstrates precise tumor cell specificity and integrates two independent, highly effective mechanisms. Upon target binding, IMAB362 primarily mediates cell killing via ADCC and CDC. Therefore, IMAB362 can efficiently lyse CLDN18.2-positive cells, including human gastric cancer cell lines both in vitro and in vivo. The antitumor efficacy of IMAB362 has been demonstrated in mouse models of xenografts of CLDN18.2-positive cancer cell lines.

IgG1 antibodies typically activate the cellular immune system by interacting with Fcγ receptors (FcγRs) expressed on various immune cells, including natural killer cells, through their Fc domain. However, IgG1 monoclonal antibodies (mAbs) that trigger ADCC face several limitations, including the widespread distribution of low-affinity Fc receptor variants in the population (up to 80%) and the potential for in vivo IgG1 modification to reduce mAb potency (Chames et al., 2009, British Journal of Pharmacology, 157(2):220-233). Therapeutic antibodies also need to compete with IgG in the patient's body, leading to the need for higher doses of mAbs in vivo. Furthermore, therapeutic antibodies may interact with FcγRIIb (an inhibitory Fcγ receptor expressed by B cells, macrophages, dendritic cells, and neutrophils), thereby generating a negative signal and reducing their efficacy.

One objective of this invention is to provide novel cancer treatment methods.

This invention relates essentially to the treatment of an individual, including administration of a bispecific binding agent as described herein, comprising two CLDN18.2 binding domains (in Fab format) targeting cancer cells and a CD3 binding domain (in scFv format) targeting the T cell-specific antigen CD3, allowing binding to T cells and drawing them into the complex, thereby targeting the cytotoxicity of T cells to cancer cells. The formation of this complex can induce cytotoxic T cell signaling, leading to the release of cytotoxic mediators, whether alone or in combination with helper cells. When using the bispecific binding agents described herein for treatment, they can also be combined with other treatment methods, including administration of one or more drugs that stabilize or increase CLDN18.2 performance.

This invention demonstrates that treatment methods involving the bispecific binder and agents that stabilize or increase CLDN18.2 expression described herein can induce a potent antitumor effect.

This invention generally provides a combination therapy involving a bispecific binding agent that binds CLDN18.2 and CD3 and an agent that stabilizes or increases CLDN18.2 expression. In some embodiments, the bispecific binding agent is a bispecific tetramer binding agent. In some embodiments, the agent that stabilizes or increases CLDN18.2 expression is gemcitabine, paclitaxel, or a combination thereof.

This invention provides a composition or pharmaceutical preparation comprising: (a) a bispecific binding agent comprising a first binding domain for human CLDN18.2, a second binding domain for human CLDN18.2, and a third binding domain for human CD3, wherein the binding agent comprises four polypeptide chains, wherein (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 80% sequence identity with 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 having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 28; and (iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 28. (iv) an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 29; and (b) an agent that stabilizes or enhances the performance of CLDN18.2.

In some embodiments, the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 29.

In some embodiments, 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 with the amino acid sequence of SEQ ID NO: 29.

In some embodiments, (i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 80% sequence identity with 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 having at least 80% sequence identity with 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 having at least 80% sequence identity with 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 with the amino acid sequence of SEQ ID NO: 29.

The present invention also provides a composition or pharmaceutical preparation comprising: (a) a bispecific binding agent comprising a first binding domain for human CLDN18.2, a second binding domain for human CLDN18.2, and a third binding domain for human CD3, wherein the binding agent comprises four polypeptide chains, wherein (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27; (ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28; (iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and (b) a medicament for stabilizing or increasing the expression of CLDN18.2.

In some embodiments, the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27.

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

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

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

In some embodiments, (i) the first polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 27; (ii) the second polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 28; (iii) the third polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 29.

The present invention also provides a composition or pharmaceutical preparation comprising: (a) a bispecific binding agent comprising a first binding domain for human CLDN18.2, a second binding domain for human CLDN18.2, and a third binding domain for human CD3, wherein the binding agent is encoded by one or more nucleic acid molecules comprising: (i) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 27; (ii) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 28; and (iii) a third nucleic acid sequence encoding a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 29; and (b) a medicament for stabilizing or increasing the expression of CLDN18.2.

In some embodiments, the one or more nucleic acid molecules are a group of nucleic acids.

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

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

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

In some embodiments, the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 29.

In some embodiments, the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 29.

In some embodiments, (i) the first polypeptide chain includes the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 27 includes the deletion of lysine at position 447 of SEQ ID NO: 27; (ii) the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 28 includes the deletion of lysine at position 720 of SEQ ID NO: 28; (iii) the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 29.

In some embodiments, (i) the first polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 27 includes the deletion of lysine at position 447 of SEQ ID NO: 27; (ii) the second polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 28 includes the deletion of lysine at position 720 of SEQ ID NO: 28; (iii) the third polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 29.

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

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

In some embodiments, the first polypeptide chain interacts with the second and third polypeptide chains, and the second polypeptide chain interacts with the fourth polypeptide chain.

In some embodiments, the first polypeptide chain includes, from the N-terminus to the C-terminus: (i) a variable region (VH) of the heavy chain derived from an immunoglobulin that binds to human CLDN18.2 (VH(CLDN18.2)), (ii) a first constant region (CH1) of the heavy chain derived from an immunoglobulin or a functional variant thereof, (iii) a second constant region (CH2) of the heavy chain derived from an immunoglobulin or a functional variant thereof, and (iv) a third constant region (CH3) of the heavy chain derived from an immunoglobulin or a functional variant thereof.

In some embodiments, the second polypeptide chain includes, from the N-terminus to the C-terminus: (i) a heavy chain variable region (VH) derived from an immunoglobulin binding to human CLDN18.2 (VH(CLDN18.2)), (ii) a first fixed region (CH1) of the heavy chain derived from an immunoglobulin or a functional variant thereof; (iii) a light chain variable region (VL) derived from an immunoglobulin binding to human CD3 (VL(CD3)), (iv) a heavy chain variable region (VH) derived from an immunoglobulin binding to human CD3 (VH(CD3)), (v) a second fixed region (CH2) of the heavy chain derived from an immunoglobulin or a functional variant thereof, and (vi) a third fixed region (CH3) of the heavy chain derived from an immunoglobulin or a functional variant thereof.

In some embodiments, the third polypeptide chain includes, from the N-terminus to the C-terminus: (i) a variable light chain region (VL) (VL(CLDN18.2)) derived from an immunoglobulin that binds to human CLDN18.2, and (ii) a constant light chain region (CL) derived from an immunoglobulin or a functional variant thereof.

In some embodiments, the fourth polypeptide chain includes, from the N-terminus to the C-terminus: (i) a variable light chain region (VL) (VL(CLDN18.2)) derived from an immunoglobulin that binds to human CLDN18.2, and (ii) a constant light chain region (CL) derived from an immunoglobulin or a functional variant thereof.

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

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

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

In some embodiments, the VH (CLDN18.2) includes CDR1 containing the amino acid sequence SYWIN (SEQ ID NO: 10), CDR2 containing the amino acid sequence NIYPSDSYTNYNQKFQG (SEQ ID NO: 11), and CDR3 containing the amino acid sequence SWRGNSFDY (SEQ ID NO: 12).

In some embodiments, the VL (CLDN18.2) includes CDR1 containing the amino acid sequence KSSQSLLNSGNQKNYLT (SEQ ID NO: 13), CDR2 containing the amino acid sequence WASTRES (SEQ ID NO: 14), and CDR3 containing the amino acid sequence QNDYSYPFT (SEQ ID NO: 15).

In some embodiments, the VH(CD3) includes CDR1 containing the amino acid sequence TYAMN (SEQ ID NO: 18), CDR2 containing the amino acid sequence RIRSKANNYATYYADSVKG (SEQ ID NO: 23), and CDR3 containing the amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO: 19).

In some embodiments, the VL (CD3) includes CDR1 containing the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 20), CDR2 containing the amino acid sequence GTNKRAP (SEQ ID NO: 21), and CDR3 containing the amino acid sequence ALWYSNHWV (SEQ ID NO: 22).

In some embodiments, 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 some embodiments, CH1 on the first polypeptide chain interacts with CL on the third polypeptide chain.

In some embodiments, CH1 on the second polypeptide chain interacts with CL on the fourth polypeptide chain.

In some embodiments, the immunoglobulin is IgG1. In some embodiments, the IgG1 is human IgG1.

In some embodiments, the VH (CLDN18.2) comprises SEQ ID NO: 16 or consists of the amino acid sequence it represents.

In some embodiments, the VL (CLDN18.2) comprises SEQ ID NO: 17 or consists of the amino acid sequence it represents.

In some embodiments, the VL(CD3) includes SEQ ID NO: 24 or is composed of the amino acid sequence it represents.

In some embodiments, the VH(CD3) includes SEQ ID NO: 25 or is composed of the amino acid sequence it represents.

In some embodiments, the VH (CLDN18.2) comprises SEQ ID NO: 16 or is composed of the amino acid sequence it represents, the VL (CLDN18.2) comprises SEQ ID NO: 17 or is composed of the amino acid sequence it represents, the VH (CD3) comprises SEQ ID NO: 25 or is composed of the amino acid sequence it represents, and the VL (CD3) comprises SEQ ID NO: 24 or is composed of the amino acid sequence it represents.

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

In some embodiments, CH1 on the first polypeptide chain is linked to CH2 via a peptide linker L1. In some embodiments, the peptide linker L1 comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 6) or a functional variant thereof.

In some embodiments, the VL(CD3) is linked to CH1 via a peptide linker L2. In some embodiments, the peptide linker L2 comprises an amino acid sequence ( _G4S ) _x or a functional variant thereof, wherein x is 2, 3, 4, 5, or 6. In some embodiments, the peptide linker L2 comprises an amino acid sequence ( _G4S ) ₂ (SEQ ID NO: 5) or a functional variant thereof.

In some embodiments, the VH(CD3) is linked to CH2 via a peptide linker L4. In some embodiments, the peptide linker L4 comprises an amino acid sequence ( _G4S ) _x or a functional variant thereof, wherein x is 2, 3, 4, 5, or 6. In some embodiments, the peptide linker L4 comprises an amino acid sequence ( _G4S ) ₂ (SEQ ID NO: 5) or a functional variant thereof.

In some embodiments, VH(CD3) is linked to CH2 via a peptide bond linking L4. In some embodiments, the peptide bond linking L4 comprises an amino acid sequence (G4S)x or a functional variant thereof, wherein x is 2, 3, 4, 5, or 6. In some embodiments, the peptide bond linking L4 comprises an amino acid sequence (G4S)2 (SEQ ID NO: 5) or a functional variant thereof.

In some embodiments, the CH1, CH2, and/or CH3 regions of the bispecific binder described herein include one or more amino acid modifications, particularly substitutions and/or deletions, corresponding to positions of human IgG1 according to EU designation. In some embodiments, the CH1 region on the first and/or second polypeptide chain includes an aspartic acid residue at position 208 according to EU designation. In some embodiments, the CH1 region on the first polypeptide chain includes an aspartic acid residue at position 208 according to EU designation, while the CH1 region on the second polypeptide chain includes an aspartic acid residue at position 208 according to EU designation.

Furthermore, in some embodiments, the binders described herein do not substantially bind (e.g., are undetectable) to human FcγRI, IIa, IIb, and/or IIIa. In some embodiments, the CH2 region on the first and/or polypeptide chain includes an amino acid sequence comprising one or more of the following: a proline residue at position 233, a volate residue at position 234, an alanine residue at position 235, a deletion at position 236, an lysine residue at position 267, and a glutamic acid residue at position 295 (according to EU designation). In some embodiments, the CH2 regions on the first and second polypeptide chains include amino acid sequences comprising: a proline residue at position 233, a volate residue at position 234, an alanine residue at position 235, a deletion at position 236, and an lysine residue (according to EU designation) at position 267, wherein the CH2 region on the first polypeptide chain further includes a glutamate residue (according to EU designation) at position 295, and the CH2 region on the second polypeptide chain further includes a glutamine residue (according to EU designation) at position 295.

In some embodiments, the CH3 region of the first and/or second polypeptide chain includes an amino acid sequence comprising one or more of the following: a glutamic acid 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 designations. In some embodiments, the CH3 region of the first polypeptide chain includes 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 E U-number, and the CH3 region of the second polypeptide chain includes a glutamic acid residue at position 357, a lysine residue at position 364, a leucine residue at position 368, a lysine 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 number.

In some embodiments, the first polypeptide chain includes an amino acid sequence represented by SEQ ID NO: 7.

In some embodiments, the second polypeptide chain includes an amino acid sequence represented by SEQ ID NO: 8.

In some embodiments, the third and/or fourth polypeptide chain includes an amino acid sequence represented by SEQ ID NO: 9.

In some embodiments, CD3 is expressed on the surface of T cells. In some embodiments, the bispecific binder described herein binds to the ε-chain of CD3. In some embodiments, the binding of the binder to CD3 on the surface of T cells leads to T cell proliferation and/or activation. In some embodiments, T cell proliferation and/or activation includes the proliferation and/or activation of CD4 and/or CD8 T cells, preferably CD107a+ T cells. In some embodiments, these proliferating and/or activated T cells have a degranulation effect. In some embodiments, these activated T cells release cytotoxic factors, such as perforin and granzyme, and induce cytolysis and/or apoptosis of cancer cells.

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, a bispecific binder binds to the extracellular portion of CLDN18.2. In some embodiments, the binder induces T cell-mediated cytotoxicity against cancer cells expressing CLDN18.2.

In some embodiments, cancer cells are selected from a group of cancers consisting of gastric cancer (especially gastric adenocarcinoma), esophageal cancer, gastroesophageal junction cancer (GEJ), especially GEJ adenocarcinoma, pancreatic cancer (especially pancreatic cancer), lung cancer (such as 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 their metastases, Crugenburg tumor, peritoneal metastases and/or lymph node metastases.

In some embodiments, the bispecific binding agent includes one or more post-translational modifications. In some embodiments, the bispecific binding agent is derived from one or more post-translational modifications of the 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), lysine deletion at the C-terminus of the first polypeptide chain, and lysine deletion at the C-terminus of the second polypeptide chain.

In some embodiments, the bispecific binding agent is provided in the form of a nucleic acid encoding the binding agent. In some embodiments, the bispecific binding agent is provided in the form of a set of nucleic acids that commonly encode the binding agent. In some embodiments, the nucleic acid or the set of nucleic acids may express the bispecific binding agent.

In some embodiments, the vector comprises the nucleic acid or the group of nucleic acids. In some embodiments, a group of vectors comprises the group of nucleic acids. In some embodiments, each nucleic acid in the group of nucleic acids is included in the vector in the group of vectors. In some embodiments, the vector or the group of vectors may express a bispecific binding agent.

For example, expressing the nucleic acid or group of nucleic acids in an individual, or the vector or group of vectors, can provide the bispecific binding agent.

In some embodiments, nucleic acids are operatively linked to any number of regulatory elements (such as promoters, replication initiation sites, selective markers, ribosome binding sites, inducing agents, etc.). The vector can be a performance vector and can be an extrachromosomal or integrative vector. In some embodiments, the nucleic acids encoding the binding agents described herein are individually contained in a single performance vector. These nucleic acids can be controlled by different or the same promoters. In this embodiment, different vector ratios can be used to drive the formation of the binding agents described herein.

In some embodiments, the agents that stabilize or increase CLDN18.2 performance include chemotherapy agents.

In some embodiments, the agents that stabilize or increase the performance of CLDN18.2 include cytotoxic and/or cell growth inhibitors.

In some embodiments, the agents that stabilize or enhance the performance of CLDN18.2 include the group consisting of anthracycline antibiotics, nucleoside analogs, platinum compounds, camptothecin analogs and taxanes, their prodrugs, their salts, and combinations thereof.

In some embodiments, the drug that stabilizes or enhances CLDN18.2 performance includes nucleoside analogues, their prodrugs, or their salts.

In some embodiments, the drug that stabilizes or enhances CLDN18.2 performance includes paclitaxel derivatives, their prodrugs, or their salts.

In some embodiments, the drug that stabilizes or increases the performance of CLDN18.2 includes (i) gemcitabine, its prodrug, or salts thereof, (ii) paclitaxel, its prodrug, or salts thereof, or (iii) a combination of gemcitabine, its prodrug, or salts thereof, and paclitaxel, its prodrug, or salts thereof.

In some embodiments, the composition or pharmaceutical preparation includes: (i) the bispecific conjugate; (ii) oxaliplatin; (iii) 5-fluorouracil or a precursor thereof; and optionally (iv) folic acid.

In some embodiments, the composition or pharmaceutical formulation further includes pembrolizumab or irinotecan.

In some embodiments, the composition or pharmaceutical formulation includes: (i) the bispecific conjugate; (ii) paclitaxel; and (iii) ramucirumab.

In some embodiments, the composition or pharmaceutical formulation is a pharmaceutical composition. In some embodiments, the pharmaceutical composition further includes 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 binder and the preparation that stabilizes or enhances CLDN18.2 performance are stored separately in vials.

In some embodiments, the composition or pharmaceutical preparation further includes the bispecific binder and instructions for use of the preparation for the treatment or prevention of cancer that stabilize or enhance CLDN18.2 performance.

This invention also provides for the medicinal use of the compositions or pharmaceutical preparations described herein.

In some embodiments, the medical use includes the treatment or prevention of disease or condition.

In some implementations, the treatment or prevention of disease or condition includes the treatment or prevention of cancer.

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

In some embodiments, the cancer is selected from a group consisting of gastric cancer (especially gastric adenocarcinoma), esophageal cancer, gastroesophageal junction cancer (GEJ) (especially gastroesophageal junction adenocarcinoma), pancreatic cancer (especially pancreatic cancer), lung cancer (such as 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 its metastases, Crugenburg tumor, peritoneal metastases and/or lymph node metastases.

In some embodiments, the compositions or pharmaceutical preparations described herein are intended for administration to humans.

The present invention also provides a method for treating or preventing cancer in an individual, comprising administering to an individual: (a) a bispecific binding agent described herein; and (b) an agent that stabilizes or increases CLDN18.2 performance.

In some embodiments, the bispecific binding agent is a bispecific binding agent in the composition or pharmaceutical preparation described herein. In some embodiments, the agent that stabilizes or increases CLDN18.2 performance is an agent that stabilizes or increases CLDN18.2 performance as described herein, such as in the composition or pharmaceutical preparation described in the context herein.

In some implementations, this system is human.

This invention also provides the compositions or pharmaceutical preparations described herein for use in the methods described herein.

In some embodiments, CLDN18.2 preferably has an amino acid sequence such as SEQ ID NO: 1.

In some embodiments, the first polypeptide chain does not include VL (CLDN18.2). In some embodiments, the second polypeptide chain does not include VL (CLDN18.2). In some embodiments, the third polypeptide chain does not include VH (CLDN18.2). In some embodiments, the fourth polypeptide chain does not include VH (CLDN18.2).

In some embodiments, the CLDN18.2 binding domain of the bispecific binding agent is in the form of a Fab fragment. In some embodiments, the CD3 binding domain of the bispecific binding agent is in the form of an scFv fragment.

In some embodiments, the bispecific binder does not bind to CLDN18.1. In some embodiments, the bispecific binder does not bind to CLDN18.1 in humans, mice, or cynomolgus monkeys. In some embodiments, the bispecific binder does not bind to CLDN9, such as human CLDN9.

In some embodiments, the bispecific binder binds to CLDN18.2 of more than one species, such as human, mouse and cynomolgus monkey CLDN18.2.

In some embodiments, treatment of an individual (e.g., a patient) with the bispecific binding agent described herein and an agent that stabilizes or increases CLDN18.2 expression prolongs that individual's survival. In some embodiments, treatment of patients with the bispecific binding agent described herein and an agent that stabilizes or increases CLDN18.2 expression reduces, preferably significantly reduces, tumor growth and/or volume in an individual (e.g., a patient).

In some embodiments, the bispecific binder described herein can redirect T cells to attack cancer cells, thus exerting its effect through redirected T cytotoxicity (RTCC). In some embodiments, the bispecific binder cannot or substantially cannot induce ADCC. In some embodiments, the binder cannot or substantially cannot induce CDC.

In some embodiments, the bispecific binding agent is manufactured by a method comprising transfecting a host cell with a nucleic acid, a set of nucleic acids, a vector, or a set of vectors encoding a bispecific binding agent polypeptide chain. In some embodiments, the host cell expresses the nucleic acid, the set of nucleic acids, the vector, or the set of vectors. In some embodiments, the host cell co-expresses a nucleic acid encoding a first polypeptide chain, a nucleic acid encoding a second polypeptide chain, a nucleic acid encoding a third polypeptide chain, and a nucleic acid encoding a fourth polypeptide chain of the bispecific binding agent. In some embodiments, the nucleic acid is contained in a vector or a set of vectors. In some embodiments, the host cell expresses the entire polypeptide chain of the bispecific binding agent. In some embodiments, the transfected host cells preferably produce the bispecific binder under suitable binder-producing conditions, such as those described herein or known in the art. In some embodiments, the bispecific binder may be obtained from the host cells.

Therefore, in some embodiments, a method for manufacturing a bispecific binding drug includes the following steps: transfecting a nucleic acid encoding a first polypeptide chain of the bispecific binding drug, a nucleic acid encoding a second polypeptide chain of the bispecific binding drug, a nucleic acid encoding a third polypeptide chain of the bispecific binding drug, and a nucleic acid encoding a fourth polypeptide chain of the bispecific binding drug into a host cell, expressing these nucleic acids in the host cell, and obtaining the bispecific binding drug. In some embodiments, the host cell is a mammalian cell, preferably selected from a population including CHO cells, BHK cells, HeLa cells, COS cells, HEK293 cells, and HEK293 T cells. In some embodiments, the host cell is a bacterial cell, yeast cell, fungal cell, plant cell, or insect cell. In some embodiments, the conjugated drug is manufactured in vitro. In some embodiments, the conjugated drug is manufactured in vivo, for example, in an individual requiring treatment, such as someone with a disease, particularly a disease associated with cells expressing CLDN18.2 (such as cancer). In some embodiments, the different polypeptide chains of the bispecific binding agent are manufactured in two or more different host cells. In some embodiments, the entire polypeptide chain of the bispecific binding agent is manufactured in the same host cell.

In some embodiments, the polypeptide chains of the bispecific binder are linked to each other, for example, covalently. In some embodiments, the polypeptide chains of the bispecific binder are manufactured as a single polypeptide comprising all polypeptide chains of the bispecific binder. In some embodiments, at least two polypeptide chains of the bispecific binder are linked together and manufactured as a single polypeptide. In some embodiments, the polypeptide chains of the bispecific binder are manufactured separately, i.e., as separate polypeptides, for example, in the same or different cells, and interact during or after manufacturing to form the bispecific binder, for example, intracellularly or extracellularly. In some embodiments, the polypeptide chains are generated as separate polypeptides, namely, a first polypeptide chain as a polypeptide, a second polypeptide chain as a polypeptide, a third polypeptide chain as a polypeptide, a fourth polypeptide chain as a polypeptide, and these polypeptide chains interact to form a bispecific binder.

Other features and advantages of this invention will become apparent from the following detailed description and claims.

Although the present invention will be described in detail below, it should be understood that the invention is not limited to the specific methods, protocols, and reagents described herein, as variations are possible. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to limit the scope of the invention to 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.

The requirements of this invention will be described below. These requirements list specific embodiments, but it should be understood that they can be combined in any way and in any number to create more embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This specification should be understood to support and include embodiments that combine the explicitly described embodiments with any number of disclosed and/or preferred requirements. Furthermore, unless the context otherwise requires, any permutation and combination of all requirements described in this application shall be deemed to be disclosed in this specification.

Preferably, the terms used in this article should be understood according to the definitions in the Multilingual Biotechnology Terminology Dictionary (IUPAC Recommendation), edited by H.G.W. Leuenberger, B. Nagel and H. Kölbl, Helvetic Chimica Acta, Basel, Switzerland, CH-4010 (1995).

The implementation of this invention will employ conventional chemical, biochemical, cell biological, immunological, and recombinant DNA techniques, all of which are described in detail in the literature in this field (see, for example, Molecular Cloning: A Laboratory Manual, 2nd edition, J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

In this specification and the following claims, unless the context otherwise requires, the word "comprising" and its variations such as "comprising" and "including" shall be understood to include the listed components, integers, or steps, or combinations of components, integers, or steps, but do not exclude the presence of other components, integers, or steps. In some embodiments, other components, integers, or steps may be excluded, i.e., the subject matter is to include the listed members, integers, or steps, or combinations of members, integers, or steps. As a specific embodiment of this disclosure, the word "comprising" also includes the possibility that no other components are present, i.e., in this embodiment, "comprising" shall be understood as "consisting of" or "substantially consisting of".

In describing this invention (particularly in the context of the claim), the use of terms such as “a,” “an,” and “the” should be understood to cover both the singular and plural forms, unless otherwise specified herein or clearly inconsistent with the context.

The range of values mentioned herein is only a brief, individual reference to each value within the range. Unless otherwise specified herein, each individual value is included in the specification as if individually mentioned.

Unless otherwise specified herein or clearly inconsistent with the context, all methods described in the manual may be performed in any suitable order.

All examples or illustrative language provided herein (e.g., "such") are provided only to better illustrate the invention and are not intended to limit the scope of the invention. Nothing in this specification should be construed as indicating that any requirement not claimed in the claims is necessary for the implementation of the invention.

In some embodiments, “about” means approximately or close to, and in the context of numerical values or ranges, it means ±20%, ±10%, ±5%, or ±3% of the numerical value or range mentioned or requested.

This specification cites numerous references (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.). All such references, whether mentioned above or below this document, are incorporated herein by reference in their entirety. Nothing in this specification should be construed as an endorsement that this invention could not have been published before the date of such prior art. Bispecific binder

The first target molecule of the bispecific binding agent described in this article is CLDN18.2.

Neminins are a class of proteins and the most important components of tight junctions, forming paracellular barriers between cells and controlling the flow of molecules between epithelial cells. Neminins are transmembrane proteins, crossing the membrane four times, with their N-terminus and C-terminus located in the cytoplasm. The first outer loop (called EC1 or ECL1) consists of an average of 53 amino acids, while the second outer loop (called EC2 or ECL2) consists of approximately 24 amino acids. Cell surface proteins in the neminin family, such as CLDN18.2, are present in tumors of multiple origins. Due to their selective expression (absence in toxic-associated normal tissues) and location on the cell membrane, they are particularly well-suited as target structures for antibody-mediated cancer immunotherapy.

CLDN18.2 has been identified as differentially expressed in tumor tissues, with the stomach being the only normal tissue to express CLDN18.2. 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, particularly gastric cancer, especially gastric adenocarcinoma, esophageal cancer, gastroesophageal junction (GEJ) cancer, especially GEJ adenocarcinoma, pancreatic cancer, especially pancreatic cancer, lung cancer such as 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 their metastatic cancers, especially gastric cancer metastases such as Kukenberg tumor, peritoneal metastases and lymph node metastases. The term "CLDN18" or "close-linked protein 18" refers to close-linked protein 18, including any variants, including close-linked protein 18 splice variant 1 (close-linked protein 18.1 (CLDN18.1)) and close-linked protein 18 splice variant 2 (close-linked protein 18.2 (CLDN18.2)).

The terms "claudin 18.2" or "CLDN18.2" preferably refer to human CLDN18.2, and more particularly to proteins comprising the amino acid sequence listed in SEQ ID NO: 1 or variations thereof, or composed thereof. The first extracellular loop of CLDN18.2 preferably comprises amino acid sequences from positions 27 to 81, more preferably from positions 29 to 78. The second extracellular loop of CLDN18.2 preferably comprises amino acid sequences from positions 140 to 180 or from positions 144 to 167. The first and second extracellular loops preferably constitute the extracellular portion of CLDN18.2. Generally, the antibodies described herein are capable of binding to the extracellular portion of human CLDN18.2.

The second target molecule of the bispecific binding agent described in this article is CD3 (differentiation group 3), particularly CD3ε. The CD3 complex refers to an antigen expressed on mature T cells, thymocytes, and a group of natural killer cells as part of the multimolecular T cell receptor (TCR) complex. The T cell receptor is a protein complex composed of four distinct chains. In mammals, this complex includes the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains bind to a molecule called the T cell receptor (TCR) and the ζ chain, generating activation signals in T lymphocytes. The TCR, the ζ chain, and the CD3 molecule together constitute the TCR complex.

Human CD3ε is accessed in GenBank under the number NM_000733. Human CD3γ is accessed in GenBank under the number NM_000073. Human CD3δ is accessed in GenBank under the number NM_000732. CD3 is responsible for TCR signaling. As described by Lin and Weiss in the Journal of Cell Science, Vol. 114, pp. 243-244 (2001), the TCR complex is activated by binding to specific antigenic epitopes presented by the MHC, which leads to phosphorylation of immune receptor tyrosine activating motifs (ITAMs) by Src family kinases, thereby triggering the recruitment of more kinases and ultimately leading to T cell activation, including the release of Ca2 ⁺ . CD3 accumulation on T cells, for example through immobilized anti-CD3 antibodies, leads to T cell activation, which is independent of the type specificity of T cell receptor selection.

As used herein, "CD3" includes human CD3, referring to an antigen expressed on human T cells as part of a multimolecular T cell receptor complex. Regarding CD3, the binding agent described in this invention preferably recognizes the ε-chain of CD3. In some embodiments, it recognizes an epitope corresponding to the first 27 amino terminal amino acids of the CD3 ε-chain, or a functional segment of these 27 amino acid segments.

According to the present invention, "CLDN18.2 positive cancer" or similar terms refer to cancers involving the expression of CLDN18.2 on cancer cells, preferably cancers whose surface expresses CLDN18.2.

"Cell surface" is used in the conventional sense in this field and therefore includes extracellular regions that can bind to proteins and other molecules.

If CLDN18.2 is located on the cell surface and can bind to newly added CLDN18.2-specific antibodies, then CLDN18.2 is considered to be expressed on the cell surface.

If CD3 is located on the cell surface and can bind to newly added CD3-specific antibodies, then CD3 is considered to be expressed on the cell surface.

In the context of this invention, "extracellular portion" refers to the portion of a molecule (such as a protein) that faces the extracellular space, and preferably this portion can be contacted from outside the cell by, for example, antigen-binding molecules (such as antibodies). Preferably, the term refers to one or more extracellular loops or regions, or segments thereof.

The terms "part" and "fragment" are used interchangeably herein to refer to any portion of a structure (such as an amino acid sequence) smaller than the complete structure. In some embodiments, a part of a structure refers to a continuous component of that structure. In some embodiments, a part of a structure refers to more than one continuous component in the structure that can be linked. A part, portion, or fragment of a structure preferably includes one or more functional features of the structure. A part or fragment of an amino acid sequence preferably includes 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 continuous amino acids in a protein sequence.

In the context of amino acid sequences (peptides or proteins), a "fragment" refers to a portion of an amino acid sequence, specifically a truncated amino acid sequence at the N-terminus and/or C-terminus. A C-terminal truncated fragment (N-terminal fragment) can be obtained by transcribing a truncated open reading frame lacking the 3' end. An N-terminal truncated fragment (C-terminal fragment) can be obtained by transcribing a truncated open reading frame lacking the 5' end, provided that the truncated open reading frame includes a start codon to initiate transcribing. An amino acid sequence fragment may, for example, comprise 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.

The term "variant" or similar expression as used in this document refers to a sequence that has at least one amino acid modification compared to the parent amino acid sequence. The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or a modified version of the wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, for example, from 1 to about 20 amino acid modifications, and more preferably from 1 to about 10 or from 1 to about 5 amino acid modifications compared to the parent.

In this article, "wild-type," "WT," or "natural" are used to refer to amino acid sequences found in nature, including allelic variations. Wild-type amino acid sequences, peptides, or proteins refer to sequences that have not been intentionally modified.

In this disclosure, "variant" refers to an insertion, addition, deletion, and/or substitution variant of an amino acid sequence (peptide, protein, or polypeptide). The term "variant" includes all mutants, splice variants, post-translational modifications, conformations, allelic variants, species variants, and species homologs, especially naturally occurring variants. Specifically, the term "variant" includes segments of amino acid sequences.

Amino acid insertion variants involve inserting one or two or more amino acids into a specific amino acid sequence. In variants with inserted amino acid sequences, one or more amino acid residues can be inserted at a specific position in the amino acid sequence, although random insertion and appropriate screening of the resulting product are also possible.

New amino acid variants include fusions of amino and/or carboxyl terminals, such as adding 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, such as the removal of amino acids 1, 2, 3, 5, 10, 20, 30, 50, or more. These deletions can occur at any location in a protein. If the deletion occurs at the N-terminus and/or C-terminus of a protein, this amino acid deletion variant is also called an N-terminal and/or C-terminal truncated variant.

Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and its replacement by another residue. Preferably, these modifications occur at non-conserved positions between homologous proteins or peptides, and/or are replaced by other amino acids with similar properties. Preferably, the amino acid changes in peptide and protein variants are conserved amino acid changes, i.e., the substitution of an amino acid with the same charge or without a charge. Conserved amino acid changes involve the substitution of amino acids from the same family that are similar in their side chains.

In the context of this invention, conservative substitutions can be defined by the amino acid categories reflected in the following table: acidic residues Asp(D) and Glu(E) alkaline residues Lys(K), Arg(R), and His(H) Hydrophilic uncharged residue Ser(S), Thr(T), Asn(N), and Gln(Q) Aliphatic uncharged residues Gly(G), Ala(A), Val(V), Leu(L), and Ile(I) Nonpolar uncharged residue Cys(C), Met(M), and Pro(P) Fangzu uncharged residue Phe(F), Tyr(Y), and Trp(W)

Preferably, the similarity, or identity, between the given amino acid sequence and a variant thereof 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%. In this regard, the term "at least 80% sequence identity" as used herein includes amino acid sequences that have at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the given amino acid sequence. The degree of similarity or identity preferably refers to 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 full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably evaluated for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 consecutive amino acids. In some embodiments, the degree of similarity or identity is evaluated for the full length of the reference amino acid sequence. For determining sequence similarity, preferably sequence identity alignment, tools known in the art can be used, preferably the best sequence alignment tool, such as Align with standard settings, or preferably EMBOSS::needle, matrix: Blosum62, gap open: 10.0, gap extension: 0.5.

"Sequence similarity" refers to the percentage of amino acids that are the same or represent conserved amino acid substitutions in two amino acid sequences. "Sequence consistency" between two amino acid sequences refers to the percentage of amino acids that are exactly the same in the two sequences. "Sequence consistency" between two nucleic acid sequences refers to the percentage of nucleotides that are exactly the same in the two sequences.

"% identical," "% identical," or similar terms refer to the percentage of identical nucleotides or amino acids in two sequences to be compared after optimal alignment. This percentage is only a statistical value; differences between two sequences may, but are not necessarily, randomly distributed across the entire sequence length. Comparisons of two sequences are typically performed after optimal alignment, in units of a segment or "comparison window," to identify regions of corresponding sequences. The best alignment can be performed manually, or using the local homology calculus algorithms described by Smith and Waterman in *Ads App. Math.*, Vol. 2, p. 482 (1981), Needleman and Wunsch in *J. Mol. Biol.*, Vol. 48, p. 443 (1970), and Pearson and Lipman in *Proceedings of the National Academy of Sciences*, Vol. 88, p. 2444 (1988), or using computer programs of these calculus algorithms (such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetic Software Package). In some embodiments, the percentage identity of two sequences is determined using the BLASTN or BLASTP algorithm available on the National Center for Biotechnology Information (NCBI) website (e.g., accessible at blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expected threshold of 10; (ii) a word size of 28; (iii) a maximum number of matches within the query range of 0; (iv) match/non-match scores of 1 and -2; (v) linear interval costs; and (vi) the use of a low-complexity region filter. In some implementations, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) the expected threshold is set to 10; (ii) the word size is set to 3; (iii) the maximum number of matches in the query range is set to 0; (iv) the matrix is set to BLOSUM62; (v) the interval cost is set to Existence: 11 and Extension: 1; and (vi) conditional component number adjustment is performed.

The percentage of consistency is obtained by determining the number of identical positions between the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and then multiplying the result by 100.

In some embodiments, the similarity or identity percentage is given for a region comprising 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 full length of the reference sequence. For example, if the reference nucleotide sequence consists of 200 nucleotides, the identity is given for at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or about 200 consecutive nucleotides. In some embodiments, the similarity or identity percentage is given for the full length of the reference sequence.

According to this disclosure, the homologous amino acid sequence exhibits at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and more preferably at least 95%, at least 98%, or at least 99% similarity to the amino acid residues of the reference sequence.

The amino acid sequence variants described in this disclosure can be easily prepared by those skilled in the art, for example, by manipulating recombinant DNA. Detailed methods for manipulating DNA sequences to prepare polypeptides or proteins with substitutions, additions, insertions, or deletions can be found in the work of Sambrook et al. (1989). Furthermore, the polypeptides and amino acid variants described in this disclosure can also be easily prepared using known polypeptide synthesis techniques, such as solid-phase synthesis.

In some embodiments, fragments or variants of amino acid sequences (peptides or proteins) are preferably “functional fragments” or “functional variants.” As used herein, a “functional fragment” or “functional variant” refers to any fragment or variant that exhibits the same or similar functional properties as its derived amino acid sequence, i.e., a functional equivalent. For antigen-binding domains including functional VH and VL variants, a specific function is the retention of the binding capacity of the binding domain. As used herein, “functional fragment” or “functional variant” specifically refers to a molecule or sequence that includes one or more amino acid changes compared to the parent molecule or sequence, and still performs one or more or all of the functions of the parent molecule or sequence, such as forming a binding domain for a specific antigen. For example, binding domains including functional VH and VL variants or functional CDR variant sequences have the same or similar binding properties compared to the parent molecule. In some embodiments, modifications to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the properties of the molecule or sequence. In different embodiments, the properties of molecules including functional fragments or functional variants, such as the binding strength of binding domains, may be reduced but still significantly present; for example, the binding properties of binding domains including functional variants may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of those of the parent molecule or sequence. For example, functional variants may have 1, 2, 3, 4, 5, or more amino acid insertions, additions, substitutions, and/or deletions compared to the parent molecule. However, in other embodiments, the properties of molecules including functional variants or functional fragments, such as the binding properties of binding domains including functional fragments or functional variants, may be enhanced compared to the parent molecule. In some embodiments, a “functional variant” refers to a “functional fragment”, for example, whose N-terminus and/or C-terminus are shortened compared to the parent molecule, but which still retains or substantially retains one or more or all of the functions of the parent molecule, as described above, and in particular, a parent molecule functional equivalent.

The term "functional variant of an amino acid sequence" includes the "functional" segment of the amino acid sequence.

An amino acid sequence (peptide, protein, or polypeptide, such as VH, VL, CH1, CH2, or CH3) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide, such as VH, VL, CH1, CH2, or CH3) refers to the source of the first amino acid sequence. Preferably, the amino acid sequence derived from a specific amino acid sequence has the same, substantially the same, or homologous amino acid sequence as that specific sequence or a fragment thereof. The amino acid sequence derived from a specific amino acid sequence may be a variant of that specific sequence or a fragment thereof, preferably a functional variant described herein, including functional fragments. For example, those skilled in the art will understand that suitable amino acid sequences used herein can be modified to differ sequentially from their naturally occurring or native sequences, including amino acid insertion, deletion, addition, and/or substitution, while maintaining or substantially maintaining 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 chain of the conjugates described herein are derived from the amino acid sequences of the VH, VL, CH1, CH2, and/or CH3 domains of immunoglobulins, but may differ from their source domains. For example, according to the present invention, the amino acid sequences included in the VH or VL derived from immunoglobulins may be identical to the amino acid sequences of their source VH or VL, or differ from their parental VH or VL sequences at one or more positions. For example, the VH domain of the conjugates described herein may include one or more amino acid insertions, additions, deletions, and/or substitutions compared to the amino acid sequence of their source VH domain. For example, the VL domain of the conjugates described herein may include one or more amino acid insertions, additions, deletions, and/or substitutions compared to the amino acid sequence of their source VL domain. Preferably, a VH or VL having a functional variant of the parental VH or VL amino acid sequence can provide the same or substantially the same function as the parental VH or VL amino acid sequence, such as in binding specificity, binding strength, etc. However, those skilled in the art will understand that in some embodiments, it is preferred to provide an amino acid sequence, such as a VH or VL functional variant, that has different properties compared to the amino acid sequence of the parent molecule. The same consideration also applies to, for example, CDR amino acid sequences, and other amino acid sequences such as CH1, CH2, CH3 and/or CL domains.

When describing a bispecific binding agent comprising a VH "derived from" an immunoglobulin and a VL "derived from" the same or different immunoglobulins, the term "derived from" indicates that the bispecific binding agent is generated by recombinating the VH and VL of the immunoglobulins using any known method to produce a bispecific antibody. In this context, "recombination" is not limited to any particular recombination method and therefore includes all methods of producing bispecific binding agents described herein or known in the art, such as recombination at the nucleic acid level and/or recombination of molecules that co-express different molecules in the same cell.

In this specification, the term "bispecific" used to describe agents such as antibodies, antibody derivatives, or any other drugs refers to an agent having two distinct antigen-binding domains defined by different amino acid sequences. In some embodiments, these distinct antigen-binding domains bind to different epitopes on the same antigen, or, as discussed below, to the same epitope on the same antigen. However, in preferred embodiments, these distinct antigen-binding domains bind to different target antigens. The binding agent can bind to each distinct antigen or epitope by one, two, or more binding domains, i.e., monovalent, bivalent (or divalent), trivalent, tetravalent, or even higher valence states. For example, in the case of trivalent bispecific antibodies, two binding domains bind to one target (which may be the same epitope or different epitopes), while the other binding domain binds to a second target.

If the expression level is lower than that in gastric cells or gastric tissue, CLDN18.2 is not substantially expressed. Preferably, the expression level is lower than 10% in gastric cells or gastric tissue, more preferably lower than 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05%, or even lower. Preferably, if the expression level is no more than 2-fold, more preferably 1.5-fold, and more preferably no more than the expression level in non-cancerous tissue outside the stomach, CLDN18.2 is not substantially expressed. Preferably, if the expression level is lower than the detection limit and/or if the expression level is too low to allow the newly added CLDN18.2-specific antibody to bind to cells, CLDN18.2 is not substantially expressed.

CLDN18.2 is expressed in the cell if its expression level is more than 2-fold, preferably 10-fold, 100-fold, 1000-fold, or 10000-fold compared to non-cancerous tissue outside the stomach. Preferably, CLDN18.2 is expressed in the cell if its expression level is above the detection limit and/or sufficiently high to allow newly generated CLDN18.2-specific antibodies to bind to the cell. Preferably, CLDN18.2 expressed in the cell is expressed on or exposed on the cell surface.

According to this invention, the term "disease" refers to any pathological condition, including cancer, especially the form of cancer described herein. Any reference to cancer or a particular form of cancer in this invention also includes its metastasis. In a preferred embodiment, the disease treated according to this teaching involves cells expressing CLDN18.2.

"Diseases involving cells expressing CLDN18.2," "diseases associated with cells expressing CLDN18.2," or similar expressions, as used herein, refer to the expression of CLDN18.2 in cells of diseased tissues or organs. In some embodiments, the expression level of CLDN18.2 in diseased tissues or organs is increased by at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or more than that in healthy tissues or organs. In some embodiments, expression is found only in diseased tissues and is suppressed in healthy tissues. According to the present invention, diseases associated with cells expressing CLDN18.2 include cancer. According to the present invention, the preferred cancer is cancer cells that express CLDN18.2.

As used in this invention, "cancer" or "carcinoma" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and/or migration. "Cancer cells" are defined as abnormal cells that proliferate and grow rapidly and uncontrolledly, continuing to grow even after the initial stimulus has ceased. Preferably, "cancer" is characterized by cells expressing CLDN18.2, and cancer cells express CLDN18.2. Cells expressing CLDN18.2 are preferably cancer cells, especially the cancers described herein.

According to this invention, "cancer" 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, colorectal cancer, stomach cancer, intestinal cancer, head and neck cancer, bile duct cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, gastroesophageal junction cancer (GEJ), colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, and lung cancer and their metastases. For example, lung cancer, breast cancer, prostate cancer, colon cancer, kidney cancer, cervical cancer, or metastases of the above-mentioned cancer types or tumors. According to this invention, cancer terminology also includes cancer metastasis.

According to this invention, "cancer" is a malignant tumor that originates from epithelial cells. This group of cancers is the most common type of cancer, including common breast cancer, prostate cancer, lung cancer, and colon cancer.

Adenocarcinoma is a type of cancer that originates from glandular tissue, which falls within the larger category of epithelial tissue. Epithelial tissue includes the skin, glands, and various other tissues along body cavities and organs. Epithelial cells are embryologically derived from the ectoderm, endoderm, and mesoderm. To be classified as adenocarcinoma, cells do not necessarily have to be glandular, as long as they have a secretory function. This type of cancer can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas usually resemble the glandular tissue from which they originate, while undifferentiated ones may not. By staining biopsy cells, pathologists can determine whether a tumor is adenocarcinoma or another type of cancer. Due to the ubiquity of glands in the body, adenocarcinoma can occur in multiple tissues throughout the body. Although each gland secretes different substances, any cell with exocrine function is considered glandular tissue, and its malignant form is called adenocarcinoma. Malignant adenocarcinoma can invade other tissues and may metastasize within a sufficient time. Ovarian adenocarcinoma is the most common type of ovarian cancer, and it includes serous and mucinous adenocarcinoma, clear cell adenocarcinoma, and endometrial adenocarcinoma.

"Metastasis" refers to the spread of cancer cells from their primary site to other parts of the body. The formation of metastasis is a highly complex process, depending on the detachment of malignant cells from the primary tumor, their invasion of the extracellular matrix, their penetration of the endothelial basement membrane into body cavities and blood vessels, and their subsequent infiltration into the target organ via the bloodstream. Finally, the growth of the new tumor at the target site depends on angiogenesis. Even after the removal of the primary tumor, metastasis can still occur because tumor cells or components may remain and develop metastatic potential. In some embodiments, the term "metastasis" in this invention refers to "distant metastasis," that is, metastasis that is distant from the primary tumor and the regional lymphatic system. In some embodiments, the term "metastasis" in this invention refers to lymph node metastasis. The treatment methods or approaches of this invention are particularly suitable for metastatic forms originating from the primary site of gastric cancer. In preferred embodiments, such gastric cancer metastasis includes Clukenberg's tumor, peritoneal metastasis, and/or lymph node metastasis.

Clukenberg's disease is a rare metastatic ovarian tumor, accounting for 1% to 2% of all ovarian tumors. The prognosis for Clukenberg's disease remains very poor, and there is currently no established treatment. Clukenberg's disease is a metastatic cyclic adenocarcinoma of the ovary. In most cases of Clukenberg's disease (approximately 70%), the stomach is the primary site. The colon, appendix, and breast (primarily invasive lobular carcinoma) are the next most common primary sites. In addition, there are rare cases reported where Clukenberg's tumors originate from the gallbladder, bile ducts, pancreas, small intestine, duodenal papilla, cervix, and bladder or urethra.

"Treatment" refers to the administration of a compound or combination of compounds or combinations to an individual to prevent, alleviate or eliminate disease, including reducing the size or number of tumors in an individual; stopping or slowing the progression of disease in an individual; inhibiting or slowing the development of new disease in an individual; reducing the frequency and/or severity and recurrence of symptoms in an individual who currently has or previously had a disease; and/or prolonging the lifespan of an individual, i.e., increasing their lifespan.

Specifically, “treatment of disease” includes curing, shortening the duration of disease, relieving, preventing, slowing or inhibiting the progression or worsening of disease, or preventing or delaying the onset of disease or its symptoms.

In the context of this invention, terms such as “protection,” “prevention,” “preventive,” “protective,” or “protective” are related to the prevention or treatment of the occurrence and/or spread of disease in an individual, particularly to minimizing the individual’s likelihood of developing the disease or delaying its development. For example, individuals at higher risk of cancer would be candidates for preventative cancer treatment.

"At risk" refers to individuals identified as having a higher chance of developing a certain disease, especially cancer, compared to the general population. Furthermore, individuals who have had or currently have a certain disease, particularly cancer, are at increased risk because they are more likely to develop the disease. Individuals who currently have or have had cancer also have a higher risk of cancer metastasis.

The terms “individual” and “subject” as used herein are used interchangeably. They refer to a person or other mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) who may have or be susceptible to a disease or condition (e.g., cancer), but may not have the disease or condition. Unless otherwise stated, the terms “individual” and “subject” do not refer to a specific age and therefore include adults, the elderly, children, and newborns. In several embodiments disclosed herein, “individual” and “subject” refer to a “patient.”

The term "patient" in this invention refers to a subject receiving treatment, especially a subject suffering from a disease, including humans, non-human primates or other animals, particularly mammals such as cattle, horses, pigs, sheep, goats, dogs, cats, or rodents such as mice and rats. In a preferred embodiment, "patient" refers to humans.

"Target cells" refers to any unwanted cells, such as cancer cells. In a preferred embodiment, target cells exhibit CLDN18.2.

In this invention, "activation" or "stimulation" refers to a state in which immune effector cells (e.g., T cells) have been adequately stimulated to induce detectable cell proliferation. Activation can also be associated with the initiation of signaling pathways, induction of cytokine production, and detectable effector function. The term "activated immune effector cells" includes immune effector cells undergoing cell division.

The term "primitive immunization" as used in this invention refers to the process by which immune effector cells (such as T cells) are first exposed to their specific antigens and thus differentiate into effector cells (such as effector T cells).

The term "selective proliferation" or "proliferation" as used in this invention refers to an increase in the number of a specific organism. In the context of this disclosure, this term is preferably used to describe an immune response in which immune effector cells proliferate upon stimulation by an antigen, amplifying specific immune effector cells that recognize that antigen. Preferably, selective proliferation leads to the differentiation of immune effector cells.

As used in this invention, "interaction" refers to a physical bond between two molecular species (such as two polypeptide chains or portions thereof). This bond, referred to as an interaction, can involve non-covalent and/or covalent interactions, preferably non-covalent interactions, such as charge-charge interactions, charge-dipole interactions, dipole-dipole interactions, van der Waals forces, hydrogen bonds, and/or hydrophobic interactions.

As used herein, "binding" or "binding force" refers to a non-covalent interaction with a target molecule. In some embodiments, "binding" or "binding force" refers to specific binding. As used herein, "specific binding" or "specifically binding" refers to a molecule (such as an antibody) that recognizes a particular target molecule but does not actually recognize or bind to other molecules in a sample or individual. For example, an antibody that specifically binds to an antigen may also bind to the same antigen in one or more other species. However, this cross-species cross-reactivity does not change the specificity classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, this cross-reactivity itself does not change the specificity classification of the antibody.

In some cases, the terms "specifically binding" or "specifically binding" can be used to describe the interaction between an antibody, protein, or peptide and a second chemical substance, meaning that the interaction depends on the presence of a specific structure on the chemical substance (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure, rather than a protein in general. If an antibody specifically binds to epitope "A," then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A binding to the antibody.

This disclosure provides binding molecules and binding agents that bind CLDN18.2 and/or CD3. Such binding molecules and binding agents can form complexes with CLDN18.2 and/or CD3.

As used herein, "binding agent" refers to any agent capable of binding to a desired antigen. Binding agents may also include synthetic, modified, or non-naturally occurring portions. For example, these portions may link to a desired antigen-binding function or region, such as an antibody or antibody fragment. In some embodiments, a binding agent is a synthetic construct including an antigen-binding CDR or variable region. In some embodiments, the binding agents disclosed herein include bispecific or multispecific binding agents, such as bispecific antibody-derived binding agents, which include a first binding domain and a second binding domain, wherein the first binding domain is capable of binding CLDN18.2 and the second binding domain is capable of binding CD3.

As used herein, the term "binding domain" or "antigen-binding domain" refers to any region, group, functional group, or domain that interacts with an antigen. In some embodiments, the term "binding domain" or "antigen-binding domain" refers to the site in the conjugate described herein that binds to an antigen, including the antigen-binding portion of the conjugate. In some embodiments, the binding domain is or includes an antibody, an antibody fragment, or any other binding protein, or a combination thereof. The binding domain may consist of heavy chain variable domains and light chain variable domains (VH and VL), each domain comprising four conserved frame regions (FRs) and three cored receptors (CDRs). The CDRs have distinct sequences and determine specificity for a particular antigen. The VH and VL domains together may form binding sites, for example, sites that specifically bind to a particular antigen. The term "binding domain" for an antigen refers to a binding domain, such as the binding domain of the binder described herein, that can bind to the antigen and bind to it specifically and preferably.

According to the present invention, an agent such as an antibody is considered to bind to a predetermined target if it has a significant affinity for that target and can bind to the target in a standard test. "Affinity" or "binding affinity" is typically measured by the equilibrium dissociation constant ( _KD ). Preferably, the term "significant affinity" refers to a binding dissociation constant ( _KD ) of ^10⁻⁵ M or lower, ^10⁻⁶ M or lower, ^10⁻⁷ M or lower, ^10⁻⁸ M or lower, ^10⁻⁹ M or lower, ^10⁻¹⁰ M or lower, ^10⁻¹¹ M or lower, or ^10⁻¹² M or lower.

If a drug cannot (substantially) bind to a target, it has no significant affinity for that target and will not bind significantly to the target in standard tests, in particular, its binding to the target will not be detected. Preferably, when the drug concentration is as high as 2, preferably 10, more preferably 20, especially 50 or 100 µg/ml or higher, the drug will not be detected as binding to the target. Preferably, the drug has no significant affinity for the target if its _KD value for binding to the target is at least 10, 100, ^10³ , ^10⁴ , ^10⁵ , or ^10⁶ times that for binding to a pre-defined _, bindable target. For example, if the binding _KD value of a drug to a target it can bind to is ^10⁻⁷ M, then the binding _KD value of the drug to a target to which it has no significant affinity should be at least ^10⁻⁶ M, ^10⁻⁵ M, ^10⁻⁴ M, ^10⁻³ M, ^10⁻² M, or ^10⁻¹ M.

A conjugating agent (such as an antibody) is considered specific to the predetermined target if it can bind to the target but not to other targets; that is, it has no significant affinity for other targets in standard tests and does not significantly bind to other targets. According to the present invention, if a conjugating agent can bind to CLDN18.2 but cannot (or substantially cannot) bind to other targets, the conjugating agent is considered specific to CLDN18.2. Preferably, a conjugating agent is considered specific to CLDN18.2 if its affinity and binding capacity to other targets do not significantly exceed that to proteins unrelated to CLDN18.2 (such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or transmembrane proteins of non-closing proteins, such as MHC molecules or transferrin receptors, or any other specified polypeptides). More preferably, a conjugating agent is considered specific to the predetermined target if its binding affinity (K_{_D} ) to the target is at least 10, 100, 10³, 10 ^⁴ , 10^⁵, or 10 ^⁶ lower than its affinity for non-specific targets. For example, if a binding affinity ( _KD ) of a binding agent to its specific target is ^10⁻⁷ mol, then its binding affinity to non-specific targets should be at least ^10⁻⁶ mol, ^10⁻⁵ mol, ^10⁻⁴ mol, ^10⁻³ mol, ^10⁻² mol, or ^10⁻¹ mol.

The term " _kd " ( ^sec⁻¹ ) used in this invention refers to a specific dissociation rate constant for antibody-antigen interactions. This value is also known as the _koff value.

The term " _KD " (M) used in this invention refers to a specific dissociation equilibrium constant of antibody-antigen interactions.

The binding of the binder to the target can be experimentally determined by any suitable method; for example, see Berzofsky et al., “Antibody-Antigen Interactions” (in Basic Immunology, eds. Paul, WE, Raven Press, New York, NY (1984)), and Kuby, “Immunology”, WH Freeman and Company, New York, NY (1992), and the method described in this invention. Affinity can be easily determined by conventional techniques, such as by balanced dialysis; using a BIAcore 2000 instrument and following the manufacturer’s general procedure; by radioimmunoassay using a radiolabeled target antigen; or by other methods known to those skilled in the art. For example, the method described by Scatchard et al. in the Bulletin of the New York Academy of Sciences (1949) can be used as a reference for analyzing affinity data. The measured affinity of a specific interaction between the binder and the antigen may differ depending on the conditions measured, such as salt concentration and pH. Therefore, the measurement of affinity and other antigen-binding parameters (such as _KD , _IC50 ) is preferably performed using standardized binder and antigen solutions and standard buffers.

The term "competitive" refers to the competition between two binding agents (such as antibodies) for binding to a target antigen. If two binding agents do not block each other when binding to a target antigen, these binding agents are non-competitive, indicating that they do not bind to the same part of the target antigen, i.e., the epitope. Those skilled in the art are familiar with how to test the binding competitiveness of binding agents to a target antigen. An example of this method is called a cross-competition test, which can be performed, for example, by ELISA or flow cytometry.

Two binders (such as antibodies) are considered to have "the same specificity" if they bind to the same antigen and the same epitope. In some embodiments, binders that bind to the same epitope are considered to bind to the same amino acid on the target molecule. An articulator can determine whether an antibody binds to the same epitope of the target antigen using a standard alanine scan or antibody-antigen crystallization assay.

The competitive binding ability of a conjugate to an antigen indicates whether it may bind to the same epitope region of the antigen or stereotype the binding to certain specific epitope regions when binding to other epitopes. Competitive conjugates can be identified by standard binding assays (such as surface plasmon resonance analysis, ELISA, or flow cytometry) (see WO 2013/173223). For example, competition between conjugates can be detected by cross-blocking assays. For instance, a competitive ELISA can be performed by coating the target antigen in the wells of a microplate and then adding the antigen conjugate and candidate competitive assay conjugates. The amount of antigen bound by the antigen-binding agent in the well is indirectly related to the binding ability of competing candidate competitive test conjugates. Specifically, the greater the affinity of a candidate competitive test conjugate for the same epitope, the less antigen-binding agent is bound. The amount of antigen-binding agent bound in the well can be measured by labeling the conjugate with a detectable or measurable tag. As known in WO 2013/173223 and in the art, surface plasmon resonance analysis (e.g., using a Biacore instrument) can be used to identify overlapping or different epitope regions identified by the conjugate. Alternatively, biolayer interferometry can be used to determine competitiveness.

A binding agent that competes with another binding agent for binding antigen, such as a binding agent comprising variable regions of the heavy and light chains as described in this application, or a binding agent having the same antigen specificity as another binding agent, such as a binding agent comprising variable regions of the heavy and light chains as described in this application, such as an antibody, may be a binding agent comprising variants of the variable regions of the heavy chain and/or light chains as described in this application, such as modifications in the CDR and/or a certain degree of homology as described in this application.

The term "antigen" refers to a molecule such as a protein or peptide that includes an epitope, against which a binder can be directed and/or pre-directed, preferably to induce an immune response. In some embodiments, an antigen or its processed product (such as a T-cell epitope) can bind to T-cell or B-cell receptors, or to immunoglobulin molecules (such as antibodies). Thus, an antigen or its processed product can specifically react with antibodies or T lymphocytes (T cells). In a preferred embodiment, the antigen is a tumor-associated antigen, such as CLDN18.2, which is a component of cancer cells and may originate from the cytoplasm, cell surface, and nucleus, especially those antigens that are produced in large quantities within cancer cells or as surface antigens.

In the context of this invention, the term "tumor-associated antigen" or "cancer-associated antigen" preferably refers to a protein that is specifically expressed in a limited number of tissues and/or organs under normal conditions or at a specific developmental stage, and is expressed or abnormally expressed in one or more tumors or cancerous tissues. In the context of this invention, the tumor-associated antigen is preferably associated with the cell surface of cancer cells and is preferably not expressed or only rarely expressed in normal tissues.

The term "epitaph" refers to an antigenic determinant in a molecule, such as a portion of the molecule that is recognized by the immune system (e.g., by antibodies). For example, an epitope is a discrete three-dimensional structural site on an antigen that is recognized by the immune system. Epitopes are typically composed of chemically active surface groups of a molecule, such as amino acids or sugar side chains, and usually have specific three-dimensional structural features and specific charge characteristics. Epitopes can be classified as conformational epitopes and non-conformational epitopes, of which only the former, not the latter, loses their binding ability in the presence of denaturing solvents. Protein epitopes preferably include continuous or discontinuous portions of the protein, and preferably have a length of 5 to 100, more preferably 5 to 50, more preferably 8 to 30 amino acids, and most preferably 10 to 25 amino acids. For example, the length of the epitope can preferably be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids.

The term "immunoglobulin" refers to proteins in the immunoglobulin superfamily, more specifically to antigen receptors such as antibodies or B cell receptors (BCRs). Immunoglobulins are characterized by the presence of structural domains, known as immunoglobulin domains (Ig folds). The term encompasses both membrane-bound immunoglobulins and soluble immunoglobulins. Soluble immunoglobulins are often referred to as antibodies. Immunoglobulins typically consist of multiple chains, usually comprising two identical heavy chains and two identical light chains linked together by disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as the VL (variable light chain) domain, CL (static light chain) domain, VH (variable heavy chain) domain, and CH (static heavy chain) domains CH1, CH2, CH3, and CH4. There are five types of mammalian immunoglobulin heavy chains: α, δ, ε, γ, and µ. These five heavy chains constitute different classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Immunoglobulin classes are also called "isotypes" (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM), which refers to the class of immunoglobulins encoded by genes in constant regions of the heavy chain. When a specific isotype, such as IgG1, is mentioned in this article, the term is not limited to a specific isotype sequence, such as a specific IgG1 sequence, but rather indicates that the antibody sequence is more closely similar to that isotype, such as IgG1, and differs significantly from other isotypes. Unlike the heavy chains of soluble immunoglobulins, the heavy chains of membrane-bound or surface immunoglobulins include a transmembrane domain and a short intracellular domain at their C-terminus. In mammals, there are two types of light chains: λ and κ. Immunoglobulin chains consist of variable and constant regions. The constant regions remain largely unchanged across different isotypes of immunoglobulins, while the variable regions are highly diverse and responsible for antigen recognition.

The term "antibody" includes immunoglobulins comprising at least two heavy chains (H) and two light chains (L), linked by disulfide bonds, or their antigen-binding portions. The term "antibody" also includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and chimeric antibodies. Each heavy chain comprises a variable region (referred to herein as VH) and a constant region (according to EU designation, amino acid residues 118-447 of human IgG1), the constant region comprising CH1, CH2, and CH3 domains, wherein CH1 is typically linked to CH2-CH3 via peptide linkers (also referred to as "hinges"). Each light chain comprises a variable region (referred to as VL in this invention) and a constant region (referred to as CL in this invention). The terms "domain" and "structural domain" are used interchangeably herein. The VH and VL domains can be further divided into highly variable regions, referred to as complementary determining regions (CDRs), and more conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see Chothia and Lesk, J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or required by context, the CDR sequences in this invention are identified according to the Kabat numbering system, while references to amino acid positions in the constant regions of the antibody are made according to the EU numbering system (Edelman et al., (1969) Proc. Natl. Acad. Sci. USA 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edit. 1991 NIH Publication No. 91-3242). Variable regions of the heavy and light chains include binding domains that interact with antigens. The constant regions of the antibody may 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 pathway.

As used in this invention, the term "amino acid corresponding to position..." refers to the amino acid position number in the human IgG1 chain, particularly the IgG1 heavy chain. The corresponding amino acid positions in other immunoglobulins can be found by comparison with human IgG1. Therefore, "corresponding" of an amino acid or fragment in one sequence to an amino acid or fragment in another sequence means that an alignment using a standard sequence alignment program such as ALIGN, ClustalW, or similar (usually by default) shows at least 50%, at least 80%, at least 90%, or at least 95% homology with the human IgG1 heavy chain. Those skilled in the art understand how to align sequences or fragments within sequences to determine the corresponding positions of amino acid positions in this invention.

As used herein, the term "IgG Fc ligand" refers to a molecule, preferably a polypeptide, that binds to the Fc region of IgG immunoglobulin to form an Fc/Fc ligand complex. Fc ligands include, but are not limited to, FcγRI, FcγRII, FcγRIII, FcRn, C1q, C3, mannose-binding lectin, mannose receptors, Staphylococcus aureus protein A, group A streptococcus protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), which are a class of Fc receptors homologous to FcγR (see Davis et al., (2002) Immunol. Rev. 190:123-136). Specific IgG Fc ligands include FcRn and Fc gamma receptors. The "Fc ligand" used herein can be derived from any organism, such as mice, humans, and cynomolgus monkeys.

"Fcγ receptor", "FcγR" or "FcgammaR" refers to any member of the protein family that can bind to the Fc region of IgG and is encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), including its isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including its isoforms FcγRIIa (including the H131 and R131 alleles), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including its isoforms FcγRIIIa (including the V158 and F158 alleles) and FcγRIIIb (including the FcγRIIb-NA1 and FcγRIIb-NA2 alleles) (Jefferis et al., (2002) Immunol. Lett. 82:57-65). FcγR can originate from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. FcγR in mice includes, but is not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2).

As used in this specification, "FcRn" or "newborn Fc receptor" refers to a protein that binds to the Fc region of IgG and is at least partially encoded by the FcRn gene. FcRn can originate from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Functional FcRn proteins consist of two polypeptides, commonly referred to as the heavy chain (encoded by the FcRn gene) and the light chain (β2-microglobulin). Unless otherwise stated, "FcRn" or "FcRn protein" refers to the complex of the FcRn heavy chain and β2-microglobulin. As used in this specification, "FcRn variant" refers to a variant that increases binding to the FcRn receptor and may increase its half-life in serum.

As used in this invention, "Fc," "Fc region," or "Fc domain" refers to a polypeptide comprising the CH2 and CH3 domains of an IgG molecule, and optionally a peptide linker, including all or part of the stranded region. According to European Notation, the CH2-CH3 domain of human IgG1 includes amino acid positions 231-447, while the stranded region includes positions 216-230. Therefore, when referring to IgG, the "Fc domain" as used in this invention includes amino acid positions 231-447 (CH2-CH3) and 216-447 (stranded-CH2-CH3) according to European Notation, as well as its functional variations and functional fragments. The "Fc fragment" may contain fewer amino acids, such as N-terminal or C-terminal truncated variants, but still retains the ability to form a dimer with another Fc domain or Fc fragment. This can be detected using standard methods, such as size-based methods (e.g., non-denaturing chromatography, size exclusion chromatography, etc.), and is therefore a functional variant. According to the present invention, the Fc domain of IgG is preferably the Fc domain of human IgG, including Fc domains derived from human IgG1, IgG2, or IgG4.

As used in this invention, "stranded region," "antibody stranded region," or "stranded domain" refers to the peptide linker between the CH1 and CH2 domains in an immunoglobulin (e.g., IgG). In naturally occurring IgG molecules, such as IgG1, according to European numbering, the CH1 domain ends at amino acid position 215, and the CH2 domain begins at amino acid position 231. Therefore, for IgG, the stranded region includes amino acid positions 216 to 230 according to European numbering.

A "variant Fc segment" includes amino acid modifications compared to the parental Fc segment. Thus, for example, a "variant IgG1 Fc segment" (such as a variant human IgG1 Fc segment) includes amino acid modifications (e.g., amino acid substitutions and/or deletions) at positions corresponding to an IgG1 Fc segment (e.g., a human IgG1 Fc segment), and preferably a functional variant of the parental Fc segment. Such variant IgG Fc segments retain at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to the corresponding parental human IgG Fc segment. Optionally, the variant Fc segment may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the parental Fc segment. Optionally, the variant Fc segment may have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the parental Fc segment. Preferably, the variant Fc segment retains the ability to form a dimer with another Fc segment, as measured by techniques known in this specification or in the art (e.g., non-denaturing gel electrophoresis). Fc Modification

The conjugate described in this disclosure comprises three distinct polypeptide chains, wherein two polypeptide chains, such as a first polypeptide chain and a second polypeptide chain, include CH2-CH3 regions, preferably derived from IgG, such as IgG1, particularly human IgG1. The CH2-CH3 regions in the first and second polypeptide chains can interact, for example, dimerize, to form a heterodimer of the conjugate comprising the first and second polypeptide chains. Preferably, the CH2-CH3 regions in the conjugate are derived from IgG1, more preferably from human IgG1, although CH2 and CH3 regions from other subtypes may also be used, as described herein. Furthermore, as discussed herein, the conjugate can self-assemble, such as within the host cell of production. For example, it is anticipated that the CH2 region on the first polypeptide chain interacts with the CH2 region on the second polypeptide chain, and/or the CH3 region on the first polypeptide chain interacts with the CH3 region on the second polypeptide chain, thereby forming Fc fragments. These Fc fragments may include one or more amino acid modifications, or "Fc modifications," as mentioned in the discussion of human IgG1 herein, to facilitate the interaction of the CH2 and CH3 regions between the first and second polypeptide chains, and/or to facilitate the purification of heteropolymers (such as heterodimers) that include the interacting first and second polypeptide chains, rather than homopolymers that include only one polypeptide chain, and/or to impart other beneficial functions, as described herein. The amino acid modifications discussed in this disclosure may also be present in the CH1 region, for example, in the first and/or second polypeptide chains of the described conjugates. Furthermore, peptide linkers, such as peptide linkers in single-chain variable fragments (scFv) or peptide linkers connecting other domains or binding domains of the binding agent, such as CH1 and scFv, or CH2 and scFv, or CH1 and CH2, may include one or more amino acid modifications as described herein. For example, according to EU designations, peptide linkers connecting the CH2-CH3 region to other domains or binding domains (such as VH(CD3) or CH1) may include a serine at the position corresponding to position 220 in native IgG1 (typically cysteine). Using peptide linkers that include a serine at position 220 corresponding to human IgG1 can reduce disulfide bond formation between two polypeptide chains including the CH2-CH3 region.

Therefore, the formation of the binders described in this invention is based on the use of different monomers (e.g., the first and second polypeptide chains described in this invention) comprising amino acid substitutions in the CH1, CH2, and/or CH3 regions, which may "prefer" to form heterodimers composed of these monomers rather than homodimers described in this invention, preferably in conjunction with "pI modifications" to easily purify the heterodimer from the homodimer, and optionally in conjunction with "elimination modifications" and other Fc region modifications discussed in this invention. Those skilled in the art will understand that any amino acid modifications in this invention relating to the CH1, CH2, and/or CH3 regions and peptide linkers (e.g., twisted chain variants) may be combined with other amino acid modifications discussed in this invention or known in the art. For example, any tendency modification and pI modification can independently combine with elimination modification and other Fc region modifications. Fc region modifications according to the invention can be insertions, additions, deletions, or substitutions of amino acids.

The Fc region modifications discussed in this invention are defined based on the amino acid modifications that constitute them. For example, N434S is an Fc region modification in which serine replaces aspartic acid at position 434 of the parental human IgG1 Fc polypeptide, according to the EU numbering system. If the identity of the parental amino acid is not explicitly specified, the above variant may be referred to as 434S, meaning that, according to the EU numbering system, this variant includes serine at the amino acid position corresponding to position 434 in human IgG1. pI modification

pI modification increases the isoelectric point (pI) difference between monomers, which allows for the purification of homologous and heterologous multimer proteins by isoelectric point. Generally, pI modification can increase the pI of a polypeptide chain (basic modification) or decrease the pI of a polypeptide chain (acidic modification).

As discussed herein, a pI difference of at least 0.1, such as 0.2, 0.3, 0.4, or 0.5, between two polypeptide chains can be separated by methods such as ion exchange chromatography or isoelectric focusing, which are sensitive to the isoelectric point. Therefore, pI modifications, including those that alter the pI of interacting polypeptide chains (e.g., the first and second polypeptide chains described herein), such that each polypeptide chain has a different pI, and the heteropolymers formed from these polypeptide chains also have unique pIs, facilitate the isoelectric point purification of the binder including the heteropolymers. These substitutions also help to identify and monitor the formation of any undesirable homo or heteropolymer contamination.

pI modifications can be included in one or both chains of a heteropolymer that includes a heavy-chain constant region, such as the first and second polypeptide chains of the conjugates described herein, and in the CH1, CH2, and/or CH3 regions, or in peptide linkers (e.g., linkers connecting the VH and VL regions of the scFv unit). For example, pI variants can be included in the first and/or second polypeptide chains of the conjugates described herein to reduce or prevent the formation of homopolymers. Typically, when these modifications are included in the first and second polypeptide chains, pI variants are used such that the pI of one polypeptide chain increases while the pI of the other polypeptide chain decreases. This can be achieved by replacing neutral amino acid residues with positively or negatively charged amino acid residues, or vice versa, or by converting charged amino acid residues from positive to negative, or vice versa, as discussed in this invention. Therefore, in some embodiments of this invention, a sufficient pI variation is provided in at least one polypeptide chain of the binder described herein to allow the heteropolymer (such as that of the first and second polypeptide chains) to be purified from the homopolymer. In some embodiments, this invention uses pI differences of only 0.1, 0.2, 0.3, 0.4, or 0.5 in pH units. Those skilled in the art will understand that, in order to achieve good separation, the number of pI modifications included in one or more polypeptide chains of the binder described herein depends on the initial pI value of the polypeptide chain, the pI value of the CH region, the Fv scaffold region, etc. Variations in pI can be calculated using any method known in the art, such as the method described by Sillero and Maldonado based on the CH region (Sillero, Maldonado, (2006) Comput. Biol. Med. 36(2):157–166). Alternatively, the pI of each polypeptide chain can be compared. Furthermore, heteropolymers can also be separated based on their size.

In some embodiments, the variable regions of the binder described herein do not contain pI modifications, skew modifications, additional Fc modifications, or ablation modifications.

In some embodiments, pI modifications are derived from different IgG isotypes, resulting in a change in the pI of the corresponding polypeptide chain without introducing immunogenicity (see U.S. 2014/0370013). Preferably, pI modifications are derived from human IgG isotypes to reduce the risk of introducing immunogenicity. The modifications discussed herein are described for human IgG1, but all IgG isotypes can be modified in this way, including isotype mixtures. If the heavy chain constant region is derived from IgG2-4, R133E and R133Q can be used.

IgG1 is a common isotype of therapeutic antibodies for several reasons, including its potent therapeutic function. However, the pI of IgG1 in the CH region is higher than that of IgG2. By introducing a specific IgG2 residue into the IgG1 backbone, the pI of the monomer can be decreased or increased, and serum half-life may be increased. For example, according to EU designation, human IgG1 has a glycine residue at position 137 (pI approximately 5.97), while human IgG2 has a glutamic acid residue at the corresponding position (pI approximately 3.22); replacing the glycine residue with a glutamic acid residue affects the pI of the resulting peptide. Lowering the pI of the constant region of an antibody may increase its serum half-life in vivo (see USSN 13/194,904; Ghetie and Ward, 1997, Immunol Today. 18(12): 592-598). Furthermore, lower pI of the variable region may also increase serum half-life (see Igawa et al., 2010, PEDS 23(5): 385-392).

In a preferred pI modification combination, according to EU designations, a polypeptide chain (e.g., the first polypeptide chain of the conjugate described herein, comprising VH(CLDN18.2), CH1, CH2, and CH3) contains an aspartic acid residue at position 208, a glutamic acid residue at position 295, an aspartic acid residue at position 384, a glutamic acid residue at position 418, and an aspartic acid residue at position 421 (i.e., N208D/Q295E/N384D/Q418E/N421D relative to human IgG1). Another polypeptide chain (e.g., a second polypeptide chain with the same binding agent, including VH(CLDN18.2), CH1, VL(CD3), VH(CD3), CH2, and CH3) includes a positively charged peptide linker linking VH(CD3) and VL(CD3) ("scFv linker") (e.g., a polypeptide linker including or composed of an amino acid sequence (GKPGS) ₄ or a functional variant thereof).

For polypeptide chains that do not contain the CH1 domain, since they do not include the amino acid position corresponding to EU number 208, the following negative pI modifications can be used: a glutamic acid residue at position 295, an aspartic acid residue at position 384, a glutamic acid residue at position 418, and an aspartic acid residue at position 421 (i.e., Q295E/N384D/Q418E/N421D relative to human IgG1).

In some embodiments, a polypeptide chain, such as a first polypeptide chain, includes a set of variations as described herein, and a polypeptide chain interacting therewith, such as a second polypeptide chain, includes charged scFv linkers, such as the positively charged scFv linker of SEQ ID NO: 2 or a functional variation thereof, or negatively charged scFv linkers. Skew modification

"Oblique modification" refers to spatial modifications that promote interactions between polypeptide chains, including such modifications. A spatial modification strategy known in the field is called "knobs and holes," which refers to the introduction of a knot on the first heavy-chain polypeptide (usually in the Fc region, i.e., CH2-CH3) by amino acid engineering, and a corresponding hole on the second heavy-chain polypeptide (also usually in the Fc region, i.e., CH2-CH3). In this way, the knot can be embedded in the hole at the interface of the double chain, thereby promoting the formation of heterodimers and inhibiting the formation of homodimers (see USSN 61/596,846, Ridgway et al., (1996) Protein Engineering 9(7):617; Atwell et al., (1997) Molecular Biology 270:26; US Patent No. 8,216,805). "Bumps" are constructed by replacing small amino acid side chains on the interface of the first heavy-chain polypeptide with larger side chains. Compensating "depressions" of the same or similar size as the bumps are created on the interface of the second heavy-chain polypeptide by replacing large amino acid side chains with small amino acid side chains (US Patent 5,731,168). "Bump-depression" modifications can be combined with disulfide bonds to deflect heteropolymerization, such as heterodimerization of the first and second heavy-chain polypeptides (see Merchant et al., (1998) Nature Biotechnology 16:677).

Useful skew modifications include, but are not limited to, the following double modification pairs, wherein one portion of each double modification pair will be present in one polypeptide chain of the binder described herein (e.g., the first polypeptide chain described herein), and the other portion will be present in another polypeptide chain of the binder described herein (e.g., the second polypeptide chain described herein): S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368D/K370S : S364K/E357Q; L368E/K370S : S364K; T411E/K360E/Q362E : D401K; L368D/K370S : S364K/E357L; K370S: S364K/E357Q; T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C: T366W/S354C, and according to EU designations and targeting human IgG1. Preferably, L368D/K370S: S364K/E357Q is used in the conjugates described herein.

The skew modification described in this specification may also affect pI (see Gunasekaran et al., (2010) J. Biol. Chem. 285(25):19637), and therefore also affects the purification process; thus, it can also be considered a variant of pI. Elimination of modification

In this specification, "elimination" refers to a reduction or removal of activity. "Elimination of FcγR binding" means that the FcγR binding activity of an Fc region including one or more elimination modifications is reduced by more than 50% compared to an Fc region without the specific modification. Preferably, the FcγR binding activity of an Fc region including one or more elimination modifications is reduced by more than 70%, 80%, 90%, 95%, 98%, or even higher compared to an Fc region without the specific modification. Preferably, the FcγR binding activity of an Fc region including one or more elimination modifications is below a detectable level in Biacore, SPR, or BLI assays.

As is well known, the Fc domain of human IgG1 has the strongest binding affinity to the Fcγ receptor; therefore, elimination modification can be used when the constant region of the binder is derived from IgG1. Alternatively, in addition to elimination modification, mutations at glycosylation position 297 (typically A or S) significantly reduce binding to FcγRIIIa. Human IgG2 and IgG4 naturally have lower binding affinity to the Fcγ receptor (Parren et al., 1992, J. Clin Invest. 90: 1537-1546; Bruhns et al., 2009, Blood 113: 3716-3725); therefore, the binders described herein can use CH1, CH2, and CH3 domains derived from IgG2 or IgG4, with or without elimination modification. The amino acid modification method to eliminate FcγR binding has been described in the study of Dall'Acqua WF et al. (J Immunol. 177(2):1129-1138 (2006)) and Hezareh M (J Virol.; 75(24):12161-12168 (2001)).

Therefore, the Fc portion of the binder described herein may include one or more "FcγR elimination modifications" or "Fc knockout (FcKO or KO) modifications." In some embodiments, it is desirable to reduce or eliminate the binding of the Fc region to one or more or all Fcγ receptors (such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, etc.). In some embodiments, it is desirable to eliminate or significantly reduce the FcγRIIIa binding of a monovalently CD3-binding binder (such as the binder described herein) to eliminate or significantly reduce ADCC activity. Therefore, with respect to the binder described herein, one or more polypeptide chains of the binder, such as a first polypeptide chain and a second polypeptide chain, include one or more FcγR elimination variants. In a preferred embodiment, one or more elimination variants are selected from: 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/G236d el/S267K, E233P/L234V/L235A/G236del/S239K/A327G, E233P/L234V/L235A/ G236del, S239K/S267K, 267K/P329K, E233P/L234V/L235A/G236del/S239K, E2 The group consisting of 33P/L234V/L235A/G236del/S267K, E233P/L234V/L235A/G236del/S239K/A327G, E233P/L234V/L235A/G236del/S267K/A327G, and E233P/L234V/L235A/G236del (referring to human IgG1 according to EU designation) is preferred. The first and second polypeptide chains of the conjugates described herein are modified with E233P/L234V/L235A/G236del/S267K (referring to human IgG1 according to EU designation). It should be noted that the elimination modification disclosed in this article eliminates the binding of FcγR, but generally does not affect the binding of FcRn. However, techniques for reducing or increasing the binding of the conjugate to FcRn to reduce or increase its serum half-life are known and available (see, for example, 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, in the conjugate described herein, the first polypeptide chain includes the amino acid sequence of SEQ ID NO: 7, and the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 8. Both the first and second polypeptide chains include the skew modification L368D/K370S:S364K/E357Q. The first polypeptide chain also includes the pI modification N208D/Q295E/N384D/Q418E/N421D. Both the first and second polypeptide chains further include the elimination modification E233P/L234V/L235A/G236del/S267K. For example, the first polypeptide chain includes VH (CLDN18.2) and CH1, while the second polypeptide chain includes scFv (CD3), VH (CLDN18.2), and CH1. Of course, further modifications can be incorporated into the amino acid sequence of each binder; for example, in addition to the modifications mentioned above, the binder may also include modifications to other amino acids, such as substitutions. Other Fc modifications

In addition to other modifications described in this paper, such as pI modification, skew modification and descaling modification, a variety of useful modifications can be used to alter the binding to one or more FcγR receptors, as well as the binding to FcRn receptors.

Therefore, a variety of useful amino acid substitutions can be made to the binding agents described herein to alter their binding to one or more FcγR receptors. These substitutions can result in either enhanced or reduced binding, both of which may be of practical use. For example, it is known that enhanced binding to FcγRIIIa can improve ADCC (antibody-dependent cell-mediated cytotoxicity). Similarly, reduced binding to FcγRIIb may also be beneficial. Amino acid substitutions that may be used in this invention include those listed in USSN 11/124,620, 11/174,287, 11/396,495, and 11/538,406, the entire contents of which are incorporated herein by reference. Particularly 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.

In addition, there are other modifications used to increase binding to FcRn and prolong serum half-life, as disclosed in USSN 12/341,769, the entire contents of which are incorporated herein by reference, 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.

In addition, when forming an Fc heterodimer, the CH3 region on one or two polypeptide chains, preferably on both polypeptide chains, may include M428L/N434S modification to achieve a longer serum half-life.

As will be understood by those skilled in the art, the modifications discussed herein can be combined independently with other modifications. In some embodiments, one polypeptide chain (e.g., a first polypeptide chain) of the binder described herein includes modifications of N208D, Q295E, N384D, Q418E, and N481D according to EU designations, and another polypeptide chain (e.g., a second polypeptide chain) includes positively charged scFv linkers as described herein. Preferably, the first polypeptide chain of the binder further includes K370S and L368D modifications, and the second polypeptide chain further includes E357Q and S364K modifications, both according to EU designations. Furthermore, in a preferred embodiment, both the first and second polypeptide chains further include E233P, L234V, L235A, G236del, and S267K modifications, according to EU designation. Preferably, the first polypeptide chain of the binder described herein includes N208D, E233P, L234V, L235A, G236del, S267K, Q295E, L368D, K370S, N384D, Q418E, and N481D modifications, and the second polypeptide chain includes E233P, L234V, L235A, G236del, S267K, E357Q, and S364K modifications, and optionally includes a C220S modification to remove cysteine that is normally paired with the light chain.

The term "monopolymer conjugate" as used in this application includes "monopolymer antibody" and refers to a formulation of a conjugate molecule consisting of a single molecule.

"Monoantibodies" exhibit single binding specificity and affinity for specific epitopes.

The term "recombinant binder" as used in this application includes "recombinant antibody" and includes all binders prepared, expressed, established or isolated by recombination.

The term "human binding agent" as used in this application includes "human antibody," meaning a binding agent having variable and constant regions derived from human germline immunoglobulin sequences. Human binding agents may include amino acid residues encoded by non-human germline immunoglobulin sequences (e.g., mutations introduced by random or position-specific mutations in vitro, or mutations introduced by somatic mutations in vivo).

The term "humanized binding agent" in this specification includes "humanized antibody," which refers to a molecule having an antigen-binding site primarily derived from an immunoglobulin of a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin. This can be achieved, for example, by transplanting the complementarity-determining regions (CDRs) of six non-human antibodies, which together form an antigen-binding site, onto a homologous human receptor frame region (FR) (see WO92/22653 and EP0629240). The antigen-binding site may include the complete fusion of the variable domain and the constant domain, or only the CDRs may be transplanted into the appropriate frame region within the variable domain. To fully restore the binding affinity and specificity of a parental binder, it may be necessary to replace the framework residues of the parental binder (e.g., a non-human binder, such as a mouse antibody) with those in the human framework region (reverse mutation). Structural homology modeling can help identify amino acid residues in the framework region that are important for the binder's binding properties. The antigen-binding site can be wild-type or modified by substitution of one or more amino acids, for example, to more closely resemble human immunoglobulins. Some forms of humanized binders retain all CDR sequences (e.g., humanized mouse antibodies include all six CDR sequences from mouse antibodies). Other forms have one or more CDR sequences that have been altered compared to the original binder (e.g., an antibody). Therefore, humanized binding agents may include non-human CDR sequences, primarily composed of human framework regions, and optionally include one or more non-human amino acid sequences of reverse amino acid mutations, as well as fully human normal domains.

As used in this specification, the term "chimeric binder" includes "chimeric antibody," which refers to a conjugate in which a portion of the amino acid sequence of each heavy and light chain is homologous to a corresponding sequence of a binder (e.g., an antibody) derived from a particular species or belonging to a particular class, while the remaining segments of the chain are homologous to a corresponding sequence of another species. Typically, the variable regions of the light and heavy chains mimic the variable regions of antibodies derived from a particular mammal, while the constant regions are homologous to antibody sequences derived from another species. A clear advantage of this chimeric form is that the variable regions can be readily obtained from existing sources, using readily available non-human host organ B cells or hybridomas, and binding to constant regions derived from, for example, human cell preparations. When the variable region has the advantage of easy preparation and its specificity is not affected by the source, and the constant region is human, compared to a constant region from a non-human source, the binding agent is less likely to induce a human immune response when injected. However, this definition is not limited to specific examples.

The binder or fragments thereof (e.g., variable and/or constant regions) may be derived from different species, including but not limited to mice, rats, rabbits, guinea pigs and humans.

The immunoglobulins described herein include IgA (such as IgA1 or IgA2), IgG1 (with polymorphic isotypes at amino acid positions 356 (D or E) and 358 (L or M) according to EU designations), IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the immunoglobulin is an IgG1 antibody, more particularly an IgG1, κ, or IgG1, λ 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 of the IgG1 isotype 356D/358M described herein also includes the 356E/358L isotype.

As used in this invention, "IgG subtype modification" or "isotype modification" refers to replacing a certain amino acid in one IgG isotype with the corresponding amino acid in another different, compared IgG isotype. For example, according to EU designations, since IgG1 has tyrosine at amino acid position 296 and IgG2 has phenylalanine, the F296Y substitution in IgG2 is considered an IgG subtype modification.

The term "heterologous binder" as used in this invention includes "heterologous antibody" and is defined as a term associated with binders produced by transgenic organisms. This term refers to a binder whose amino acid sequence or encoded nucleic acid sequence corresponds to a sequence in a non-transgenic organism, typically originating from a species different from the transgenic organism.

As used in this article, "heterogeneous hybrid conjugates" include "heterogeneous hybrid antibodies," which are conjugates containing light and heavy chains from different organisms. For example, antibodies containing human heavy chains and mouse light chains are heterogeneous hybrid antibodies.

The antibody-containing conjugates described herein are preferably isolated. The term "isolated" is used herein to refer to a conjugate that is substantially free of other agents with different antigenic specificities (e.g., an isolated conjugate specifically binding to CLDN18.2 and CD3 is substantially free of conjugates specifically binding to antigens other than CLDN18.2 and CD3). However, isolated conjugates specifically binding to epitopes, isomers, or variants of human CLDN18.2 may cross-react with other relevant antigens, such as antigens from other species (e.g., species homologs of CLDN18.2). Furthermore, isolated conjugates are substantially free of other cellular material and/or chemicals.

The terms "antigen-binding portion" (or simply "binding portion") of an antibody or "antigen-binding fragment" (or simply "binding fragment") refer to one or more binding fragments that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the term "antigen-binding moiety" of conjugates such as antibodies include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments consisting of two Fab fragments linked by disulfide bonds in a twisted region; (iii) Fab' fragments, derived from F(ab')2 fragments, containing a free thiohydrogen group, which can be alkylated or used to bind to enzymes, toxins, or other proteins of interest, wherein Fab' may include a small fraction of Fc; (iv) Fd fragments, consisting of VH and CH1 domains; (v) Fv fragments, consisting of the VL and VH domains of the antibody single arm; (vi) dAb fragments (Ward et al., (1989) Nature). (341:544-546), consisting only of the VH domain; (vii) separate complementary determinant regions (CDRs); (viii) combinations of two or more separate CDRs, which may be linked by synthetic linkers. Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by different genes, they can be linked by synthetic linkers using recombination methods to form a single protein chain, where the VL and VH domains pair to form a monovalent molecule (called a single-strand Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also considered to encompass the "antigen-binding fragment of the antibody" within a conjugate such as an antibody. Another example is a binding domain immunoglobulin fusion protein, which includes: (i) a binding domain polypeptide fused to an immunoglobulin stranded region polypeptide, (ii) an immunoglobulin heavy chain CH2 fixed region fused to the stranded region, and (iii) an immunoglobulin heavy chain CH3 fixed region fused to the CH2 fixed region. The binding domain polypeptide may be a variable region of the heavy chain or a variable region of the light chain. For details on binding domain immunoglobulin fusion proteins, please refer to US 2003/0118592 and US 2003/0133939. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and their effectiveness is screened in the same way as that of intact antibodies.

Single-chain variant fragments (scFvs) are fusion proteins of the heavy chain (VH) and light chain (VL) variants of immunoglobulins, typically linked by a short linker peptide consisting of about 10 to 30 amino acids, such as the scFv linker peptide (e.g., represented by SEQ ID NO: 2). This linker peptide is typically enriched with glycine to provide flexibility and serine or threonine to enhance solubility, and can link the N-terminus of the VH to the C-terminus of the VL, and vice versa. Bivalent (or bispecific) single-chain variant fragments (di-scFvs, bi-scFvs) can be designed by linking two scFvs. This can be achieved by generating a single peptide chain comprising two VH and two VL domains, thereby forming tandem scFvs. This invention also includes multispecific molecules having multiple scFv binding domains. A common flexible linker peptide is ( _G4S ) _x , where x can be 2, 3, 4, 5, or 6. Preferably, the linker peptide connecting the VH and VL domains comprises, and more preferably consists of, an amino acid sequence (GKPGS) _x or a functional variant thereof, where x can be 2, 3, 4, 5, or 6. Alternatively, the link between VH and VL can be stabilized by one or more intermolecular disulfide bonds.

Antibody fragments (such as antigen-binding fragments (Fab) of an antibody) are immunoreactive polypeptides comprising a monovalent antigen-binding domain of an antibody, comprising a polypeptide consisting of a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1) and a polypeptide consisting of a light chain variable region (VL) and a light chain constant region (wherein the CL and CH1 portions are linked by disulfide bonds, for example by cysteine residues) forming a monovalent antigen-binding domain. In the Fab fragments described herein, preferably the CH1 and CL portions are human. In some embodiments, CL is κ-type CL. In some embodiments, CH1 is derived from IgG1, preferably human IgG1.

The antibodies and antibody derivatives (such as antibody fragments) described herein for the purposes of this invention are included in the term "antibody". "Antibody derivative" refers to any modified form of an antibody, such as a complex of an antibody with another drug or antibody, or an antibody fragment. Furthermore, the antibodies and antibody derivatives described herein can be used to prepare the binding agents described herein.

Natural antibodies are typically monospecific, meaning they bind to only a single antigen. The bispecific binders described herein can bind to CD3 receptors on cytotoxic cells (such as T cells) and CLDN18.2 on target cells (such as cancer cells). The binders described in this invention can bind to at least two different types of antigens and are at least bispecific or multispecific, such as trispecific, tetraspecific, etc.

In some embodiments, the conjugates described in this invention are at least trivalent. In this invention, "valence," "valence number," "valence property," or other variations thereof refer to the number of antigen-binding sites or binding domains in the conjugate. Antigen-binding sites binding to the same antigen can identify different epitopes, or better yet, the same epitopes. The conjugates described in this invention may also have four or more valences.

The conjugates described herein are preferably artificial proteins (including protein complexes) that may consist of fragments of at least two different antibodies (these fragments of at least two different antibodies form at least two different binding domains), and thus can bind to at least two different types of antigens. The conjugates described herein are engineered to simultaneously bind to immune cells (such as immune effector cells, particularly T cells such as cytotoxic cells, for example by binding to CD3) and target cells (such as cancer cells, by binding to the tumor-associated antigen CLDN18.2) to promote their destruction.

In some embodiments, the conjugates described herein exist in the form of a Fab2-scFv architecture, which comprises two Fab fragments (each fragment comprising a VH and CH1 domain on one polypeptide chain and a corresponding VL and CL domain on another polypeptide chain, wherein the interaction of each polypeptide chain combination forms an antigen-binding domain) and an scFv group (comprising a VH and VL domain on one polypeptide chain, linked together by a polypeptide linker, wherein the VH and VL interact to form an antigen-binding domain). In some embodiments, the binder described in this invention is a tetramer composed of four polypeptide chains, wherein the first polypeptide chain includes a VH from an immunoglobulin (e.g., an immunoglobulin with first specificity), the second polypeptide chain includes a VH from an immunoglobulin (e.g., an immunoglobulin with first specificity) and an scFv group (including a VH from an immunoglobulin (e.g., an immunoglobulin with second specificity) and a VL from an immunoglobulin (e.g., an immunoglobulin with second specificity)), the third polypeptide chain includes a VL from an immunoglobulin (e.g., an immunoglobulin with first specificity), and the fourth polypeptide chain is the same as the third polypeptide chain. In some embodiments, the first and second polypeptide chains further include a CH1 domain from an immunoglobulin (e.g., located at the C-terminus of the VH domain of an immunoglobulin with first specificity), and the third and fourth polypeptide chains further include a CL domain from an immunoglobulin. In some embodiments, the first and second polypeptide chains further include CH2 and CH3 (CH2-CH3) domains from an immunoglobulin (e.g., located at the C-terminus of the Fab fragment and the scFv group, respectively). Therefore, in some embodiments, the binding agent described herein includes a first polypeptide chain VH-CH1 linked to CH2-CH3, a second polypeptide chain VH-CH1 linked to the scFv group and then linked to CH2-CH3, and the third and fourth polypeptide chains each including a VL-CL domain. In some embodiments, the first polypeptide chain interacts with the second polypeptide chain. In some embodiments, the first polypeptide chain interacts with the third polypeptide chain. In some embodiments, the second polypeptide chain interacts with the fourth polypeptide chain. In some embodiments, the first and second polypeptide chains interact, the first polypeptide chain further interacts with the third polypeptide chain, and the second polypeptide chain further interacts with the fourth polypeptide chain. In some embodiments, 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 some embodiments, VH on the first polypeptide chain interacts with VL on the third polypeptide chain to form a binding domain and/or CH1 on the first polypeptide chain interacts with CL on the third polypeptide chain. In some embodiments, VH on the second polypeptide chain (not belonging to the scFv group) interacts with VL on the fourth polypeptide chain to form a binding domain and/or CH1 on the second polypeptide chain interacts with CL on the fourth polypeptide chain. In some embodiments, a disulfide bond is formed between the cysteine residue in CL and the cysteine residue in CH1. One or two polypeptide chains comprising CH2-CH3 may include one or more amino acid modifications, such as the Fc modifications described herein, such as pI, skew, additional Fc, and ablation modifications, to promote polypeptide chain interactions. According to the present invention, the VH and VL of the scFv group are preferably linked by a peptide linker (“scFv linker”). In some embodiments, CH1 on the first and/or second polypeptide chain is linked to CH2 on the same polypeptide chain by a peptide linker. In some embodiments, scFv is linked to CH2 by a peptide linker.

In some embodiments, the VH segment on the first polypeptide chain and the VL segment on the third polypeptide chain interact to form a binding domain targeting CLDN18.2, an additional VH segment on the second polypeptide chain that does not belong to scFv interacts with the VL segment on the fourth polypeptide chain to form another binding domain targeting CLDN18.2, and the VH and VL segments of scFv on the second polypeptide chain interact to form a binding domain targeting CD3.

A "linker" is any means of connecting two different functional units (e.g., different 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 and polypeptide linkers are not limited. Preferably, peptide linkers are non-immunogenic and flexible, for example, including sequences containing serine and glycine. Linkers can be long or short depending on the specific architecture.

In a preferred embodiment, the scFv linker (i.e., the linker connecting VH and VL to form the scFv unit) preferably comprises, and more preferably, a flexible peptide linker as described herein, preferably an amino acid sequence (GKPGS) _x or a functional variant thereof, wherein x is 2, 3, 4, 5, or 6. In a more preferred embodiment, the scFv linker comprises, and more preferably, an amino acid sequence (GKPGS) ₄ (SEQ ID NO: 2) or a functional variant thereof. Preferably, the scFv unit is linked to the CH2 region 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.

According to the present invention, the linker connecting scFv and CH1 is preferably located at the C-terminus of CH1 and preferably comprises, or is preferably composed of, the following 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 CH2 and scFv is preferably located at the N-terminus of CH2 and preferably comprises, or is preferably composed of, the following amino acid sequence: ( _G4S ) _2KTHTCPPC (SEQ ID NO: 4) or a variant thereof. According to the present invention, the linker connecting CH1 and CH2 preferably comprises, or is preferably composed of, the following amino acid sequence: EPKSCDKTHTCPPCP (SEQ ID NO: 6) or a functional variant thereof. According to the present invention, CH1 and scFv (e.g., on a second polypeptide chain) are linked by a peptide linker, which preferably comprises, or is preferably composed of, the following amino acid sequence: ( _G4S ) ₂ (SEQ ID NO: 5) or a functional variant thereof. However, other linkers may also be used according to methods known in the art.

In some embodiments, the binding agents described herein include first, second, third, and fourth polypeptide chains, wherein i) the first polypeptide chain includes the following domain from N-terminus to C-terminus: VH(CLDN18.2)-CH1-CH2-CH3, ii) the second polypeptide chain includes the following domain from N-terminus to C-terminus: VH(CLDN18.2)-CH1-VL(CD3)-VH(CD3)-CH2-CH3, iii) the third polypeptide chain includes the following domain from N-terminus to C-terminus: VL(CLDN18.2)-CL, and iv) ... The fourth polypeptide chain is the same as the third polypeptide chain, preferably wherein the VH (CLDN18.2) on the first polypeptide chain interacts with the VL (CLDN18.2) on the third polypeptide chain to form a CLDN18.2 binding domain, the VH (CLDN18.2) on the second polypeptide chain interacts with the VL (CLDN18.2) on the fourth polypeptide chain to form a CLDN18.2 binding domain, the VH (CD3) and VL (CD3) interact to form a CD3 binding domain, and the domains on the polypeptide chains are preferably linked by peptide linkers described herein.

In some embodiments, the binding agent described herein includes: a) a first polypeptide chain comprising VH (CLDN18.2) and an aspartic acid residue at EU encoding position 208, a proline residue at position 233, a volate residue at position 234, an alanine residue at position 235, a deletion at position 236, an lysine residue at position 267, and a glutamine at position 295. a) Aspartic acid residue at position 368, serine residue at position 370, aspartic acid residue at position 384, glutamic acid residue at position 418, and aspartic acid residue at position 421; b) a second polypeptide chain, comprising VH (CLDN18.2) and CH1 as described herein, via charged scFv linkers (amino acid sequences (GKPGS)). ₄ (SEQ ID NO:2)) VH (CD3) and VL (CD3) linked together, and CH2 and CH3 at EU coding positions 233 (proline residue), 234 (velamine residue), 235 (alanine residue), 236 (deletion), 267 (lysine residue), 357 (glutamine residue), and 364 (lysine residue); c) a third polypeptide chain including VL (CLDN18.2) and CL; d) a fourth polypeptide chain identical to the third polypeptide chain.

In some embodiments, the binding agents described herein include first, second, third, and fourth polypeptide chains, wherein i) the first polypeptide chain includes the following domain from the N-terminus to the C-terminus: VH(CLDN18.2)-CH1-linker-CH2-CH3, ii) the second polypeptide chain includes the following domain from the N-terminus to the C-terminus: VH(CLDN18.2)-CH1-linker-VL(CD3)-linker-VH(CD3)-linker-CH2-CH3, iii) the third polypeptide chain includes the following domain from the N-terminus to the C-terminus: VL(CLDN18.2)-CL, iv) ... The fourth polypeptide chain is the same as the third polypeptide chain. Preferably, VH (CLDN18.2) on the first polypeptide chain and VL (CLDN18.2) on the third polypeptide chain interact to form a binding domain for CLDN18.2, VH (CLDN18.2) on the second polypeptide chain and VL (CLDN18.2) on the fourth polypeptide chain interact to form a binding domain for CLDN18.2, while VH (CD3) and VL (CD3) interact to form a binding domain for CD3.

In some embodiments, the conjugates described herein include first, second, third, and fourth polypeptide chains, wherein i) the first polypeptide chain comprises, from N-terminus to C-terminus: VH(CLDN18.2)-CH1-linker 1-CH2-CH3, ii) the second polypeptide chain comprises, from N-terminus to C-terminus: VH(CLDN18.2)-CH1-linker 2-VL(CD3)-linker 3-VH(CD3)-linker 4-CH2-CH3, iii) the third polypeptide chain comprises, from N-terminus to C-terminus: VL(CLDN18.2)-CL, and iv) the fourth polypeptide chain is identical to the third polypeptide chain, wherein linker 1 comprises the amino acid sequence EPKSCDKTHTCPPCP or a functional variant thereof, and linker 2 comprises the amino acid sequence ( _G4S ). _x or a functional variant thereof, wherein x is 2, 3, 4, 5 or 6, preferably x is 2, linker 3 includes an amino acid sequence (GKPGS) _x or a functional variant thereof, wherein x is 2, 3, 4, 5 or 6, preferably x is 4, linker 4 includes an amino acid sequence ( _G4S ) ₂ KTHTCPPCP or a functional variant thereof, and preferably wherein VH (CLDN18.2) on the first polypeptide chain and VL (CLDN18.2) on the third polypeptide chain interact to form a binding domain targeting CLDN18.2, VH (CLDN18.2) on the second polypeptide chain and VL (CLDN18.2) on the fourth polypeptide chain interact to form a binding domain targeting CLDN18.2, and VH (CD3) and VL (CD3) interact to form a binding domain targeting CD3.

The conjugates described herein, and/or the first, second, third, and fourth polypeptide chains of such conjugates, may also include amino acid sequences that facilitate the secretion of the conjugate or polypeptide chain, such as an N-terminal secretion signal, and/or one or more epitope tags that facilitate the binding, purification, or detection of the molecule. Preferably, the secretion signal is a signal sequence that allows the conjugate or its polypeptide chain to be secreted into the extracellular environment via a secretory pathway and/or through sufficient channels. Preferably, the secretion signal sequence is cleavable and can be removed from the mature conjugate or polypeptide chain. The secretion signal sequence should preferably be selected based on the cell or organism in which the conjugate or polypeptide chain is generated.

The amino acid sequence of the epitope tag can be inserted at any position in the amino acid sequence of the binder or polypeptide chain and can form a loop encoding a protein structure, or can be fused to the binder or polypeptide chain at the N-terminus or C-terminus. Preferably, the epitope tag is fused to the binder or polypeptide chain at the C-terminus. The epitope tag may include a cleavage site that allows the tag to be removed from the binder or polypeptide chain. The epitope tag can be any epitope tag that functions normally under natural and/or denaturing conditions, preferably a histidine tag, and most preferably a tag comprising six histidine ions.

In addition to the first, second, and third binding domains described herein, the binding agents described herein may also include one or more further binding domains that can be used, 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.

The term "post-translational modification" or similar terms refers to protein modifications that occur after protein biosynthesis, such as covalent and enzymatic modifications. As is known in the art, intracellularly expressed binding agents (such as antibodies) are typically modified post-translationally. For example, post-translational modifications of binding agents (such as antibodies) can occur at the amino side or N- or C-terminus of the heavy or light chain (as described herein in the first and/or second polypeptide chains). Post-translational modifications that may occur in the conjugates described herein include, but are not limited to: cleavage of the lysine at the C-terminus of the heavy chain (such as the first and/or polypeptide chain), for example by carboxypeptidase; modification of the glutamine or glutamate at the N-terminus of the heavy chain (such as the first and/or second polypeptide chain) to pyrrolomine by pyrrolomine acidification; modification of the glutamine or glutamate at the N-terminus of the light chain (such as the third and/or fourth polypeptide chain) to pyrrolomine by pyrrolomine acidification; glycosylation; oxidation; deamination; and glycosylation. These post-translational modifications are known to occur in various conjugates (Liu et al., 2008, J. Pharmacol. Sci. 97(7):2426-2447). Post-translational modifications such as N-terminal pyrroleic acidation and C-terminal lysine deletion generally do not affect the activity of the binder (Lyubarskaya et al., 2006, Analyt. Biochem. 348(1):24-39).

Therefore, in some embodiments, the binding agent described herein may include one or more post-translational modifications. In some embodiments, one or more post-translational modifications include pyroglutamylation at the N-terminus of one or more polypeptide chains of the binding agent. In some embodiments, one or more post-translational modifications include pyroglutamylation at the N-terminus of one or more VH (CLDN18.2). In some embodiments, one or more post-translational modifications include deletion of lysine at the C-terminus of a first polypeptide chain. In some embodiments, one or more post-translational modifications include deletion of lysine at the C-terminus of a second polypeptide chain.

In the context of this invention, the binding agents described herein are preferably capable of inducing one or more immune response functions as described herein. Preferably, these immune response functions are targeted at cells with the cancer-associated antigen CLDN18.2 on their surface.

In the context of this invention, "immune effect function" includes any function mediated by components of the immune system that results in, for example, inhibition of cancer growth and/or cancer development, including inhibition of cancer metastasis and spread. Preferably, immune effect function results in the killing of cancer cells. Immune effect functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis in cells carrying cancer-associated antigens, cytolysis of cells carrying cancer-associated antigens, and/or inhibition of proliferation of cells carrying cancer-associated antigens. The binding agents described in this article are better able to recruit and redirect T cells, such as CD4 and/or CD8 T cells, especially CD107a+ T cells, to disease-related cells (such as cancer cells), thereby exerting their effects through redirected T cell cytotoxicity (RTCC), i.e., after redirection, T cells better kill disease-related cells, such as cancer cells. The expression of CD107a is known to be associated with the cytotoxic potential of CD4 and CD8 T cells. Preferably, these CD107a+ T cells are degranulated, meaning they can release cytotoxic molecules such as perforin and granzymes, and may release one or more cytokines such as tumor necrosis factor-α (TNFα), interleukin-2 (IL2), and interferon-γ (IFNγ), thereby leading to the death of target cells (such as cancer cells). The conjugates described in this article redirect T cells. Conjugates may also work solely by binding to cancer-associated antigens on the surface of cancer cells. For example, conjugates may block the function of cancer-associated antigens, or induce apoptosis in cancer cells solely by binding to surface cancer-associated antigens.

In the context of this invention, the term "immune effector cell" or "effector cell" refers to a cell that performs an effector function in 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 and include helper T cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), which include cytolytic T cells. The term "MHC-dependent T cells" or similar terms refers to T cells that, when presented in an MHC context, are able to recognize antigens and better perform T cell effector functions (e.g., kill target cells expressing antigens).

T cells belong to a group of white blood cells called 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 a T-cell receptor (TCR). The thymus is the main organ for T cell maturation. Several different T cell subsets have been identified, each with unique functions.

Helper T cells assist other white blood cells in the immune process, including B cell maturation into plasma cells and the activation of cytotoxic T cells and macrophages. These cells are also called CD4+ T cells because they express CD4 glycoprotein on their surface. Helper T cells are typically activated when they are activated by peptide antigens presented on the surface of antigen-presenting cells (APCs) using MHC class II molecules. Once activated, they rapidly divide and secrete small proteins called cytokines, which regulate or assist the body's immune response.

Cytotoxic T cells destroy virus-infected cells and cancer cells, and play a crucial role in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells typically identify target cells by binding to MHCI-like molecules, which are present on the surface of almost every cell in the body.

All T cells possess a complex protein structure called a T cell receptor (TCR). The T cell TCR interacts with immunogenic peptides (epitopes) that bind to the major histocompatibility complex (MHC) molecule and is present on the surface of target cells. Specific binding of the TCR triggers a series of signaling pathways within the T cell, leading to its proliferation and differentiation into mature effector T cells. In most T cells, the actual T cell receptor consists of two independent peptide chains, produced by individual T cell receptor alpha and beta (TCRα and TCRβ) genes, known as the α and β TCR chains. A small subset of T cells (approximately 2% of all T cells), namely gamma delta T cells, have distinct T cell receptors (TCRs) on their surface, consisting of a gamma chain and a delta chain.

All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic precursor cells derived from hematopoietic stem cells fill the thymus and expand through cell division, generating a large number of immature thymocytes. The earliest thymocytes do not express either CD4 or CD8 and are therefore classified as double-negative (CD4-CD8-) cells. As they develop, they become double-positive thymocytes (CD4+CD8+), eventually maturing into single-positive (CD4+CD8- or CD4-CD8+) thymocytes, and are then released from the thymus into the surrounding tissues.

In this application, the term "NK cell" or "natural killer cell" refers to a type of peripheral blood lymphocyte that expresses CD56 or CD16 and lacks T cell receptors.

MHC molecules in humans are commonly referred to as HLA (human leukocyte antigen) molecules. MHC molecules are mainly divided into two classes: class I and class II. MHC class I antigens are present on almost all nucleated cells. The primary function of these MHC molecules is to display (or present) peptide fragments of intracellular proteins to CTLs (cytotoxic T lymphocytes). Based on this display, CTLs attack cells that display MHC-binding peptides (including disease-related peptides such as cancer antigens). CD8-positive T cells are typically cytotoxic (hence the name cytotoxic T cells = CTLs), recognizing peptides of 9 to 10 amino acids in length formed by the localized treatment of intracellular proteins on the subcellular surface by MHC class I molecules. Therefore, the surface expression of MHC class I molecules plays a key role in determining the susceptibility of target cells to CTLs.

The binding agents described herein can be bound to therapeutic groups or agents, such as cytotoxins, drugs (like immunosuppressants), or radioisotopes. Cytotoxins or cytotoxic agents include any agent that is harmful to cells and, in particular, kills cells. Examples include paclitaxel, cytosolic acid B, polypeptide D, ethionyl tin bromide, rotenone, mitomycin, etoposide, teniposide, vanroxyphene, vincristine, colchicine, doxorubicin, doxorubicin, dihydroxyanthraquinone, mitoxanthraquinone, luciferin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and purines and their analogues or homologues. Suitable therapeutic agents for the formation of conjugates include, but are not limited to, antimetabolites (such as methotrexate, 6-pyridine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil deoxyfluorouridine), alkylating agents (such as dichloroethylmethylamine, thiotepachloramphenicol, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, and dibromoglycine. Mannitol, streptomycin, mitomycin C, and cis-dichlorodiamine platinum II (DDP), cisplatin, anthracycline antibiotics (such as doxorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (such as actinomycin (formerly known as actinomycin), bleomycin, mitocin, and amycin (AMC)), and antimitotic drugs (such as vancritin and vincristine). In a preferred embodiment, the treatment agent is a cytotoxic or radiotoxicant. In another embodiment, the treatment agent is an immunosuppressant. In yet another embodiment, the treatment agent is GM-CSF. In a preferred embodiment, the treatment agent is doxorubicin, cisplatin, bleomycin, sulfuric acid, carmustine, chloramphenicol, or lysine A.

The binder can also bind to radioactive isotopes (such as iodine-131, yttrium-90, or indium-111) to create cytotoxic radiopharmaceuticals.

Techniques for binding this therapeutic group to a conjugate are known; see, for example, Arnon et al., “Monoclonal Antibodies as Immunotargeting Agents in Cancer Therapy” (in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al., eds., pp. 243-256, Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies for Drug Delivery” (in Controlled Drug Release, 2nd ed., Robinson et al., eds., pp. 623-653, Marcel Dekker, Inc., 1987); Thorpe, “The Application of Antibodies as Cytotoxic Agent Carriers in Cancer Therapy: A Review” (in Monoclonal Antibodies). '84: Biological and Clinical Applications, Pinchera et al., eds., pp. 475-506, 1985); and “Efficacy Analysis, Results and Future Prospects of Radiolabeled Antibodies in Cancer Treatment” (in “Application of Monoclonal Antibodies in Cancer Detection and Treatment”, Baldwin et al., eds., pp. 303-316, Academic Press, 1985); and “Preparation of Antibody-Toxin Conjugates and Their Cytotoxic Properties”, Thorpe et al., Immunological Review, 62: 119-158 (1982).

In this invention, "isotype" refers to an antibody class (e.g., IgM or IgG1) encoded by a gene in a heavy chain constant region.

In this invention, "isotype switching" refers to the phenomenon where the class or isotype of an antibody changes from one immunoglobulin class to another.

In this invention, "rearrangement" refers to the configuration of a heavy-chain or light-chain immunoglobulin locus, in which the V segment is located directly adjacent to the D-J or J segment, forming a nearly complete VH or VL region. Rearranged immunoglobulin (antibody) loci can be identified by comparison with germline DNA; rearranged loci will have at least one recombinated heptamer/nonamer homologous element.

In this invention, when "unrearranged" or "reproductive configuration" refers to the V segment, it refers to a configuration in which the V segment is adjacent to the D or J segment without recombination.

In some embodiments, the binder of the present invention has the ability to bind to CLDN18.2, that is, to bind to, and preferably to, an epitope on CLDN18.2, preferably located in the extracellular domain of CLDN18.2, particularly the first extracellular loop, preferably amino acids 29 to 78 of CLDN18.2. In certain specific embodiments, the agent having the ability to bind to CLDN18.2 binds to an epitope on CLDN18.2 that is not present on CLDN18.1.

Drugs capable of binding to CLDN18.2 preferably bind to CLDN18.2 in humans, mice, and/or cynomolgus monkeys, but preferably do not bind to CLDN18.1 in humans, mice, and/or cynomolgus monkeys. Preferably, drugs capable of binding to CLDN18.2 do not bind to CLDN9 in humans, mice, and/or cynomolgus monkeys. Preferably, drugs capable of binding to CLDN18.2 are specific for CLDN18.2. Preferably, drugs capable of binding to CLDN18.2 can bind to CLDN18.2 expressed on the cell surface. In certain preferred embodiments, an agent having the ability to bind to CLDN18.2 can bind to the natural epitope of CLDN18.2 present on the surface of living cells.

In some embodiments, the binding domain includes an antibody fragment. The term "fragment" specifically refers to one or more complementary determinant regions (CDRs) of the heavy chain variable region (VH) and/or the light chain variable region (VL), preferably including at least the CDR3 variable region. In some embodiments, the one or more complementary determinant regions (CDRs) are selected from a set of CDR1, CDR2, and CDR3. In a particular preferred embodiment, the term "fragment" refers to the complementary determinant regions CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) and/or the light chain variable region (VL).

In some embodiments, the binding domain described herein, comprising one or more CDRs, a set of CDRs, or a combination of multiple sets of CDRs, includes the CDRs and the frame regions between them. Preferably, this portion also includes at least about 50% of the first and fourth frame regions, i.e., 50% of the C-terminus of the first frame region and 50% of the N-terminus of the fourth frame region. Binding agents constructed using recombinant DNA technology may result in the introduction of residues at the N-terminus or C-terminus of variable regions encoded by linkers, which are introduced to facilitate selection or other maneuvers, including the introduction of linkers to link the variable regions to other protein sequences, including immunoglobulin heavy chains, other variable regions, or protein tags.

In some embodiments, the binding domains described herein that include one or more CDRs, a set of CDRs, or a combination of multiple CDRs are included in the human antibody framework.

The accurate identification of CDR regions depends on the calculation method used to determine the amino acid residues involved. For example, according to the method of Kabat et al. (ibid.), the variable region typically includes amino acid residues at positions approximately 24–34 (CDR1), 50–56 (CDR2), and 89–97 (CDR3) in the VL, and amino acid residues at positions approximately 31–35 (CDR1), 50–65 (CDR2), and 95–102 (CDR3) in the VH; the variable region may also include residues forming highly variable rings (e.g., positions 26–32 (CDR1), 50–52 (CDR2), and 91–96 (CDR3) in the VL, and positions 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 precise identification of the CDR positions in the sequences disclosed in this disclosure may vary slightly depending on the numbering system used, as shown in Table 1 (see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77(2003)):

Table 1 Kabat + Chothia IMGT Kabat AbM Chothia Contact VH: CDR1 26-35 27-38 31-35 26-35 26-32 30-35 VH: CDR2 50-65 56-65 50-65 50-58 52-56 47-58 VH: CDR3 95-102 105-117 95-102 95-102 95-102 93-101 VL: CDR1 24-34 27-38 24-34 24-34 24-34 30-36 VL: CDR2 50-56 56-65 50-56 50-56 50-56 46-55 VL: CDR3 89-97 105-117 89-97 89-97 89-97 89-96

Therefore, the CDR sequence disclosed in this invention also includes variants derived according to different numbering systems. Thus, the disclosure of each VH is the disclosure of its derived CDRs (e.g., CDR1, CDR2, and CDR3) and the disclosure of each VL is the disclosure of its derived CDRs (e.g., CDR1, CDR2, and CDR3).

In this specification, when referring to the stubs of the variable regions of the CD3 or CLDN18.2 binding domain (approximately stubs 1 to 107 of the light chain and stubs 1 to 113 of the heavy chain), the Kabat numbering system is used; while for the CH1 and CH2-CH3 regions (optionally including hinged regions), the EU numbering system is used, as described herein.

In some embodiments, the CLDN18.2 binding domain of the binder described in this invention includes a VH that includes CDR1, CDR2 and/or CDR3 identified in the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the CLDN18.2 binding domain of the binder described in this invention includes a VL that includes CDR1, CDR2 and/or CDR3 identified in the amino acid sequence of SEQ ID NO: 17.

In a preferred embodiment, the binding domain of the binder described herein includes the following CDR combination: VH includes a CDR3 sequence containing the sequence shown in SEQ ID NO: 12 or a functional variant thereof, and VL includes a CDR3 sequence containing the sequence shown in SEQ ID NO: 15 or a functional variant thereof.

In some embodiments, VH further includes a CDR1 sequence containing the sequence shown in SEQ ID NO: 10 or a functional variant thereof and/or a CDR2 sequence containing the sequence shown in SEQ ID NO: 11 or a functional variant thereof, and/or VL further includes a CDR1 sequence containing the sequence shown in SEQ ID NO: 13 or a functional variant thereof and/or a CDR2 sequence containing the sequence shown in SEQ ID NO: 14 or a functional variant thereof.

In a preferred embodiment, the binding domain of the binder described herein includes the following CDR combinations: VH includes a CDR1 sequence containing the sequence shown in SEQ ID NO: 10 or a functional variant thereof, a CDR2 sequence containing the sequence shown in SEQ ID NO: 11 or a functional variant thereof, and a CDR3 sequence containing the sequence shown in SEQ ID NO: 12 or a functional variant thereof; and VL includes a CDR1 sequence containing the sequence shown in SEQ ID NO: 13 or a functional variant thereof, a CDR2 sequence containing the sequence shown in SEQ ID NO: 14 or a functional variant thereof, and a CDR3 sequence containing the sequence shown in SEQ ID NO: 15 or a functional variant thereof. Preferably, VH includes CDR1, 2, and 3 of SEQ ID NO: 10, 11, and 12; and VL includes CDR1, 2, and 3 of SEQ ID NO: 13, 14, and 15.

In some embodiments, the variable regions of the heavy and light chains include complementary determining regions dispersed within the framework regions. In some embodiments, each variable region includes three complementary determining regions (CDR1, 2, and 3) and four framework regions (FR1, 2, 3, and 4). In some embodiments, the complementary determining regions and framework regions are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

In a preferred embodiment, the CLDN18.2 binding domain of the binder described herein includes a VH (CLDN18.2) containing an amino acid sequence or a functional variant thereof as shown in SEQ ID NO: 16.

In another preferred embodiment, the CLDN18.2 binding domain of the binder described herein includes a VL (CLDN18.2) comprising the amino acid sequence represented by SEQ ID NO: 17 or a functional variant thereof.

In a preferred embodiment, the CLDN18.2 binding domain of the binder described herein includes a combination of VH(CLDN18.2) and VL(CLDN18.2): VH(CLDN18.2) includes the amino acid sequence represented by SEQ ID NO: 16 or a functional variant thereof, and VL(CLDN18.2) includes the amino acid sequence represented by SEQ ID NO: 17 or a functional variant thereof.

In a preferred embodiment, the framework regions of the VH and VL domains presented in the binder described herein may include amino acid variations, but retain at least 80%, 85%, or 90% human germline sequence identity.

In a further embodiment, the CLDN18.2 binding domain of the binder described herein includes a heavy chain and a light chain variable region of the antibody, which (i) competitively binds CLDN18.2 to an antibody including the aforementioned heavy chain and light chain variable region, and/or (ii) has CLDN18.2 specificity of an antibody including the aforementioned heavy chain and light chain variable region.

In some embodiments, the CLDN18.2 binding domain of the binder described herein has the Fab molecular format described herein. In this embodiment, VH (CLDN18.2) is part of the first and second polypeptide chains described herein, and VL (CLDN18.2) is part of the third polypeptide chain described herein and a fourth polypeptide chain identical to the third polypeptide chain.

It should be understood that the CLDN18.2 binding fields of the Fab2-scFv format of the binders described herein can be the same or substantially the same, and therefore can bind the same or substantially the same epitopes of CLDN18.2. Therefore, the two CLDN18.2 binding fields of the Fab2-scFv format of the binders described herein can correspond to or substantially correspond to one of the CLDN18.2 binding fields described herein.

Preferably, the CD3-binding domain is able to specifically recognize human CD3 in the native conformation of the TCR on activated primordial human T cells, in the presence of other TCR subunits.

In some embodiments, the CD3 binding domain of the binder described herein includes a VH containing CDR1, CDR2 and/or CDR3 identified in the amino acid sequence of SEQ ID NO: 25.

In some embodiments, the CD3 binding domain of the binder described herein includes VH, which includes the following combinations of CDR1, CDR2 and CDR3: 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.

In a preferred embodiment, the CD3 binding domain of the binder described herein includes a VL containing CDR1, CDR2 and/or CDR3 identified in the amino acid sequence of SEQ ID NO: 24.

In a preferred embodiment, the CD3 binding domain of the binder described herein includes VL, which includes the following combination of CDR1, CDR2 and CDR3: CDR1: SEQ ID NO: 20 or a functional variant thereof, CDR2: SEQ ID NO: 21 or a functional variant thereof, and CDR3: SEQ ID NO: 22 or a functional variant thereof.

In a preferred embodiment, the CD3 binding domain of the binder described herein includes 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.

In a preferred embodiment, the CD3 binding domain of the binder described herein includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 25 or a functional variant thereof.

In a preferred embodiment, the CD3 binding domain of the binder described herein includes a VL containing the amino acid sequence shown in SEQ ID NO: 24 or a functional variant thereof.

In some embodiments, the CD3 binding domain of the binder described herein includes the following VH and VL: VH includes an amino acid sequence according to SEQ ID NO: 25 or a functional variant thereof or composed thereof, and VL includes an amino acid sequence according to SEQ ID NO: 24 or a functional variant thereof or composed thereof.

In some embodiments, the CD3 binding domain includes the amino acid sequence of SEQ ID NO: 26 or a functional variant thereof or composed of the same.

In some embodiments, the CH1 domain of the first and/or second polypeptide chain of the conjugate described herein is derived from IgG, preferably IgG1, and more preferably human IgG1. In some embodiments, the CH2 and CH3 domains of the first and/or second polypeptide chain of the conjugate described herein are derived from IgG, preferably IgG1, and more preferably human IgG1.

In some embodiments, the CL of the third and/or fourth polypeptide chain of the binder described herein is derived from Igκ or Igλ, preferably Igκ, and more preferably human Igκ.

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

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

In some embodiments, the conjugate described herein includes at least two binding domains for binding CLN18.2 and at least one binding domain for binding CD3. In some embodiments, the first polypeptide chain of the conjugate described herein includes the amino acid sequence represented by SEQ ID NO: 27 or a functional variant thereof, or is composed of the amino acid sequence represented by SEQ ID NO: 28. In some embodiments, the third polypeptide chain of the conjugate described herein includes the amino acid sequence represented by SEQ ID NO: 29 or a functional variant thereof. In some embodiments, the fourth polypeptide chain of the conjugate described herein includes the amino acid sequence represented by SEQ ID NO: 29 or a functional variant thereof, or is composed of the amino acid sequence represented by SEQ ID NO: 29. In some embodiments, the binder described herein includes at least two binding domains for binding CLDN18.2 and at least one binding domain for binding CD3, the binder comprising combinations of first, second, third and fourth polypeptide chains according to SEQ ID NO: 27, 28, 29 and 29.

It should be understood that the conjugating agents described herein can be administered to a patient by delivering a nucleic acid (such as RNA) encoding the drug to the patient and/or by delivering a host cell containing a nucleic acid (such as RNA) encoding the drug to the patient. If the conjugating agent comprises multiple polypeptide chains, different polypeptide chains may be encoded by the same nucleic acid or different nucleic acids, such as a set of nucleic acids. Therefore, the nucleic acid to be delivered may be a mixture of different nucleic acid molecules, such as a set of nucleic acids. The nucleic acid or set of nucleic acids encoding the conjugating agent (e.g., when delivered to an individual such as a patient) may be present in a naked form, or in the form of a suitable delivery vector, such as liposomes, nanoparticles, or viral particles, or within a host cell. The provided nucleic acid or set of nucleic acids may produce the drug in a prolonged manner over a longer period of time, at least partially alleviating the instability observed in therapeutic antibodies. Nucleic acids or nucleic acid sets delivered to patients can be produced through combinatorial methods. If the nucleic acids or nucleic acid sets are administered to a patient in the absence of host cells, it is preferable that these nucleic acids be taken up by the patient's cells to express the drug-binding agent encoded by that nucleic acid. If the nucleic acids or nucleic acid sets are administered to a patient while already within host cells, it is preferable that these cells express the drug-binding agent encoded by that nucleic acid within the patient's body.

In the context of this invention, the term "recombination" means "manufactured by genetic engineering." Preferably, the "recombinant objects" (such as recombinant nucleic acids) in the context of this invention do not exist naturally.

As used in this article, "naturally occurring" means that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses) and can be isolated from natural sources without intentional modification in a laboratory are naturally occurring.

As used in this application, "nucleic acid" is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinated molecules, and chemically synthesized molecules. Nucleic acids may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.

Nucleic acids or sets of nucleic acids may be included in a vector. Nucleic acid combinations may also be included in vector combinations, such that each nucleic acid in the nucleic acid combination is included in a vector. As used herein, "vector" includes any vector known to those skilled in the art, including plasmid vectors, granule vectors, phage vectors (such as λ phage), viral vectors (such as adenovirus vectors or baculovirus vectors), or artificial chromosome vectors (such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). These vectors include expression and selection vectors. Expression vectors include plasmid and viral vectors and typically contain the desired coding sequence and appropriate DNA sequences necessary for expression of the coding sequence to be operatively linked in a specific host organism (such as bacteria, yeast, plants, insects, or mammals) or in vitro expression system. Selection vectors are typically used to engineer and enhance specific desired DNA fragments and functional sequences that may be lacking for the expression of the desired DNA fragment.

In the context of this invention, the term "RNA" refers to a molecule that includes ribonucleotide residues, and preferably consists entirely or primarily of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofurano group. This term includes double-stranded RNA, single-stranded RNA, isolated RNA (such as partially purified RNA, substantially pure RNA), synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA, modifications which may include the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such modifications may include the addition of non-nucleotide material, such as addition to the ends or interior of RNA, for example, at one or more nucleotide positions in RNA. Nucleotides in RNA molecules may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be called analogs or analogs of naturally occurring RNA.

According to the present invention, the term "RNA" includes and preferably refers to "mRNA," or "messenger RNA," which is a "transcriptional material" that can be produced using DNA as a template and encodes peptides or proteins. mRNA typically includes 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 in cells and in vitro environments. Preferably, mRNA is produced using a DNA template through in vitro transcription. In some embodiments of the present invention, RNA is obtained by in vitro transcription or chemical synthesis. Methods of in vitro transcription are well known to those skilled in the art. For example, various in vitro transcription kits are available commercially.

In some embodiments of the present invention, the RNA is self-replicating RNA, such as single-stranded self-replicating RNA. In some embodiments, the self-replicating RNA is valid 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 alpha-viral genomic RNA or RNA derived from alpha-viral genomic RNA. According to common knowledge in the art, alpha-viral RNA can serve as mRNA. 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, wherein at least one transgene encodes the binding agent described herein. In some embodiments, if the RNA is viral RNA or RNA derived from viral RNA, the transgene may partially or completely replace the viral sequence, such as a viral sequence encoding a structural protein. In some embodiments, the self-replicating RNA is RNA transcribed in vitro.

In order to improve the expression and/or stability of the RNA used according to the present invention, it can be modified, preferably without altering the sequence of the expressed peptide or protein.

The "modification" of RNA used in this invention includes any modifications that are not naturally present in the RNA.

In some embodiments of the present invention, the RNA used according to the present invention does not contain uncapped 5'-triphosphate groups. Removal of these uncapped 5'-triphosphate groups can be achieved by treating the RNA with a phosphatase.

The RNA according to the present invention may contain modified natural or synthetic nucleotides to enhance its stability and/or reduce its cytotoxicity and/or immunogenicity. For example, in some embodiments, the cytidine in the RNA used according to the present invention may be partially or completely replaced by 5-methylcytidine, preferably completely replaced. Alternatively, or additionally, the uridine in the RNA used according to the present invention may be partially or completely replaced by pseudouridine, preferably completely replaced.

In some embodiments, the term "modification" refers to providing RNA with a 5'-cap or a 5'-cap analogue. A 5'-cap structure refers to the cap structure at the 5' end of an mRNA molecule, typically consisting of a guanylic acid nucleotide linked to the mRNA by an unusual 5' to 5' triphosphate bond. In some embodiments, the guanylic acid is methylated at the 7-position. In this invention, "common 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably 7-methylguanylic acid-terminated (m7G). In the context of this invention, 5'-caps include 5'-cap analogues that resemble RNA cap structures, modified to possess the ability to stabilize RNA when linked, preferably in vivo and/or intracellularly.

Providing RNA with a 5' cap structure or a 5' cap analogue can be achieved by in vitro transcription of a DNA template containing the 5' cap structure or a 5' cap analogue, wherein the 5' cap structure co-transcribedly binds to the resulting RNA strand during transcription, or the RNA can be generated, for example, by in vitro transcription and then 5' capped using a capping enzyme, such as a capping enzyme of vaccinia virus.

RNA may include other modifications. For example, further modifications to the RNA used in this invention may include the elongation or truncation of naturally occurring poly(A) tails, or changes to the 5' or 3' untranslated regions (UTRs), such as the introduction of UTRs unrelated to the coding region of the RNA, for example, the insertion of one or more, preferably two, 3'-UTRs derived from globulin genes, such as α2-globulin, α1-globulin, β-globulin, preferably β-globulin, and more preferably human β-globulin.

Therefore, to improve the stability and/or performance of the RNA used in this invention, it can be modified to bind a poly-A sequence, preferably 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, and particularly 100 to 150 adenosine residues. In a particularly preferred embodiment, the poly-A sequence is approximately 120 adenosine residues long. Furthermore, incorporating two or more 3' untranslated regions (UTRs) into the 3' untranslated region of the RNA molecule can improve translation efficiency. In a specific embodiment, the 3'-UTR is derived from the human β-globulin gene.

Ideally, if RNA is delivered, it is transfected into cells, especially in the body, where it will express the proteins, peptides, or antigens it encodes.

The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. Within the scope of this invention, "transfection" also includes the introduction of nucleic acids into or the uptake of nucleic acids by such cells, wherein the cells may be present in an individual (e.g., a patient). Therefore, according to this invention, cells used for transfecting nucleic acids as described herein can be cells present in vitro or in vitro, such as cells that are part of a patient's organ, tissue, and/or organism. According to this invention, transfection can be transient or stable. For some transfection applications, it is sufficient if the transfected genetic material can be expressed temporarily. Since the nucleic acids introduced during transfection typically do not integrate into the nuclear genome, the foreign nucleic acids are diluted or degraded due to mitosis. Cells that allow for the expansion of nucleic acid episomes can significantly reduce the dilution rate. If the nucleic acid to be transfected is to actually remain in the genome of the cell and its daughter cells, stable transfection must be performed. RNA can be transfected into cells to temporarily express the protein it encodes.

In this invention, "stability" refers to the "half-life" of RNA. Half-life is the time required to eliminate half of the activity, quantity, or molecular weight of RNA. Within the context of this invention, the half-life of RNA can serve as an indicator of its stability. The half-life of RNA may affect its "performance time." It can be expected that RNA with a longer half-life will have a longer performance time.

In the context of this invention, "transcription" refers to a process in which genetic codes in a DNA sequence are transcribed into RNA. Subsequently, the RNA may be translated into proteins. According to this invention, "transcription" includes "in vitro transcription," which refers to a process of synthesizing RNA, particularly mRNA, in a cell-free line system, preferably using appropriate cell extracts. Preferably, selection vectors are used to generate transcription products. These selection vectors are commonly referred to as transcription vectors and, according to this invention, are encompassed within the scope of the term "vector."

According to the present invention, "translation" refers to a process within the cellular ribosome in which a sequence of mRNA directs the assembly of amino acid sequences to generate peptides or proteins.

As used in this invention, the term "performance" is used in its broadest sense, including the production of RNA and/or peptides or proteins, for example, through transcription and/or translation. Regarding RNA, the term "performance" or "translation" specifically refers to the production of peptides or proteins. It also includes partial performance of nucleic acids. Furthermore, performance can be transient or stable. According to this invention, the term "performance" also includes "abnormal performance" or "non-standard performance."

According to the present invention, "abnormal manifestation" or "abnormal performance" refers to a change, preferably an increase, in performance compared to a reference state, such as a state when the individual does not have a disease associated with abnormal or abnormal performance of a specific protein (e.g., tumor antigen). An increase in performance is defined as an increase of at least 10%, particularly at least 20%, at least 50%, or at least 100% or more. In some embodiments, the performance is found only in diseased tissue, while performance in healthy tissue is suppressed.

The term "specific expression" refers to a protein being expressed primarily only in a specific tissue or organ. For example, a tumor antigen specifically expressed in the gastric mucosa means that the protein is expressed mainly in the gastric mucosa and not expressed or expressed at significantly lower levels in other tissue or organ types. Therefore, if a protein is expressed uniquely only in gastric mucosal cells and expressed at significantly lower levels in other tissues such as the testes, it is considered to be specifically expressed in gastric mucosal cells. In some embodiments, a tumor antigen may normally be specifically expressed in more than one tissue type or organ, such as in two or three tissue types or organs, but preferably no more than three different tissue or organ types. In this case, the tumor antigen will be specifically expressed in these organs. For example, if a tumor antigen is normally expressed primarily in the lungs and stomach (ideally to almost the same degree), then the tumor antigen will be specifically expressed in the lungs and stomach.

According to this invention, the term "RNA coding" refers to the ability of RNA, in a suitable environment, preferably within a cell, to be expressed in order to produce the proteins or peptides it encodes.

Some aspects of this invention rely on the acceptor transfer of host cells transfected with nucleic acids (such as RNA encoding the drug-binding agents described herein) in vitro to a recipient (such as a patient), preferably after in vitro amplification from a low precursor frequency to a clinically relevant number of cells. The host cells used for treatment according to this invention can be autologous, allogeneic, or syngeneic to the treatment recipient.

The term "autologous" is used to describe any substance derived from the same individual. For example, "autologous transplantation" refers to the transplantation of tissues or organs derived from the same individual. This type of surgery has advantages because it can overcome immune barriers that would otherwise lead to rejection.

The term "alien" is used to describe any substance that originates from different individuals of the same species. When two or more individuals have different genes at one or more gene loci, they are called allogeneic.

The term "homogeneous" is used to describe any substance that originates from an individual or tissue with the same genotype, such as identical twins or animals or tissues of the same inbred strain.

In this invention, the term "heterogeneous" is used to describe something composed of multiple different components. For example, transferring bone marrow from one person to another constitutes a heterologous transplant. Heterogeneous genes refer to genes originating from outside the individual.

According to this invention, the term "peptide" includes oligopeptides and polypeptides, referring to a substance consisting of two or more, preferably three or more, more preferably four or more, more preferably six or more, more preferably eight or more, more preferably nine or more, more preferably ten or more, more preferably thirteen or more, more preferably sixteen or more, more preferably twenty or more, with a maximum and preferably eight, ten, twenty, thirty, forty or fifty, particularly 100 amino acids covalently linked by peptide bonds. The term "protein" refers to larger peptides, preferably peptides containing more than 100 amino acid residues, but generally, the terms "peptide" and "protein" are synonymous and used interchangeably in this invention.

For specific amino acid sequences (e.g., those listed in the sequence listing), the teachings herein should be understood to also involve variations of these specific sequences to form sequences functionally equivalent to that specific amino acid sequence, such as amino acid sequences exhibiting the same or similar properties as those specific amino acid sequences. An important property is maintaining the ability to bind to a target or maintaining the effect function. Preferably, a sequence is a variation of a specific sequence that, when substituted in an antibody, maintains the binding ability of the antibody to CLDN18.2 and/or CD3 and preferably retains the antibody function described herein. Furthermore, preferably, a sequence is a variation of a specific sequence that, when substituted in a binding agent, still maintains the binding ability of the binding agent to CLDN18.2 and/or CD3 and preferably retains the binding agent function described herein, such as cytotoxic T cell-mediated lysis.

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

Those skilled in the art will understand that sequences, particularly CDRs, highly variable regions, and variable regions, can be modified without losing their ability to bind to CLDN18.2 and/or CD3. For example, CDR regions will be identical to or highly homologous to the regions specified herein. "Highly homologous" means that there can be 1 to 5, preferably 1 to 4, substitutions in the CDR region, such as 1 to 3 or 1 or 2. Furthermore, highly variable and variable regions can also be modified to show substantial homology to the regions specifically disclosed herein. In some embodiments, the variable region sequence deviates from the variable region sequence specifically disclosed herein only in the frame region sequence.

The binding agents described herein can be produced intracellularly (e.g., in the cytoplasm, periplasm, or inclusion bodies), then isolated from host cells and optionally further purified; or they can be produced extracellularly (e.g., in a culture medium containing host cells), then isolated from the culture medium and optionally further purified. Methods and reagents for recombinant protein production, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc., are known in the art. Similarly, protein isolation and purification techniques are well known to those skilled in the art.

The term "cell" or "host cell" preferably refers to an intact cell, that is, a cell with an intact membrane that has not released its normal intracellular components (such as enzymes, organelles, or genomes). Preferably, an intact cell is a living cell, that is, a living cell capable of performing its normal metabolic functions. According to the present invention, the terminology preferably refers to any cell that can be transfected with a foreign nucleic acid. Preferably, when a cell is transfected with a foreign nucleic acid and transferred to a receptor, the nucleic acid can be expressed in the receptor. The term "cell" includes bacterial cells; other useful cells include yeast cells, fungal cells, or mammalian cells. Suitable bacterial cells include Gram-negative strains such as *Escherichia coli*, *Providencebringus*, and *Pseudomonas*, as well as Gram-positive strains such as *Bacillus subtilis*, *Streptococcus*, *Staphylococcus*, and *Lactococcus*. Suitable fungal cells include cells of the genera *Trichoderma*, *Cytomyces*, and *Aspergillus*. Suitable yeast cells include *Bacillus cerevisiae* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces pombe*, *Hansenula* (e.g., *Pichia pastoris* and *Pichia methanolica*), and *Henochia* cells. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, and 293HEK cells. However, amphibian cells, insect cells, plant cells, and any other cells used to express heterologous proteins can also be used. For adaptive transfer, mammalian cells, such as those from humans, mice, hamsters, pigs, goats, and primates, are particularly preferred. Cells can originate from a variety of tissue types, including primary cells and cell lines, such as those in 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 on their surface via the major histocompatibility complex (MHC). T cells can recognize this complex using their T cell receptors (TCRs).

The ability of antibodies and other binding agents to bind to antigens can be determined by standard binding assays (e.g., ELISA, Western blot, immunofluorescence, and flow cytometry).

The conjugates described herein can also be tested in in vivo models (e.g., in immunodeficient mice carrying xenograft tumors, using cell lines expressing CLDN18.2) to determine their effectiveness in controlling the growth of CLDN18.2-expressing tumor cells.

As used in this specification, “reduce,” “lower,” or “inhibit” refers to the ability to reduce or cause a reduction in the whole, preferably at a level of 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more, such as cell expression or proliferation levels.

Terms such as "increase" or "enhance" preferably refer to an increase or enhancement of at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, best at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or even higher. Antibody-dependent cell-mediated cytotoxicity.

ADCC describes the cytotoxic capacity of effector cells, particularly lymphocytes, as described in this article, which preferably require target cells to be labeled with antibodies.

ADCC preferably occurs when an antibody binds to an antigen on a tumor cell and the antibody's Fc domain interacts with an Fc receptor (FcR) on the surface of an immune response cell. Several Fc receptor families have been identified, and specific cell populations typically exhibit well-defined Fc receptors. ADCC can be viewed as a mechanism for directly inducing varying degrees of immediate tumor destruction, leading to antigen presentation and a tumor-directed T-cell response. Preferably, in vivo induction of ADCC will result in both a tumor-directed T-cell response and a host-derived antibody response. Antibody-dependent cell-mediated phage activity.

ADCP is one of the mechanisms of action in many antibody therapies. It is defined as a highly regulated process in which the Fc region of an antibody binds to a specific receptor on a phage, eliminating the bound target cell and triggering phage activity. ADCP can be mediated by monocytes, macrophages, neutrophils, and dendritic cells via FcγRIIa, FcγRI, and FcγRIIIa, with FcγRIIa (CD32a) on macrophages being the primary pathway.

ADCP occurs most effectively when nonspecific phages expressing Fcγ receptors recognize antibodies that bind to target cells (such as disease cells, including tumor cells), subsequently leading to phagocytosis of these target cells. ADCP can also stimulate downstream adaptive immune responses by promoting antigen presentation or stimulating the secretion of inflammatory mediators. Simultaneous in vivo treatment with immunomodulatory drugs can improve ADCP. The Fc receptor-dependent function of ADCP provides a mechanism for clearing viruses and virus-infected cells, and for stimulating downstream adaptive immune responses by promoting antigen presentation or stimulating the secretion of inflammatory mediators. Complement-dependent cytotoxicity.

CDC is another antibody-induced cell killing mechanism. IgM is the most potent complement activator. IgG1 and IgG3 are also potent complement activators via the classical complement initiation pathway. Preferably, in this cascade reaction, the formation of the antigen-antibody complex exposes multiple C1q binding sites in the CH2 region of the participating antibody molecule (such as IgG molecule) (C1q is one of the three subcomponents of complement C1). These exposed C1q binding sites preferably transform the originally low-affinity C1q−IgG interaction into a high-affinity interaction, triggering a cascade of events involving other complement proteins, and leading to effector cytokinesis and the proteolytic release of activators C3a and C5a. Preferably, the complement cascade reaction ultimately forms a membrane attack complex, which creates pores in the cell membrane, allowing water and solutes to freely enter and exit the cell. Chimerism

Unlabeled mouse antibodies exhibit high immunogenicity when repeatedly applied to humans, leading to reduced therapeutic efficacy. This primary immunogenicity is mediated by the heavy chain constant region. Chimeric or humanized conjugates derived from mouse antibodies can reduce or completely eliminate their immunogenicity in humans. Chimeric conjugates are conjugates composed of different parts derived from different animal species, such as variable regions derived from mouse antibodies and constant regions from human immunoglobulins. Chimerism of conjugates is achieved by linking the variable regions of the heavy and light chains of mouse antibodies with the constant regions of the heavy and light chains of humans (e.g., the method described by Kraus et al. in the *Molecular Biology Methods Series*, ISBN-0-89603-918-8). In one preferred embodiment, the chimeric conjugate is generated by linking a constant region of the human κ light chain with a variable region of the mouse light chain. In yet another preferred embodiment, the chimeric conjugate can be generated by linking a constant region of the human λ light chain with a variable region of the mouse light chain. Preferred constant regions of the heavy chain for generating the chimeric conjugate include IgG1, IgG3, and IgG4. Other preferred constant regions of the heavy chain include IgG2, IgA, IgD, and IgM. Humanization

Binding agents (such as antibodies) primarily interact with target antigens through amino acid residues located in the complementary determinant regions (CDRs) of the six heavy and light chains. Therefore, the amino acid sequences within the CDRs exhibit greater diversity among individual antibodies than those outside the CDRs. Since CDR sequences lead to most antibody-antigen interactions, these sequences can be constructed as expression vectors containing CDR sequences of specific natural antibodies and then grafted onto the backbone sequences of other antibodies with different properties. This allows for the expression of recombinant binders (such as antibodies) that mimic the properties of specific natural antibodies (see, for example, Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U. S. A. 86: 10029-10033). These backbone sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences differ from mature antibody gene sequences because they do not include the fully assembled variable region genes, which are formed during B cell maturation via V(D)J recombination. Germline gene sequences also differ from the individual locations within the variable region of high-affinity secondary library antibodies. Drugs that stabilize or increase the expression of clathrin 18.2 (CLDN18.2) are also relevant.

This article demonstrates that administration of a drug that stabilizes or increases CLDN18.2 performance can support the efficacy of the bispecific conjugates described herein. In some embodiments, a combination therapy for the effective treatment and/or prevention of cancer is provided, comprising administering to a patient the bispecific conjugates described herein and a drug that stabilizes or increases CLDN18.2 performance. This drug may be administered before, simultaneously with, or after the bispecific conjugate, or in combination thereof.

Certain chemotherapy agents, such as gemcitabine, platinum oxide, and 5-fluorouracil, have been shown to upregulate the expression level of existing CLDN18.2 in cancer cells (WO2013/174510; Türeci Ö. et al. (2019) OncoImmunology, 8:1).

The term "agent that stabilizes or increases CLDN18.2 expression" refers to a drug or combination of drugs that, when administered to cells, results in increased levels of CLDN18.2 RNA and/or protein, preferably increased levels of CLDN18.2 protein on the cell surface, compared to when the drug or combination of drugs is not administered. Preferably, these cells are cancer cells, particularly cancer cells expressing CLDN18.2. The term "agent that stabilizes or increases CLDN18.2 expression" also specifically refers to a drug or combination of drugs that, when administered to cells, results in a higher density of CLDN18.2 on the cell surface than when the drug or combination of drugs is not administered to the cells. "Stable CLDN18.2 performance" specifically includes medications or combinations of medications that prevent or reduce the decrease in CLDN18.2 performance. For example, CLDN18.2 performance may decrease when no medication or combination of medications is provided, and providing medications or combinations of medications prevents or reduces the decrease in CLDN18.2 performance. "Increasing the performance of CLDN18.2" specifically includes the increase in the amount of CLDN18.2 performance by a drug or drug combination. For example, when no drug or drug combination is provided, the amount of CLDN18.2 performance decreases, remains essentially unchanged, or increases, while when a drug or drug combination is provided, the amount of CLDN18.2 performance is higher than when no drug or drug combination is provided, thus resulting in a higher final performance, compared to the decrease, remaining essentially unchanged, or increasing of CLDN18.2 performance when no drug or drug combination is provided.

In some embodiments, "agents that stabilize or increase CLDN18.2 expression" include chemotherapy agents or combinations of chemotherapy agents, such as cell growth inhibitors. In some embodiments, agents that stabilize or increase CLDN18.2 expression may be cytotoxic agents and/or cell growth inhibitors.

In some embodiments, the term "agent that stabilizes or increases CLDN18.2 performance" refers to an agent or combination of agents, such as a cell growth inhibitory compound or a combination of cell growth inhibitory compounds, which, when administered to cells, particularly cancer cells, cause arrest or accumulation of cells at one or more stages of the cell cycle, preferably stages other than G1 and G0, more preferably stages other than G1, preferably one or more stages of G2 or S of the cell cycle, or a combination of S or G2 and G1, such as G1/G2, S/G2, G2 or S of the cell cycle. The term "cell arrest or accumulation at one or more stages of the cell cycle" refers to an increase in the percentage of cells in one or more stages of the cell cycle. Each cell goes through a four-stage cycle to replicate itself. The first stage is called G1 phase, during which the cell prepares to replicate its chromosomes. The second stage is called S phase, during which DNA synthesis and replication occur. Next is G2 phase, during which RNA and proteins replicate. Finally, there is M phase, which is the actual cell division stage. In the final stage, the replicated DNA and RNA separate and move to opposite ends of the cell, and the cell eventually divides into two identical, functional cells. Typically, chemotherapy drugs that cause DNA damage lead to cell accumulation in the G1 and/or G2 phases. Chemotherapy drugs that interfere with DNA synthesis and thus block cell growth, such as antimetabolites, usually lead to cell accumulation in the S phase. Examples of these drugs include 6-phenylpurine and 5-fluorouracil.

In some embodiments, agents used to stabilize or increase CLDN18.2 performance may include agents selected from the following groups: anthracycline antibiotics, nucleoside analogs, platinum compounds, camptothecin analogs and taxanes, their prodrugs, their salts, and combinations thereof. The anthracycline drug may be selected from the group comprising epirubicin, doxorubicin, doxorubicin, idarubicin, pentorubicin, their prodrugs and their salts. The anthracycline drug may be selected from the group comprising epirubicin, its prodrugs and their salts. Preferably, the anthracycline drug is epirubicin. The nucleoside analog may be selected from the group comprising gemcitabine, 5-fluorouracil, their prodrugs and their salts. The platinum compound may be selected from the group consisting of oxaliplatin, cisplatin, its prodrug, and its salts. The camptothecin analogue may be selected from the group consisting of irinotecan, topfuratec, its prodrug, and its salts. The paclitaxel analogue may be selected from the group consisting of paclitaxel, docetaxel, its prodrug, and its salts.

In some embodiments, the designation of an agent used to stabilize or enhance CLDN18.2 performance, such as anthracyclines, nucleoside analogs, platinum compounds, camptothecin analogs, or taxanes, such as gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan, or paclitaxel, includes any prodrug, such as an ester, salt, or derivative, such as a conjugate of the agent. Examples include conjugates of the agent with a carrier, such as protein-bound paclitaxel, such as albumin-bound paclitaxel. Preferably, the salts of the agent are pharmaceutically acceptable.

In some embodiments, “agents that stabilize or increase the performance of CLDN18.2” include “agents that induce immunogenic cell death”.

Under certain circumstances, cancer cells can enter a lethal stress pathway associated with a combination of spatiotemporally defined messages decoded by the immune system to activate a tumor-specific immune response (Zitvogel L. et al. (2010) Cell 140: 798–804). In this case, cancer cells are triggered to emit messages perceived by innate immune effector cells (such as dendritic cells), thereby triggering a homologous immune response involving CD8+ T cell and IFN-γ signaling, enabling tumor cell death to elicit an effective antitumor immune response. These signals include pre-apoptotic exposure of the endoplasmic reticulum (ER) molecule catechin calcium reticulum (CRT) on the cell surface, pre-apoptotic secretion of ATP, and post-apoptotic release of the nuclear protein HMGB1. These processes collectively constitute the molecular determinants of immunogenic cell death (ICD). Anthracyclines, oxaliplatin, and gamma radiation can induce all the signals defining ICD, while drugs such as cisplatin, which lack the ER-induced translocation of CRT from the ER to the surface of dying cells (a process requiring ER stress), require supplementation with ER stress inducers such as thioguanidine.

The term "anti-immunogenic cell death agents" as used in this article refers to agents or combinations of agents that, when administered to cells (especially cancer cells), induce a lethal stress pathway, ultimately leading to a tumor-specific immune response. Specifically, when administered to cells, anti-immunogenic cell death agents cause the cells to emit a spatiotemporally defined combination of signals, including pre-apoptotic exposure of the endoplasmic reticulum (ER) molecule ligand calcium reticulum (CRT) on the cell surface, pre-apoptotic secretion of ATP, and post-apoptotic release of the nuclear protein HMGB1.

In this invention, the term "drugs that induce immune cell death" includes anthraquinone drugs such as epirubicin and oxaliplatin.

The term "nucleoside analogue" refers to a structural analogue of a nucleoside, which includes purine analogues and pyrimidine analogues.

Specifically, the term "gemcitabine" refers to a nucleoside analogue with the following formula:

Specifically, the term refers to the compound: 4-amino-1-(2-deoxy-2,2-difluoro-β-D-furanoribosyl)pyridin-2(1H)-one or 4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oxacyclopentan-2-yl]-1,2-dihydropyridin-2-one.

The term "nucleotide analogue" includes fluoropyrimidine derivatives, such as 5-fluorouracil and its prodrugs. The terms "fluorouracil" or "5-fluorouracil" (5-FU or f5U) (marketed under names such as Adrucil, Carac, Efudix, Efudex, and Fluoroplex) are base analogues having the following formula:

Specifically, the term refers to the compound: 5-fluoro-1H-pyridine-2,4-dione.

The term "capecitabine" (Xeloda, Roche) refers to a chemotherapy drug, which is a prodrug that is converted into 5-FU in tissues. Orally administered capecitabine has the following formula:

Specifically, the term refers to the compound: pentyl[1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl] carbonate.

Floxuridine (5-fluorodeoxyuridine) is an oncology drug that is rapidly metabolized to 5-fluorouracil, which is the active form of the drug. Floxuridine has the following formula:

Tegafur (5-fluoro-1-(oxacyclopentan-2-yl)pyrimidine-2,4(3H,5H)-dione) is a pro-chemotherapeutic agent of 5-fluorouracil. After metabolism, it is converted to 5-fluorouracil. Tegafur has the following formula:

Doxifluridine (5'-deoxy-5-fluorouridine) is a fluoropyrimidine derivative of 5-fluorouracil. This second-generation nucleoside analog prodrug is used as a cell growth inhibitor in chemotherapy in several Asian countries, including China and South Korea. Intracellularly, pyrimidine nucleoside phosphatase or thymidine phosphatase metabolizes doxifluridine to 5-fluorouracil. It is also a metabolite of capecitabine. Orally administered doxyfluridine has the following formula:

"Carmofur" (INN) or "HCFU" refers to 1-hexylcarbonate-5-fluorouracil.

This compound is a pyrimidine analog used as an antitumor drug. It is a derivative of fluorouracil and a lipophilic-masked 5-fluorouracil analog. Once inside the cell, carmoflu prodrug is converted to 5-fluorouracil.

"Platinum compounds" refers to compounds whose structure includes platinum, such as platinum complexes, including compounds such as cisplatin, carboplatin, and oxaliplatin.

"Cisplatin (or cisplatinum)" refers to trans-dichlorodiamine platinum(II) (CDDP), whose chemical formula is:

"Carboplatinum" refers to trans-diamine (1,1-cyclosuccinic acid)platinum(II), whose chemical formula is:

"Oxaliplatinum" refers to a platinum compound complexed with a diaminocyclohexane support ligand, and its chemical formula is:

Specifically, "oxaliplatinum" refers to [(1R,2R)-cyclohexane-1,2-diamine](oxalic acid O,O')platinum(II). Oxaliplatinum for injection is also marketed under the trade name Eloxatine.

Paclitaxels are a class of diterpenoids extracted from natural sources such as plants in the genus *Taxus*, although some have been synthesized artificially. The main mechanism of action of paclitaxel drugs is to interfere with microtubule function, thereby inhibiting cell division. Paclitaxel drugs include docetaxel (Taxotere) and taxol.

"Docetaxel" refers to a compound having the following formula:

Specifically, "docetaxel" refers to 1,7β,10β-trihydroxy-9-oxo-5β,20-epoxytaxane-11-ene-2α,4,13α-triol 4-acetate 2-benzoate 13-((2R,3S)-3-(tert-butoxycarbonyl)amino-2-hydroxy-3-phenylpropionate).

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

Specifically, the term "paclitaxel" refers to the compound (2α,4α,5β,7β,10β,13α)-4,10-diethoxy-13-[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropionic]oxy-1,7-dihydroxy-9-oxo-5,20-epoxytaxane-11-ene-2-ylbenzoate.

The term "camptothecin analogue" refers to derivatives of the compound camptothecin (CPT; (S)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolo[1,2-b]quinoline-3,14(4H,12H)-dione). Preferably, the term "camptothecin analogue" refers to compounds comprising the following structures:

Preferred camptothecin analogues are inhibitors of the DNase topoisomerase I (topo I). Preferred camptothecin analogues include irinotecan and toponotecan.

Irinotecan is a drug that prevents DNA unwinding by inhibiting topoisomerase I. Chemically, it is a semi-synthetic natural alkaloid analogue of camptothecin, with the following formula:

Specifically, the term "irinotecan" refers to the compound (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo-1H-pyrano[3',4':6,7]indolo[1,2-b]quinoline-9-yl-[1,4’-bispiperidine]-1’-carboxylic acid ester.

Topotecan is a topotecan isomerase inhibitor with the following formula:

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

Anthracyclines are a class of antibiotics commonly used in cancer chemotherapy. Structurally, all anthracycline drugs share a common tetracyclic 7,8,9,10-tetrahydrotetrabenzo-5,12-quinone structure, and usually require glycosylation at specific positions.

Anthracycline drugs preferably achieve one or more of the following mechanisms of action: 1) by intercalating between base pairs in the DNA/RNA chain, thereby blocking the synthesis of DNA and RNA and thus preventing the replication of rapidly growing cancer cells; 2) by inhibiting topological isomerase II, preventing the unwinding of supercoiled DNA and thus blocking DNA transcription and replication; 3) by generating iron-mediated free radical groups, which damage DNA and cell membranes.

The term "anthracycline drugs" is better used to refer to a type of drug, preferably an anticancer drug used to induce apoptosis, preferably by inhibiting the rebinding of DNA in topoisomerase II.

Preferably, "anthracycline drugs" generally refers to a class of compounds having the following cyclic structure: This includes analogues and derivatives, pharmaceutically acceptable salts, hydrates, esters, conjugates, and prodrugs.

For example, anthracycline antibiotics and their analogues include, but are not limited to: daunomycin, doxorubicin, epirubicin, idarubicin, rhodomycin, pirarubicin, valrubicin, and N-trifluoroacetyl-daunorubicin-14-phenylacetic acid. The list includes doxorubicin-14-valerate, aclacinomycin, morpholino-DOX, cyano-morpholino-DOX, 2-pyrrolino-doxorubicin (2-PDOX), 5-iminodaunomycin, mitoxantrone, and aclarubicin A. Mitoxantrone belongs to the anthraquinone class of compounds and is an anthraquinone antibiotic analogue without a glycosidic group, but still retains a planar polycyclic aromatic structure that can be inserted into DNA.

The best anthracene ring compounds are those having the following formula:

in

_R1 is the group consisting of free H and OH, _R2 is the group consisting of free H and OMe, _R3 is the group consisting of free H and OH, and _R4 is the group consisting of free H and OH.

In one embodiment, _R1 is H, _R2 is OMe, _R3 is H, and _R4 is OH. In another embodiment, _R1 is OH, _R2 is OMe, _R3 is H, and _R4 is OH. In another embodiment, _R1 is OH, _R2 is OMe, _R3 is OH, and _R4 is H. In another embodiment, _R1 is H, _R2 is H, _R3 is H, and _R4 is OH.

The specific anthracycline considered is epirubicin. Epirubicin is an anthracycline drug with the following chemical structure: In the United States, this drug is marketed under the brand name Ellence, while in other regions it is marketed as Pharmorubicin or Epirubicin Ebewe. Specifically, the term "epirarubicin" refers to the compound (8R,10S)-10-[(2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyl-oxacyclohexane-2-yl]oxy-6,11-dihydroxy-8-(2-hydroxyacetyl)-1-methoxy-8-methyl-9,10-dihydro-7H-tetracyclo-5,12-dione. Epirubicin is preferred in some chemotherapy regimens compared to the most common anthracycline antibiotic, doxorubicin, because it appears to cause fewer side effects.

The bispecific conjugates described herein can be administered to an individual simultaneously or sequentially with a drug that stabilizes or enhances CLDN18.2 performance, in any order. For example, the bispecific conjugate can be administered before or after a drug that stabilizes or enhances CLDN18.2 performance.

The compositions described herein may also include other agents, and the methods described herein may also include administration of other agents. In some embodiments, the agents are used for the treatments described herein. In some embodiments, the agents are used to prevent angiogenesis. In some embodiments, the agents include antibodies that bind to human VEGFR2. In some embodiments, the agents are used to enhance T cell function. In some embodiments, the agents include immune checkpoint inhibitors. Antibodies that bind to human VEGFR2

Tumor blood vessels transport nutrients for tumor development and progression and provide pathways for tumor cell escape. Drugs targeting angiogenesis can block the tumor's nutrient supply, thereby achieving a "starvation" effect on the tumor. However, tumor angiogenesis is comprehensively regulated by multiple growth factors, receptors, and downstream signaling pathways. Among them, vascular endothelial growth factor (VEGF) and its receptor (VEGFR) play an important role in both physiological and pathological angiogenesis.

VEGFR2 (also known as FLK-1 or KDR) is a major member of the VEGFR family and a type III receptor tyrosine kinase, primarily distributed on the surface of vascular and lymphatic endothelial cell membranes. The extracellular domain of VEGFR2 comprises seven immunoglobulin-like domains (D1-D7), including domains that bind to the ligand VEGF (D2-D3) and dimerization domains (D4-D7). Its intracellular domain includes a tyrosine kinase domain. When high concentrations of VEGF dimers in the tumor microenvironment bind to VEGFR2, it induces the formation of homodimers (major) and heterodimers (minor) with VEGFR1 or VEGFR3, leading to autophosphorylation of tyrosine residues in the intracellular domain of VEGFR2, ultimately triggering tumor angiogenesis.

In some embodiments, as used herein, the term "VEGFR2" refers to a polypeptide comprising the amino acid sequence listed in SEQ ID NO: 30 or a variant thereof, for example, an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 30. In some embodiments, as used herein, the term "VEGFR2" refers to a polypeptide comprising the amino acid sequence listed in SEQ ID NO: 30. In some embodiments, as used herein, the term "VEGFR2" refers to a polypeptide with the amino acid sequence listed in SEQ ID NO: 30. VEGFR2 is also known as a kinase domain receptor (KDR). As used herein, the terms "VEGFR2 outer domain" or "VEGFR2-ECD" refer to the protein from amino acid 1 to amino acid 744 of SEQ ID NO: 30. (VEGFR2; SEQ ID NO: 30)ASVGLPSVSLDLPRLSIQKDILTIKANTTLQITCRGQRDLDWLWPNNQSGSEQRVEVTECSDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYVQDYRSPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVSLCARYPEKRFVPDGNRISWDSKK GFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVVGYRIYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFVAFGSGMESLVEATVGERVRIP AKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPQIGEKSLISPVDSYQYGTTQTLTCTVYAIPPPHHIHWYWQLEEECANEPSQAVSVTNPYPCEEWRSVEDFQGGNKIEVNKNQFALIEGKNKTVSTLV IQAANVSALYKCEAVNKVGRGERVISFHVTRGPEITLQPDMQPTEQESVSLWCTADRSTFENLTWYKLGPQPLPIHVGELPTPVCKNLDTLWKLNATMFSNSTNDILIMELKNASLQDQGDYVCLAQDRKTKKRHCVVRQLTVLERVAPTITGNLENQTTSIGESIEV SCTASGNPPPQIMWFKDNETLVEDSGIVLKDGNRNLTIRRVRKEDEGLYTCQACSVLGCAKVEAFFIIEGAQEKTNLEIIILVGTAVIAMFFWLLLVIILRTVKRANGGELKTGYLSIVMDPDELPLDEHCERLPYDASKWEFPRDRLKLGKPLGRGAFGQVIEADA FGIDKTATCRTVAVKMLKEGATHSEHRALMSELKILIHIGHHLNVVNLLGACTKPGGPLMVIVEFCKFGNLSTYLRSKRNEFVPYKTKGARFRQGKDYVGAIPVDLKRRLDSITSSQSSASSGFVEEKSLSDVEEEEAPEDLYKDFLTLEHLICYSFQVAKGMEFLAS RKCIHRDLAARNILLSEKNVVKICDFGLARDIYKDPDYVRKGDARLPLKWMAPETIFDRVYTIQSDVWSFGVLLWEIFSLGASPYPGVKIDEEFCRRLKEGTRMRAPDYTTPEMYQTMLDCWHGEPSQRPTFSELVEHLGNLLQANAQQDGKDYIVLPISETLSMEE DSGLSLPTSPVSCMEEEEVCDPKFHYDNTAGISQYLQNSKRKSRPVSVKTFEDIPLEEPEVKVIPDDNQTDSGMVLASEELKTLEDRTKLSPSFGGMVPSKSRESVASEGSNQTSGYQSGYHSDDTDTTVYSSEEAELLKLIEIGVQTGSTAQILQPDSGTTLSSPPV

VEGFR2 antibody drugs can block the binding of VEGFR2 and VEGF.

As used herein, the terms "VEGFR2 antibody" or "anti-VEGFR2 antibody" or "anti-VEGFR2 Ab" refer to antibodies that bind to VEGFR2, preferably antibodies that bind to the outer domain of VEGFR2.

The antibody will have a sufficiently strong binding affinity for VEGFR2. In some embodiments, the K_{_d} value of the antibody binding to VEGFR2 is between about 100 nM and about 1 pM. The antibody affinity can be determined, for example, by surface plasmon resonance assays (such as the BIAcore assay described in PCT application publication WO2005/012359); enzyme-linked immunosorbent assays (ELISA); and competitive assays (e.g., radioactive antigen binding assays (RIA)). In one embodiment, the K_{_d} value is measured by an RIA assay using an anti-VEGFR2 antibody, preferably ramucirumab.

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

In some embodiments, the anti-VEGFR2 antibody comprises: a heavy chain variable region (VH) of the amino acid sequence as shown in SEQ ID NO:31, and a light chain variable region (VL) of the amino acid sequence as shown in SEQ ID NO:32. (VH; SEQ ID NO:31)EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQGTMVTVSS(VL; SEQ ID NO:32)DIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLIYDASNLDTGVPSRFSGSGSGTYFTLTISSLQAEDFAVYFCQQAKAFPPTFGGGTKVDIK

In some embodiments, antiVEGFR2Ab is an antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 33 and a light chain containing the amino acid sequence of SEQ ID NO: 34.

In some embodiments, antiVEGFR2Ab is an antibody comprising a heavy chain of an amino acid sequence as shown in SEQ ID NO: 33 and a light chain of an amino acid sequence as shown in SEQ ID NO: 34.

In some implementations, the anti-VEGFR2Ab is ramucirumab.

The term "ramorumab" used in this article, also known as Cyramza®, IMC-1121b, CAS registration number 947687-13-0, refers to an anti-VEGFR2 antibody comprising two heavy chains and two light chains, wherein the amino acid sequence of each heavy chain is SEQ ID NO: 33 and the amino acid sequence of each light chain is SEQ ID NO: 34. (Heavy chain; SEQ ID NO: 33)EVQLVQSGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYYADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVT DAFDIWGQGT MVTVSSASTKGPSVLPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYSLSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PELLGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKNQVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA LHNHYTQKSL SLSPGK(light chain; SEQ ID NO: 34)DIQMTQSPSS VSASIGDRVT ITCRASQGID NWLGWYQQKP GKAPKLLIYD ASNLDTGVPSRFSGSGSGTY FTLTISSLQA EDFAVYFCQQ AKAFPPTFGG GTKVDIKRTV AAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLTLSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

Ramumarumab is a fully human monoclonal antibody targeting human vascular endothelial growth factor receptor 2 (VEGFR2). The preparation and use of ramumarumab, including its application in the treatment of supernumerary diseases such as solid and non-solid tumors, are disclosed in WO2003/075840. Furthermore, the clinical activity of ramumarumab in patients with various cancers has been reported.

In this paper, "DC101" refers to a monoclonal antibody against mouse VEGFR2 that can be used as an alternative to anti-VEGFR2 antibodies (preferably ramorumab) in mouse experiments. For example, see Witte L. et al.'s research: Monoclonal antibodies against VEGF receptor-2 (Flk1/KDR) as an anti-angiogenic therapeutic strategy. Cancer Metastasis Review, 17: 155-161, 1998; and Prewett M. et al.'s research: Monoclonal antibodies against vascular endothelial growth factor receptor (fetal liver kinase 1) inhibit angiogenesis and growth in various mouse and human tumors. Cancer Research, 59: 5209-5218, 1999.

In the combination of the present invention, the anti-VEGFR2 antibody (preferably ramumarumab) is generally effective over a broad dosage range. For example, the dosage range for every three-week cycle is generally about 6 to 10 mg/kg, preferably about 8 to 10 mg/kg, and most preferably about 10 mg/kg. In some embodiments, the anti-VEGFR2 antibody (preferably ramumarumab) is administered on day 1 at a dose of about 6 to 10 mg/kg. In some embodiments, the anti-VEGFR2 antibody (preferably ramumarumab) is administered on day 1 at a dose of about 10 mg/kg.

As used in this article, the phrase "in combination with" refers to the simultaneous administration of two drugs or their sequential administration in any order.

The bispecific conjugates described herein can be administered to individuals simultaneously with or sequentially in any order to an antibody binding to human VEGFR2. For example, the bispecific conjugate can be administered before or after the antibody binding to human VEGFR2. Immune checkpoint inhibitors.

In this invention, "immune checkpoint" refers to a regulator of the immune system, particularly a regulator of the amplitude and quality of co-stimulatory and inhibitory signals in T cell receptor recognition of antigens. In some embodiments, an immune checkpoint is an inhibitory signal. In some embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1 and/or PD-L2. In some embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86, replacing the binding of CD28. In some embodiments, the inhibitory signal is the interaction between LAG-3 and MHC class II molecules. In some embodiments, the inhibitory signal is the interaction between TIM-3 and one or more of its ligands (such as galactolectin 9, PtdSer, HMGB1, and CEACAM1). In some embodiments, the inhibitory signal is the interaction between one or more KIRs and their ligands. In some embodiments, the inhibition signal is the interaction between TIGIT and its ligands PVR, PVRL2, and PVRL3. In some embodiments, the inhibition signal is the interaction between CD94/NKG2A and HLA-E. In some embodiments, the inhibition signal is the interaction between VISTA and its binding counterpart. In some embodiments, the inhibition signal is the interaction between one or more Siglec ligands. In some embodiments, the inhibition signal is the interaction between GARP and its ligand. In some embodiments, the inhibition signal is the interaction between CD47 and SIRPα. In some embodiments, the inhibition signal is the interaction between PVRIG and PVRL2. In some embodiments, the inhibition signal is the interaction between CSF1R and CSF1. In some embodiments, the inhibition signal is the interaction between BTLA and HVEM. In some embodiments, the inhibitory signal is part of an adenosinergic pathway, such as the interaction of A2AR and/or A2BR with adenosine produced by CD39 and CD73. In some embodiments, the inhibitory signal is the interaction of B7-H3 with its receptor and/or B7-H4 with its receptor. In some embodiments, the inhibitory signal is mediated by IDO, CD20, NOX, or TDO.

"Programmed death receptor-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on previously activated T cells in vivo and binds to two ligands: PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273). The "PD-1" used in this invention includes human PD-1 (hPD-1), variants, isotypes, and analogs that share at least one common epitope with hPD-1. The "programmed death ligand-1 (PD-L1)" used in this invention is one of the two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. As used in this invention, "PD-L1" includes human PD-L1 (hPD-L1), variants, isotypes, and analogs that share at least one common epitope with hPD-L1. As used in this invention, "PD-L2" includes human PD-L2 (hPD-L2), variants, isotypes, 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 either PD-L1 or PD-L2 leads to downregulation of T cell activation. Cancer cells expressing PD-L1 and/or PD-L2 can shut down PD-1-expressing T cells, thereby suppressing the antitumor immune response. The interaction between PD-1 and its ligands leads to a decrease in tumor-infiltrating lymphocytes, reduced T-cell receptor-mediated proliferation, and immune escape by cancer cells. Immunosuppression can be reversed by inhibiting the regional interaction between PD-1 and PD-L1, and the effects are additive when the interaction between PD-1 and PD-L2 is blocked simultaneously.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) (also known as CD152) is a T-cell surface molecule belonging to the immunoglobulin superfamily. This protein downregulates the immune system by binding to CD80 (B7-1) and CD86 (B7-2). In this article, "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms, and analogs that share at least one common epitope with hCTLA-4. CTLA-4 is a homolog of the stimulatory checkpoint protein CD28, but its binding affinity to CD80 and CD86 is much higher than that of CD28. CTLA-4 is expressed on the surface of activated T cells, while its ligands are expressed on the surface of specialist antigen-presenting cells. After CTLA-4 binds to its ligand, it blocks the co-stimulatory signal of CD28 and generates an inhibitory signal. Therefore, CTLA-4 can downregulate T cell activation.

"T cell immune receptors with Ig and ITIM domains" (TIGIT, also known as WUCAM or Vstm3) are immune receptors expressed on T cells and natural killer cells (NK cells). They bind to PVR (CD155), PVRL2 (CD112; nectin-2), and PVRL3 (CD113; adhesion protein-3) on dendritic cells, macrophages, etc., and regulate T cell-mediated immune responses. The term "TIGIT" as used herein includes human TIGIT (hTIGIT), variants, isoforms, and species homologs of hTIGIT, and analogs that share at least one common epitope with hTIGIT. The term "PVR" as used herein includes human PVR (hPVR), variants, isoforms, and analogs that share at least one common epitope with hPVR. The term "PVRL2" as used in this paper includes human PVRL2 (hPVRL2), variants, isomers, and homologs of hPVRL2, and analogs that share at least one common epitope with hPVRL2. The term "PVRL3" as used in this paper includes human PVRL3 (hPVRL3), variants, isomers, and homologs of hPVRL3, and analogs that share at least one common epitope with hPVRL3.

The "B7 family" refers to inhibitory ligands with unidentified receptors. The B7 family includes B7-H3 and B7-H4, both of which are upregulated in tumor cells and tumor-infiltrating cells. As used in this article, "B7-H3" and "B7-H4" include human B7-H3 (hB7-H3) and human B7-H4 (hB7-H4), their variants, isomers and species homologs, and analogs that share at least one common epitope with B7-H3 and B7-H4, respectively.

B and T lymphocyte attenuating factor (BTLA, also known as CD272) is a member of the TNFR family that is expressed in Th1 but not Th2 cells. BTLA expression is induced during T cell activation, particularly on the surface of CD8+ T cells. As used in this article, "BTLA" includes human BTLA (hBTLA), its variants, isoforms, and species homologs of hBTLA, as well as analogs that share at least one common epitope with hBTLA. BTLA expression is gradually downregulated during the differentiation of human CD8+ T cells into effector cell phenotypes. Tumor-specific CD8+ T cells exhibit high levels of BTLA. BTLA binds to herpesvirus entry mediators (HVEM, also known as TNFRSF14 or CD270) and participates in T cell suppression. As used in this article, "HVEM" includes human HVEM (hHVEM), its variants, isoforms, and species homologs of hHVEM, as well as analogs sharing at least one common epitope with hHVEM. The BTLA-HVEM complex has a negative regulatory effect on T cell immune responses.

Killer cell immunoglobulin analogues (KIRs) are MHCI-type molecular receptors on NK T cells and NK cells, involved in the differentiation between healthy and diseased cells. KIRs bind to human leukocyte antigens (HLA) A, B, and C, inhibiting the activation of normal immune cells. The term "KIR" as used herein includes human KIR (hKIR), variants, isotypes, and analogues sharing at least one common epitope with hKIR. The term "HLA" as used herein includes HLA variants, isotypes, HLA species homologs, and analogues sharing at least one common epitope with HLA. In this invention, it specifically refers to KIR2DL1, KIR2DL2, and/or KIR2DL3.

Lymphocyte activation gene-3 (LAG-3) (also known as CD223) is an inhibitory receptor that binds to MHC class II molecules and is associated with the suppression of lymphocyte activity. This receptor enhances the function of Treg cells and inhibits the function of CD8+ effector T cells, thereby leading to the suppression of the immune response. LAG-3 is expressed on activated T cells, NK cells, B cells, and dendritic cells (DCs). The term "LAG-3" as used in this article includes human LAG-3 (hLAG-3), variants, isotypes, hLAG species homologs, and analogs that share at least one common epitope with hLAG-3.

T-cell membrane protein-3 (TIM-3) (also known as HAVcr-2) is an inhibitory receptor that suppresses lymphocyte activity by inhibiting Th1 cell responses. Its ligand is galactagogue 9 (GAL9), which is upregulated in various cancers. Other ligands for TIM-3 include phosphatidylserine (PtdSer), high-mobility group protein 1 (HMGB1), and carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM1). The term "TIM-3" as used herein includes human TIM3 (hTIM-3), variants, isotypes, and species homologs of hTIM-3, as well as analogs sharing at least one common epitope. The term "GAL9" as used herein includes human GAL9 (hGAL9), variants, isotypes, and species homologs of hGAL9, as well as analogs sharing at least one common epitope. As used in this paper, "PdtSer" includes variants and analogs with at least one common epitope. "HMGB1" as used in this paper includes human HMGB1 (hHMGB1), variants, isotypes, and homologs of the hHMGB1 species, as well as analogs with at least one common epitope. "CEACAM1" as used in this paper includes human CEACAM1 (hCEACAM1), variants, isotypes, and homologs of the hCEACAM1 species, as well as analogs with at least one common epitope.

"CD94/NKG2A" is primarily an inhibitory receptor on the surface of natural killer cells and CD8+ T cells. The "CD94/NKG2A" used in this article includes human CD94/NKG2A (hCD94/NKG2A), variants, isotypes, and species homologs of hCD94/NKG2A, as well as analogs sharing at least one common epitope. The CD94/NKG2A receptor is a heterodimer composed of CD94 and NKG2A. It may inhibit natural killer cell activation and CD8+ T cell function by binding to ligands such as HLA-E. CD94/NKG2A restricts cytokine release and cytotoxic responses from natural killer cells (NK cells), natural killer T cells (NK-T cells), and T cells (α/β and γ/δ). NKG2A is frequently expressed in tumor-infiltrating cells, and HLA-E is overexpressed in many cancers.

Tryptophan 2,3-dioxygenase (IDO) is an immunosuppressive tryptophan metabolite. As used in this article, "IDO" includes human IDO (hIDO), variants, isomers, species homologs of hIDO, and analogs sharing at least one common epitope. IDO is a key rate-limiting enzyme in tryptophan degradation, catalyzing its conversion to kynurenine. Therefore, IDO participates in the depletion of essential amino acids. It is known to participate in the suppression of T and NK cells, the generation and activation of Treg and myeloid-derived suppressor cells, and the promotion of tumor angiogenesis. IDO is overexpressed in many cancers and has been shown to promote immune escape of tumor cells and contribute to the progression of chronic tumors under the influence of regional inflammation.

In the "adenosine pathway" or "adenosine signaling pathway" used in this manual, ATP is converted to adenosine by the exonuclease activity of CD39 and CD73. Adenosine then generates an inhibitory signal by binding to one or more inhibitory adenosine receptors: the adenosine A2A receptor (A2AR, also known as ADORA2A) and the adenosine A2B receptor (A2BR, also known as ADORA2B). Adenosine is a nucleoside with immunosuppressive properties, present in high concentrations in the tumor microenvironment, limiting the invasion, cytotoxicity, and cytokine production of immune cells. Therefore, adenosine signaling is an important strategy for cancer cells to avoid clearance by the host immune system. Adenosine signaling via A2AR and A2BR is a crucial marker in cancer treatment, as it is typically activated by high concentrations of adenosine 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-receptor-mediated immune cell activation, leading to an increase in Treg cell numbers and a decrease in the activation of dendritic cells (DCs) and effector T cells. In this article, "CD39" includes human CD39 (hCD39), variants, isotypes, and species homologs of hCD39, as well as analogs with at least one common epitope. The term "CD73" as used in this paper includes human CD73 (hCD73), variants, isomorphs, and species homologs of hCD73, and analogs with at least one common epitope. The term "A2AR" as used in this paper includes human A2AR (hA2AR), variants, isomorphs, and species homologs of hA2AR, and analogs with at least one common epitope. The term "A2BR" as used in this paper includes human A2BR (hA2BR), variants, isomorphs, and species homologs of hA2BR, and analogs with at least one common epitope.

"V-domain T-cell activation inhibitory immunoglobulin" (VISTA, also known as C10orf54) shares homology with PD-L1 but exhibits a unique pattern of expression, limited to the hematopoietic system. In this article, "VISTA" includes human VISTA (hVISTA), variants, isotypes, and species homologs of hVISTA, as well as analogs sharing at least one common epitope. VISTA induces T-cell suppression and is expressed by leukocytes within tumors.

Members of the sialic acid-binding immunoglobulin-type lectin (Siglec) family recognize sialic acid and participate in distinguishing between "self" and "non-self" sialic substances. As used in this article, "Siglec" includes human Siglec (hSiglec), variants, isotypes, species homologs of hSiglec, and analogs sharing at least one common epitope with one or more hSiglecs. The human genome contains 14 Siglecs, many 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 carbohydrates, but differ in the chemistry and spatial distribution of the binding regions that recognize sialic acid residues. The phenotypes of this family members also vary. One or more Siglecs are overexpressed in a wide range of malignant tumors.

"CD20" is an antigen expressed on the surface of B cells and T cells. High expression of CD20 can be found in certain cancers, such as B-cell lymphoma, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma stem cells. In this article, "CD20" includes human CD20 (hCD20), variants, isotypes, species homologs of hCD20, and analogs sharing at least one common epitope.

Glycoprotein A repeat priority (GARP) plays a role in immune tolerance and the ability of tumors to evade the patient's immune system. The term "GARP" as used herein includes human GARP (hGARP), variants, isoforms, species homologs of hGARP, and analogs sharing at least one common epitope. GARP is expressed in peripheral blood lymphocytes (including Treg cells) and tumor-infiltrating T cells at the tumor site. It may bind to the potential transforming growth factor β (TGF-β). Interference with GARP signaling in Treg cells reduces tolerance and inhibits Treg cell migration into the intestine, as well as increasing the proliferation of cytotoxic T cells.

CD47 is a transmembrane protein that binds to the ligand SIRPα (signal regulator protein α). The term "CD47" as used herein includes human CD47 (hCD47), variants, isoforms, species homologs of hCD47, and analogs sharing at least one common epitope. The term "SIRPα" as used herein includes human SIRPα (hSIRPα), variants, isoforms, species homologs of hSIRPα, and analogs sharing at least one common epitope with hSIRPα. CD47 signaling is involved in various cellular processes, including apoptosis, proliferation, adhesion, and migration. CD47 is overexpressed in many cancers and acts as a "don't eat me" signal, preventing macrophages from phagocytosing cancer cells. By using inhibitory anti-CD47 or anti-SIRPα antibodies to block CD47 signaling, macrophages can phagocytose cancer cells and promote the activation of cancer-specific T lymphocytes.

The poliovirus receptor-associated immunoglobulin domain-containing protein (PVRIG, also known as CD112R) binds to poliovirus receptor-associated protein 2 (PVRL2). PVRIG and PVRL2 are overexpressed in various cancers. PVRIG expression also induces TIGIT and PD-1 expression, while PVRL2 and PVR (a ligand of TIGIT) are co-overexpressed in multiple cancers. Therefore, blocking the PVRIG signaling pathway can enhance T cell function and CD8+ T cell responses, thereby reducing immunosuppression and increasing interferon responses. In this article, "PVRIG" includes human PVRIG (hPVRIG), variants, isoforms, species homologs of hPVRIG, and analogs that share at least one common epitope with hPVRIG. The term "PVRIG" used in this article includes hPVRL2, as defined above.

According to this disclosure, the "colony-stimulating factor 1" pathway is another targetable checkpoint. CSF1R is a myeloid growth factor receptor that binds to CSF1. Blocking the CSF1R signaling pathway can functionally redesign macrophage responses, thereby enhancing antigen presentation and anti-tumor T cell responses. The term "CSF1R" as used herein includes human CSF1R (hCSF1R), variants, isomers, and species homologs of hCSF1R, as well as analogs sharing at least one common epitope with hCSF1R. The term "CSF1" as used herein includes human CSF1 (hCSF1), variants, isomers, and species homologs of hCSF1, as well as analogs sharing at least one common epitope with hCSF1.

"Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase" refers to an enzyme in the NOX enzyme family found in myeloid cells that produces immunosuppressive reactive oxygen species (ROS). Five NOX enzymes (NOX1 to NOX5) have been identified as involved in cancer development and immunosuppression. Elevated ROS levels have been detected in almost all cancers, contributing to multiple aspects of tumor development and progression. NOX-generated ROS can weaken the function of NK cells and T cells, and inhibiting NOX in myeloid cells can improve the antitumor function of peripheral NK cells and T cells. The term "NOX" as used in this invention includes human NOX (hNOX), variants, isotypes, species homologs of hNOX, and analogs that share at least one common epitope with hNOX.

According to this disclosure, another targetable immune checkpoint is tryptophan 2,3-dioxygenase (TDO)-mediated signaling. TDO is another pathway for tryptophan degradation and is involved in immunosuppression. Since tumor cells may metabolize tryptophan via TDO rather than IDO, TDO may represent an additional target for checkpoint blockade. Indeed, several cancer cell lines have been found to upregulate TDO expression, and TDO may complement the inhibitory effect of IDO. The term "TDO" as used in this invention includes human TDO (hTDO), variants, isotypes, species homologs of hTDO, and analogs that share at least one common epitope with hTDO.

Many immune checkpoints are regulated by interactions between specific receptor-ligand pairs, as described above. Therefore, immune checkpoint proteins mediate immune checkpoint signal transduction. For example, checkpoint proteins directly or indirectly regulate T cell activation, proliferation, and/or function. Cancer cells often utilize these checkpoint pathways to protect themselves from immune system attacks. Therefore, the functions of checkpoint proteins regulated according to this disclosure are generally related to T cell activation, T cell proliferation, and/or T cell function. Immune checkpoint proteins thus regulate and maintain the duration and magnitude of self-tolerance and physiological immune responses. Many immune checkpoint proteins belong to the B7:CD28 family or the tumor necrosis factor receptor (TNFR) superfamily. By binding to specific ligands, they activate signaling molecules recruited to the cytoplasmic domain (Suzuki et al., 2016, Jap J Clin Onc, 46:191-203).

In this invention, the term "immune checkpoint regulator" or "checkpoint modulator" refers to a molecule or compound that modulates the function of one or more checkpoint proteins. Immune checkpoint regulators typically modulate the magnitude and/or duration of self-tolerance and/or immune responses. Preferably, the immune checkpoint regulator used according to this disclosure modulates one or more human checkpoint proteins and is therefore a "human checkpoint regulator." In a preferred embodiment, the human checkpoint regulator used in this invention is an immune checkpoint inhibitor.

As used herein, "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule or molecule that can completely or partially reduce, inhibit, interfere with, or negatively regulate the expression of one or more checkpoint proteins, or a molecule that can completely or partially reduce, inhibit, interfere with, or negatively regulate the expression of one or more checkpoint proteins. In some embodiments, the immune checkpoint inhibitor binds to one or more checkpoint proteins. In some embodiments, the immune checkpoint inhibitor binds to one or more molecules that regulate checkpoint proteins. In some embodiments, the immune checkpoint inhibitor binds to precursors of one or more checkpoint proteins, for example, at the DNA or RNA level. Any agent that functions as a checkpoint inhibitor according to this disclosure may be used.

In this instruction manual, "partial" means at a level of 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%, for example, the level of inhibition of checkpoint proteins.

In some embodiments, the immune checkpoint inhibitors suitable for use herein are signal antagonists, such as antibodies targeting PD-1, PD-L1, CTLA-4, LAG-3, B7-H3, B7-H4, or TIM-3. For a summary of these ligands and receptors, see Pardoll, D., Nature. 12: 252-264, 2012. This paper describes other immune checkpoint proteins that can be targeted according to this disclosure.

In some embodiments, immune checkpoint inhibitors prevent inhibitory signals associated with immune checkpoints. In some embodiments, an immune checkpoint inhibitor is an antibody or a fragment thereof that interferes with inhibitory signals associated with immune checkpoints. In some embodiments, an immune checkpoint inhibitor is a small molecule inhibitor that interferes with inhibitory signals. In some embodiments, an immune checkpoint inhibitor is a peptide inhibitor that interferes with inhibitory signals. In some embodiments, an immune checkpoint inhibitor is an inhibitory nucleic acid molecule that interferes with inhibitory signals.

In some embodiments, the immune checkpoint inhibitor is an antibody, a fragment thereof, or an antibody mimic that prevents interactions between checkpoint blocking proteins, such as 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, a fragment thereof, or an antibody mimic that prevents the interaction between CTLA-4 and CD80 or CD86. In some embodiments, the immune checkpoint inhibitor is an antibody, a fragment thereof, or an antibody mimic that prevents the interaction between LAG-3 and its ligand, or prevents the interaction between TIM-3 and its ligand. In some embodiments, the immune checkpoint inhibitor prevents the interaction of adenosine via CD39 and/or CD73 and/or A2AR and/or A2BR. In some embodiments, immune checkpoint inhibitors prevent the interaction of B7-H3 with its receptor and/or B7-H4 with its receptor. In some embodiments, immune checkpoint inhibitors prevent the interaction of BTLA with its ligand HVEM. In some embodiments, immune checkpoint inhibitors prevent the interaction of one or more KIRs with their corresponding ligands. In some embodiments, immune checkpoint inhibitors prevent the interaction of LAG-3 with one or more of its ligands. In some embodiments, immune checkpoint inhibitors prevent the interaction of TIM-3 with one or more of its ligands (such as galactagogue-9, PtdSer, HMGB1, and CEACAM1). In some embodiments, immune checkpoint inhibitors prevent the interaction of TIGIT with one or more of its ligands PVR, PVRL2, and PVRL3. In some embodiments, immune checkpoint inhibitors prevent the interaction between CD94/NKG2A and HLA-E. In some embodiments, immune checkpoint inhibitors prevent the interaction between VISTA and one or more of its binding partners. In some embodiments, immune checkpoint inhibitors prevent the interaction between one or more Siglec molecules and their corresponding ligands. In some embodiments, immune checkpoint inhibitors prevent CD20 signaling. In some embodiments, immune checkpoint inhibitors prevent the interaction between GARP and one or more of its ligands. In some embodiments, immune checkpoint inhibitors prevent the interaction between CD47 and SIRPα. In some embodiments, immune checkpoint inhibitors prevent the interaction between PVRIG and PVRL2. In some embodiments, immune checkpoint inhibitors prevent the interaction between CSF1R and CSF1. In some embodiments, immune checkpoint inhibitors prevent NOx signaling. In some embodiments, immune checkpoint inhibitors prevent IDO and/or TDO signaling.

As described herein, inhibition or blockade of immune checkpoint signaling can prevent or reverse immunosuppression against cancer cells and establish or enhance T cell immune responses. In one embodiment, inhibition of immune checkpoint signaling as described herein can reduce or suppress the dysfunction of the immune system. In one embodiment, inhibition of immune checkpoint signaling as described herein can reduce or suppress the dysfunctional state of dysfunctional immune cells. In one embodiment, inhibition of immune checkpoint signaling as described herein makes dysfunctional T cells less dysfunctional.

In this article, the term "disabled" refers to a state of reduced immune response to antigen stimulation. This term encompasses the common characteristics of exhaustion and/or non-response, where antigen recognition may occur, but the subsequent immune response is ineffective in controlling infection or tumor growth. Disabledness also includes a state of slowed antigen recognition due to immune cell dysfunction.

The term "disabled" in this article refers to a reduced immune response of immune cells to antigen stimulation. Disabled includes no response to antigen recognition and an impaired ability to translate antigen recognition into downstream T cell effector functions (such as proliferation, cytokine production (e.g., IL-2) and/or target cell killing).

In this article, "unresponsiveness" refers to the state of not responding to antigen stimulation due to incomplete or insufficient signaling by T cell receptors (TCRs). T cell unresponsiveness can also occur due to antigen stimulation in the absence of co-stimulation, resulting in the cells remaining unresponsive to subsequent antigen stimulation even with co-stimulation. This unresponsive state can usually be overcome by the presence of IL-2. Unresponsive T cells do not undergo selective proliferation and/or acquire effector function.

As used in this application, the term "exhaustion" refers to a state of immune cell depletion, such as T cell depletion, a state of T cell dysfunction that occurs in many chronic infections and cancers due to prolonged T cell receptor (TCR) signaling. It is distinguished from unresponsiveness because it is not due to incomplete or insufficient signaling, but rather to prolonged signaling. Characteristics of exhaustion include impaired effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector T cells or memory T cells. Exhaustion states can hinder optimal control of diseases (e.g., infections and tumors). Depletion can be caused by external negative regulatory pathways (e.g., immunomodulatory cytokines) and intracellular negative regulatory pathways (such as the inhibitory immune checkpoint pathway described herein).

"Enhancing T cell function" refers to inducing, causing, or stimulating T cells to possess a sustained or amplified biological function, or reactivating or reactivating depleted or inactive T cells. Examples of enhancing T cell function include increased secretion of gamma-interferon by CD8+ T cells, enhanced proliferative capacity, and increased responsiveness to antigens relative to pre-intervention levels (e.g., tumor clearance). In one embodiment, the degree 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 higher. Methods for measuring such enhancement are well known to those skilled in the art.

Immune checkpoint inhibitors can be a type of inhibitory nucleic acid molecule. In this invention, "inhibitory nucleic acid" or "inhibitory nucleic acid molecule" refers to a nucleic acid molecule, such as DNA or RNA, that can completely or partially reduce, inhibit, interfere with, or negatively regulate one or more checkpoint proteins. Inhibitory nucleic acid molecules include, but are not limited to, oligonucleotides, siRNA, shRNA, antisense DNA or RNA molecules, and adaptors (e.g., DNA or RNA adaptors).

In this article, "oligonucleotide" refers to a nucleic acid molecule that reduces protein expression (especially checkpoint proteins, such as those described herein). Oligonucleotides are typically short DNA or RNA molecules, usually consisting of 2 to 50 nucleotides. Oligonucleotides can be single-stranded or double-stranded. Checkpoint repressor oligonucleotides can be antisense oligonucleotides. Antisense oligonucleotides are single-stranded DNA or RNA molecules that complement a specific sequence (especially a nucleic acid sequence or fragment thereof of a checkpoint protein). Antisense RNA is often used to prevent the protein translation of mRNA (such as mRNA encoding checkpoint proteins) by binding to mRNA. Antisense DNA is often used to target specific complementary (coding or non-coding) RNA. If binding occurs, this DNA/RNA hybrid can be degraded by the enzyme RNase H. Furthermore, morpholine antisense oligonucleotides can be used for gene knockout in vertebrates. For example, Kryczek et al. designed specific morphogenetic oligonucleotides targeting B7-H4 in 2006 (J Exp Med, 203:871-81), which specifically blocked B7-H4 expression in macrophages, thereby increasing T cell proliferation and reducing tumor volume in mice with tumor-associated antigen (TAA)-specific T cells.

In this invention, the terms "siRNA," "small interfering RNA," or "small repressor RNA" are used interchangeably to refer to a typical double-stranded RNA molecule of 20-25 base pairs in length that interferes with the expression of a specific gene (such as a gene encoding a checkpoint protein) through complementary nucleotide sequences. In one embodiment, siRNA interferes with mRNA, thereby blocking translation, for example, blocking the translation of immune checkpoint proteins. Transfection with exogenous siRNA can be used for gene knockout, but its effect may be temporary, especially in rapidly dividing cells. Stable transfection can be achieved by RNA modification or by using expression vectors. Useful modifications and vectors for stable transfection of siRNA in cells are known in the art. siRNA sequences can also be modified to introduce a short circular structure between the two strands, forming "hairpin RNA" or "shRNA". shRNA can be processed into functional siRNA by the Dicer enzyme. shRNA has relatively low degradation and conversion rates. Therefore, immune checkpoint inhibitors can be a type of shRNA.

As used in this invention, "aptamer" refers to a single-stranded nucleic acid molecule, such as DNA or RNA, typically 25-70 nucleotides in length, capable of binding to a target molecule (such as a polypeptide). In one embodiment, the aptamer can bind to an immune checkpoint protein, such as the immune checkpoint protein described herein. For example, the aptamer according to this 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 of aptamers and their therapeutic uses are well documented in the art (see, for example, US 5,475,096).

The terms "small molecule inhibitor" or "small molecule" as used herein are used interchangeably and refer to a low molecular weight organic compound, typically below 1000 Eardons, that can completely or partially reduce, inhibit, interfere with, or negatively regulate one or more of the checkpoint proteins described herein. These small molecule inhibitors are typically synthesized through organic chemistry, but can also be isolated from natural sources, such as plants, fungi, and microorganisms. The low molecular weight allows small molecule inhibitors to diffuse rapidly across the cell membrane. For example, various A2AR antagonists known in the art are organic compounds with molecular weights below 500 Eardons.

Immune checkpoint inhibitors can be antibodies, their antigen-binding fragments, antibody mimics, or fusion proteins comprising an antibody moiety with an antigen-binding fragment possessing desired specificity. Antibodies or their antigen-binding fragments are as described herein. Antibodies or their antigen-binding fragments as immune checkpoint inhibitors particularly include those antibodies or fragments capable of binding to immune checkpoint proteins (such as immune checkpoint receptors or immune checkpoint receptor ligands). Antibodies or their antigen-binding fragments may also bind to other moieties, as described herein. Specifically, these antibodies or antigen-binding fragments can be chimeric antibodies, humanized antibodies, or human antibodies. Preferably, the immune checkpoint inhibitor antibody or its antigen-binding fragment is an antagonist of an immune checkpoint receptor or immune checkpoint receptor ligand.

In a preferred embodiment, the antibody of the immune checkpoint inhibitor is an isolated antibody.

The immune checkpoint inhibitor antibody or its antigen-binding fragment according to this disclosure may also be an antibody that competitively binds to an antigen with any known immune checkpoint inhibitor antibody. In some embodiments, the immune checkpoint inhibitor antibody competes with one or more immune checkpoint inhibitor antibodies described herein. The ability of antibodies to competitively bind to antigens suggests that these antibodies may bind to the same epitope region of the antigen or spatially block the binding of a known immune checkpoint inhibitor antibody to that particular epitope region when binding to another epitope. These competitive antibodies may have functional properties very similar to their competitors because they are expected to block the binding of immune checkpoints to their ligands by binding to the same epitope or spatially blocking the binding of the ligand. Inter-competitive antibodies can be identified based on their ability to inter-competite one or more known antibodies by standard binding tests such as surface plasmon resonance (SPR) analysis, ELISA assays, or flow cytometry (see, for example, WO 2013/173223).

In some embodiments, an antibody or antigen-binding fragment that competes with one or more known antibodies for a given antigen or binds to the same epitope region of a given antigen, such as one or more known antibodies, is a monoclonal antibody. When these competing antibodies are administered to human patients, they may be chimeric antibodies, humanized antibodies, or human antibodies. These chimeric, humanized, or human monoclonal antibodies can be prepared and isolated using methods known in the art.

Checkpoint inhibitors can also be in soluble form of molecules (or variants thereof), such as soluble PD-L1 or PD-L1 fusion proteins.

In this disclosure, more than one checkpoint inhibitor may be used, wherein the more than one checkpoint inhibitor may target different checkpoint paths or the same checkpoint path. Preferably, the more than one checkpoint inhibitor is a different checkpoint inhibitor. Preferably, if more than one different checkpoint inhibitor is used, particularly at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different checkpoint inhibitors are used, more preferably 2, 3, 4 or 5 different checkpoint inhibitors are used, more preferably 2, 3 or 4 different checkpoint inhibitors are used, even more preferably 2 or 3 different checkpoint inhibitors are used, and most preferably 2 different checkpoint inhibitors are used. Preferred examples of combinations of different checkpoint inhibitors include: combinations of PD-1 signaling inhibitors and CTLA-4 signaling inhibitors; combinations of PD-1 signaling inhibitors and TIGIT signaling inhibitors; combinations of PD-1 signaling inhibitors and B7-H3 and/or B7-H4 signaling inhibitors; and combinations of PD-1 signaling inhibitors and BTLA signaling inhibitors. Combinations of PD-1 signaling inhibitors with KIR signaling inhibitors; combinations of PD-1 signaling inhibitors with LAG-3 signaling inhibitors; combinations of PD-1 signaling inhibitors with TIM-3 signaling inhibitors; combinations of PD-1 signaling inhibitors with CD94/NKG2A signaling inhibitors; combinations of PD-1 signaling inhibitors with IDO signaling inhibitors. Combinations, combinations of PD-1 signaling inhibitors and adenosine signaling inhibitors, combinations of PD-1 signaling inhibitors and VISTA signaling inhibitors, combinations of PD-1 signaling inhibitors and Siglec signaling inhibitors, combinations of PD-1 signaling inhibitors and CD20 signaling inhibitors, and combinations of PD-1 signaling inhibitors and GARP signaling inhibitors. Combinations of PD-1 signaling inhibitors with CD47 signaling inhibitors, combinations of PD-1 signaling inhibitors with PVRIG signaling inhibitors, combinations of PD-1 signaling inhibitors with CSF1R signaling inhibitors, combinations of PD-1 signaling inhibitors with NOX signaling inhibitors, and combinations of PD-1 signaling inhibitors with TDO signaling inhibitors.

In some embodiments, the inhibitory immunomodulator (immune checkpoint blocker) is a component of the PD-1/PD-L1 or PD-1/PD-L2 signaling pathway. Therefore, some embodiments of this disclosure provide a checkpoint inhibitor of the PD-1 signaling pathway for administration to an individual. In some embodiments, 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 its antigen-binding moiety that interferes with the interaction between the PD-1 receptor and one or more of its ligands (PD-L1 and/or PD-L2). Antibodies capable of binding to PD-1 and interfering with its interaction with one or more ligands are known in the art. In some embodiments, the antibody or its antigen-binding moiety specifically binds to PD-1. In some embodiments, the antibody or its antigen-binding moiety specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby enhancing immune activity. In some embodiments, the antibody or its antigen-binding moiety specifically binds to PD-L2 and inhibits its interaction with PD-1, thereby enhancing immune activity.

In some embodiments, the immunomodulator is a component of the CTLA-4 signaling pathway. Therefore, certain embodiments of this disclosure provide for the administration of a checkpoint inhibitor of the CTLA-4 signaling pathway to an individual. 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.

In some embodiments, an immunomodulatory agent is a component of the TIGIT signaling pathway. Therefore, certain embodiments of this disclosure provide checkpoint inhibitors for administering the TIGIT signaling pathway to an individual. 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.

In some embodiments, inhibitory immunomodulators are components of the B7 family signaling pathway. In some embodiments, B7 family members are B7-H3 and B7-H4. Some embodiments of this disclosure provide administration of checkpoint inhibitors of B7-H3 and/or B7-H4 to an individual. Therefore, some embodiments of this disclosure provide administration of antibodies or antigen-binding moieties targeting B7-H3 or B7-H4 to an individual. The B7 family does not have well-defined receptors, but these ligands are upregulated in tumor cells or tumor-infiltrating cells. Preclinical mouse models have shown that blocking these ligands can enhance antitumor immune responses.

In some embodiments, an immunosuppressive immunomodulator is a component of the BTLA signaling pathway. Therefore, some embodiments of this disclosure provide a checkpoint inhibitor of the BTLA signaling pathway for administration to an individual. 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.

In some embodiments, the suppressive immunomodulator is a component of one or more KIR signaling pathways. Therefore, some embodiments of this disclosure provide checkpoint inhibitors of one or more KIR signaling pathways for administration to an individual. 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, the KIR inhibitor according to this disclosure may be an anti-KIR antibody that binds to KIR2DL1, KIR2DL2, and/or KIR2DL3.

In some embodiments, the immunomodulator is a component of the LAG-3 signaling pathway. Therefore, certain embodiments of this disclosure provide for the administration of a checkpoint inhibitor of the LAG-3 signaling pathway to an individual. 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.

In some embodiments, the suppressive immunomodulator is a component of the TIM-3 signaling pathway. Therefore, certain embodiments of this disclosure provide for the administration of a checkpoint inhibitor of the TIM-3 signaling pathway to an individual. 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.

In some embodiments, the immunomodulator is a component of the CD94/NKG2A signaling pathway. Therefore, certain embodiments of this disclosure provide for the administration of a checkpoint inhibitor of the CD94/NKG2A signaling pathway to an individual. In some embodiments, the checkpoint inhibitor of the CD94/NKG2A signaling pathway is a CD94/NKG2A inhibitor. In some embodiments, the checkpoint inhibitor of the CD94/NKG2A signaling pathway is a CD94/NKG2A ligand inhibitor.

In some embodiments, inhibitory immunomodulators are components of the IDO signaling pathway. Therefore, some embodiments of this disclosure provide checkpoint inhibitors, such as IDO inhibitors, that deliver the IDO signaling pathway to an individual.

In some embodiments, suppressive immunomodulators are components of the adenosine signaling pathway. Therefore, some embodiments of this disclosure provide the administration of a checkpoint inhibitor of the adenosine signaling pathway to an individual. 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.

In some embodiments, inhibitory immune regulatory molecules are components of the VISTA signaling pathway. Therefore, certain embodiments of this disclosure provide a method for administering a checkpoint inhibitor of the VISTA signaling pathway to an individual. In some embodiments, the checkpoint inhibitor of the VISTA signaling pathway is a VISTA inhibitor.

In some embodiments, the inhibitory immunomodulatory molecule is a component of one or more Siglec signaling pathways. Therefore, certain embodiments of this disclosure provide a method for administering a checkpoint inhibitor of one or more Siglec signaling pathways to an individual. 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.

In some embodiments, inhibitory immunomodulatory molecules are components of the CD20 signaling pathway. Therefore, certain embodiments of this disclosure provide a checkpoint inhibitor of the CD20 signaling pathway for administration to an individual. In some embodiments, the checkpoint inhibitor of the CD20 signaling pathway is a CD20 inhibitor.

In some embodiments, inhibitory immunomodulatory molecules are components of the GARP signaling pathway. Therefore, certain embodiments of this disclosure provide a checkpoint inhibitor of the GARP signaling pathway for administration to an individual. In some embodiments, the checkpoint inhibitor of the GARP signaling pathway is a GARP inhibitor.

In some embodiments, inhibitory immunomodulatory molecules are components of the CD47 signaling pathway. Therefore, certain embodiments of this disclosure provide a method for administering a checkpoint inhibitor of the CD47 signaling pathway to an individual. 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 suppressive immune regulator is a component of the PVRIG signaling pathway. Therefore, some embodiments of this disclosure provide delivery of a checkpoint inhibitor of the PVRIG signaling pathway to an individual. 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.

In some embodiments, the inhibitory immune regulator is a component of the CSF1R signaling pathway. Therefore, some embodiments of this disclosure provide delivery of a checkpoint inhibitor of the CSF1R signaling pathway to an individual. 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 suppressive immune regulator is a component of the NOX signaling pathway. Therefore, some embodiments of this disclosure provide delivery of a checkpoint inhibitor of the NOX signaling pathway (e.g., a NOX inhibitor) to an individual.

In some embodiments, the suppressive immune regulator is a component of the TDO signaling pathway. Therefore, some embodiments of this disclosure provide delivery of a checkpoint inhibitor of the TDO signaling pathway (e.g., a TDO inhibitor) to an individual.

Exemplary PD-1 inhibitors include, but are not limited to, the following antibodies: BGB-A317 (BeiGene; see US 8,735,553, WO 2015/35606 and US 2015/0079109), cimiprimab (Regeneron; see WO 2015/112800) and ramborizumab (e.g., disclosed in WO2008/156712 as hPD109A and its humanized derivatives h409A1, h409A16 and h409A17), Ab137132 (Abcam), EH12.2H7 and RMP1-14 (#BE0146; Bioxcell Life Sciences Pvt. Ltd.), MIH4 (Affymetrix eBioscience), and nivolumab (OPDIVO, BMS-936558; Bristol Myers). Squibb (see WO 2006/121168), pembrolizumab (KEYTRUDA; MK-3475; Merck; see WO 2008/156712), pildizumab (CT-011; CureTech; see Hardy et al., Cancer Research, Vol. 54, No. 22, pp. 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; see US) 2015/0203579), JS001 (Taizhou Junshi Pharmaceutical Co., Ltd.; see Si-Yang Liu et al., 2007, Journal of Hematology and Oncology (J. Hematol. Oncol.), Vol. 70, p. 136), AMP-224 (GSK-2661380; see Li et al., 2016, International Journal of Molecular Sciences (Int J Mol Sci), Vol. 17, No. 7, p. 1151, WO 2010/027827 and WO 2011/066342), PF-06801591 (Pfizer), BGB-A317 (BeiGene; see WO 2015/35606 and US 2015/0079109), BI 754091, Sintilimab (IBI308), Camrelizumab (SHR-1210) (see WO2015/085847), and antibodies 17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 described in WO 2006/121168, INCSHR1210 (Jiangsu Hengrui Medicine; also known as Camrelizumab (SHR-1210); see WO 2015/085847), TSR-042 (Tesaro) Biopharmaceutical (also known as ANB011; see WO2014/179664), GLS-010 (Wuhan/Harbin Guangyao Pharmaceutical; also known as WBP3055; see Si-Yang et al., 2017 Journal of Hematology and Oncology, Vol. 70, p. 136), STI-1110 (Sorrento Therapeutics; see WO 2014/194302), AGN2034 (Agenus; see WO 2017/040790), MGA012 (Macrogenics; see WO 2017/19846), IBI308 (Innovent Biologics; see WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540), anti-PD-1 antibodies, as described in, for example, US 7,488,802, US 8,008,449, US 8,168,757, WO 03/042402, WO 2010/089411 (further disclosure of anti-PD-L1 antibodies), WO 2010/036959, WO 2011/159877 (further disclosure of anti-TIM-3 antibodies), WO 2011/082400, WO 2011/161699, WO 2009/014708, WO 03/099196, WO 2009/114335, WO 2012/145493 (further disclosure of anti-PD-L1 antibodies), WO 2015/035606, WO US Patents 2014/055648 (further disclosure of anti-KIR antibodies), US 2018/0185482 (further disclosure of anti-PD-L1 and anti-TIGIT antibodies), US 8,008,449, US 8,779,105, US 6,808,710, US 8,168,757, US 2016/0272708, and US 8,354,509, disclose small molecule antagonists of the PD-1 signaling pathway, as shown in, for example, the 2018 Expert Opinion on Therapeutic Patents by Shaabani et al. Pat., Vol. 28, No. 9, pp. 665-678 and Sasikumar and Ramachandra, 2018, BioDrugs, Vol. 32, No. 5, pp. 481-497. siRNA targeting PD-1 has been disclosed, for example, in WO 2019/000146 and WO 2018/103501. Soluble PD-1 protein has also been described, for example, in WO 2018/222711. Oncolytic viruses including soluble PD-1 forms have been described, for example, in WO 2018/022831.

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

Exemplary PD-1 ligand inhibitors are PD-L1 and PD-L2 inhibitors, including but not limited to anti-PD-L1 antibodies, such as MEDI4736 (dvalumab; 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), and MDX-1105 (Roche/GeneTech; see WO2013019906 and US 8,217,149). 8,217,149), STI-1014 (Sorrento; see WO2013/181634), CK-301 (Checkpoint Therapeutics), KN035 (3D Medicine/Alphamab; see Zhang et al.'s article, 2017, Cell Discovery 3:17004), atezolizumab (TECENTRIQ; Ridgeback code RG7446; MPDL3280A; R05541267; see US 9,724,413), BMS-936559 (Bristol-Myers Squibb; see US 7,943,743 and WO 2013/173223), avermectin (bavencio; see US 2014/0341917), LY3300054 (Eli Lilly), 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, anti-PD-L1 antibodies described in 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、WO 2011/066389, WO2017/034916, WO2017/020291, WO2017/020858, WO2017/020801, WO2016/111645, WO2016/1973 67. WO2016/061142, WO2016/149201, WO2016/000619, WO2016/160792, WO2016/022630, WO2016/007235, WO2015/ 179654、WO2015/173267、WO2015/181342、WO2015/109124、WO 2018/222711, WO2015/112805, WO2015/061668, WO2014/159562, WO2014/165082, WO2014/100079.

Exemplary CTLA-4 inhibitors include, but are not limited to: monoclonal antibodies such as ipilimumab (Yervoy; Bristol Myers Squibb) and trevilizumab (Pfizer/Medlmmune), trevilizumab, Agen-1884 (Agenus) and ATOR-1015, as well as 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 2002/086014, WO Anti-CTLA4 antibodies and dominant negative proteins such as abatacept (Orencia; see EP2 855 533), disclosed in US 98/42752, US 6,207,156, US 5,977,318, US 7,109,003, and US 7,132,281, which include the Fe region of IgG1 fused to the ECD of CTLA-4; and berazipcept (Nulojix; see WO 2014/207748), a second-generation high-affinity CTLA-4-Ig variant, which, in contrast to abatacept, involves two amino acid substitutions in the ECD of CTLA-4, and soluble CTLA-4 peptides such as RG2077 and CTLA4-IgG4m (see US 98/42752, US 6,207,156, US 5,977,318, US 7,109,003, and US 7,132,281, are disclosed. 6,750,334), anti-CTLA-4 aptamers and siRNAs targeting CTLA-4, such as those disclosed in US 2015/203848. Illustrative CTLA-4 ligand inhibitors are described in Pile et al.'s 2015 paper (Encyclopedia of Inflammatory Diseases, edited by M. Parnham, doi: 10.1007/978-3-0348-0620-6_20).

Examples of 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 ertiglimab (OMP-313M32; OncoMed), or antibodies disclosed in WO2017/059095, particularly those disclosed in “MAB10”, US 2018/0185482, WO 2015/009856, and US 2019/0077864.

Examples of B7-H3 checkpoint inhibitors include, but are not limited to: the Fc-optimized monoclonal antibody enotozumab (MGA271; Macrogenics; see US 2012/0294796), and the anti-B7-H3 antibodies MGD009 (Macrogenics) and pidiliterizumab (see US 7,332,582).

Examples of B7-H4 inhibitors include, but are not limited to, the antibodies described below: Dangaj et al., 2013 (Cancer Research, Vol. 73: 4820-4829) and Smith et al., 2014 (Gynecologic Oncology, Vol. 134: 181-189), WO 2013/025779 (e.g., 2D1 encoded by SEQ ID NO: 3 and 4, 2H9 encoded by SEQ ID NO: 37 and 39, and 2E11 encoded by SEQ ID NO: 41 and 43), and WO 2013/067492 (e.g., selected from SEQ ID NO: (Amino acid sequence 1-8), and morpholino antisense oligonucleotides, or soluble recombinant forms of B7-H4, as described by Kryczek et al. in 2006 (Journal of Experimental Medicine, Vol. 203: 871-881), as revealed in US 2012/0177645.

Exemplary BTLA inhibitors include, but are not limited to, the anti-BTLA antibodies described below: Crawford and Wherry, 2009 (J Leukocyte Biol 86:5-8), WO 2011/014438 (e.g., 4C7 or antibodies comprising heavy and light chains according to SEQ ID NO: 8 and 15 and/or SEQ ID NO: 11 and 18), WO 2014/183885 (e.g., antibodies stored under CNCM I-4752), and US 2018/155428.

Checkpoint inhibitors of KIR signaling include, but are not limited to, the monoclonal antibody lirelurumab (1-7F9; IPH2102; see US 8,709,411), IPH4102 (Innate Pharma; see Marie-Cardine et al., 2014, Cancer 74(21): 6060-70), and, for example, those listed in 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., including those according to SEQ ID NO: Anti-KIR antibodies (antibodies of the heavy and light chains of 2 and 3), as described in WO 2010/065939, WO 2012/071411, WO 2012/160448 and WO 2014/055648.

LAG-3 inhibitors include, but are not limited to, the following 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 (Regeron/Sanofi), BAP050 (see 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 in 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 Antibodies disclosed in WO2017/198741, WO2017/220555, WO2017/015560, WO2017/025498, WO2017/149143, WO 2018/069500, WO2018/083087, WO2018/034227, and WO2014/140180. Additionally, this includes the LAG-3 antibody proteins AVA-017 (Avacta), EP 2 205 257, and the soluble LAG-3 fusion protein IMP321 (Immutep) described by Brigone et al. (2007) in the Journal of Immunology (J. Immunol.) 179: 4202-4211. Finally, it also includes the soluble LAG-3 protein described in WO 2018/222711.

TIM-3 inhibitors include, but are not limited to, antibodies against TIM-3, such as F38-2E2 (BioLegend), cobberalumab (TSR-022; Tesaro), LY3321367 (Eli Lilly), MBG453 (Novartis), and antibodies disclosed in WO 2013/006490, WO 2018/085469 (e.g., antibodies comprising heavy and light chain sequences encoded by the nucleic acid sequences of SEQ ID NO: 3 and 4), WO 2018/106588, and WO 2018/106529 (e.g., antibodies comprising heavy and light chain sequences according to SEQ ID NO: 8-11).

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), and 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 l. l, CLB-gran-10, F34-187, T84.1, B6.2, B 1.13, YG-C94G7, 12-140-5, scFv DIATHIS1, TET-2; cCAM Antibodies described by Watt et al. in 2001 (Blood, 98: 1469-1479) and PtdSer inhibitors described in WO 2010/12557, such as baiveximab (Peregrine).

CD94/NKG2A inhibitors include, but are not limited to, monazumab (IPH2201; Innate Pharma) and the antibodies and methods of preparation thereof disclosed in US 9,422,368 (e.g., humanized Z199; see EP 2 628 753), EP 3 193 929 and WO2016/032334 (e.g., humanized Z270; see EP 2 628 753).

IDO inhibitors include, but are not limited to, exiguamine A, icardolstat (INCB024360; InCyte; see US 9,624,185), indomethacin (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), and KHK2455 (Cheong et al., 2018, Expert Opinion Ther Pat). 28(4):317-330), PF-06840003 (see WO2016/181348), navoximod (RG6078, GDC-0919, NLG919; CAS No.: 1402837-78-8), linodolin (BMS-986205; Bristol-Myers Suibb; CAS No.: 1923833-60-6), small molecules such as 1-methyl-tryptophan, 2,5-dione-pyrrolidine derivatives (see international patent application WO2015/173764), and IDO inhibitors disclosed by Sheridan in Nature Biotechnol 33:321-322 in 2015.

CD39 inhibitors include, but are not limited to, A001485 (Arcus Biosciences), PSB 069 (CAS No.: 78510-31-3), and the anti-CD39 monoclonal antibody IPH5201 (Innate Pharma; see Perrot et al., 2019, Cell Reports 8:2411-2425.E9).

CD73 inhibitors include, but are not limited to, anti-CD73 antibodies, such as CPI-006 (Corvus Pharmaceuticals), MEDI9447 (MedImmune; see WO2016075099), IPH5301 (Innate Pharma; see Perrot et al., 2019, Cell Reports 8:2411-2425.E9), and anti-CD73 antibodies described in WO2018/110555, small molecule inhibitors such as PBS 12379 (Tocris Bioscience; CAS No.: 1802226-78-3), A000830, A001190, and A001421 (Arcus Biosciences; see Becker et al., 2018, Cancer Research 78(13 Supplement): 3691-3691, doi: 10.1158/1538-7445.AM2018-3691), CB-708 (Calithera Biosciences), and a bisphosphate based on purine cytotoxic nucleotides described by Allard et al. in 2018 (Immunol Rev., 276(1):121-144).

A2AR inhibitors include, but are not limited to, the following small molecule inhibitors: such as istradefylline (KW-6002; CAS No.: 155270-99-8), PBF-509 (Palobiopharma), and CPI-444 (Corvus). Pharma/Genentech; CAS No.: 1202402-40-1), ST1535 ([2-butyl-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purine-6-ylamine]; CAS No.: 496955-42-1), ST4206 (see Stasi et al., 2015, European Pharmacy 761:353-361; CAS No.: 1246018-36-9), tozadenant (SYN115; CAS No.: 870070-55-6), V81444 (see WO 2002/055082), Preladenant (SCH420814; Merck; CAS No.: 377727-87-2), Vipadenant (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-furanyl)-(1,2,4)triazol(2,3-a)-(1,3,5)triazine-5-ylamino)ethyl)phenol; CAS No.: 139180-30-6), AZD4635 (AstraZeneca), AB928 (double A2AR/A2BR small molecule inhibitor; Arcus Biosciences) and SCH58261 (see Popoli et al., 2000, Neuropsychopharmacology 22:522-529; CAS No.: 160098-96-4).

A2BR inhibitors include, but are not limited to: AB928 (a 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; developed by Janssen Biotech) and small molecule inhibitors CA-170 (anti-PD-L1/L2 and anti-VISTA small molecule; CAS No.: 1673534-76-3).

Siglec inhibitors include, but are not limited to: anti-Siglec-7 antibodies (e.g., including antibodies of the variable heavy chain region according to SEQ ID NO: 1 and the variable light chain region according to SEQ ID NO: 15) as disclosed in US 2019/023786 and WO 2018/027203; anti-Siglec-2 antibodies inotuzumab ozogamicin (Besponsa; see US 8,153,768 and 9,642,918); anti-Siglec-3 antibodies gemtuzumab ozogamicin (Mylotarg; see US 9,359,442); and those disclosed in US 2019/062427, US 2019/023786, WO 2019/011855, WO Anti-Siglec-9 antibodies disclosed in 2019/011852 (e.g., including antibodies against CDRs according to SEQ ID NO: 171-176, 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), US 2017/306014, and EP 3 146 979.

CD20 inhibitors include, but are not limited to, anti-CD20 antibodies, such as rituximab (RITUXAN; IDEC-102; IDEC-C2B8; see US Patent 5,843,439), ABP 798 (a biosimilar of rituximab), ofamumumab (2F2; see International Patent WO2004/035607), obbinuzumab, occrizumab (2h7; see International Patent WO 2004/056312), ibrutinib, tebuconazole (Zevalin), tocimetidine, ubulituximab (LFB-R603; LFB Biotechnology), and antibodies disclosed in US 2018/0036306 (e.g., including those according to SEQ ID NO: Antibodies of light and heavy chains 1-3 and 4-6, or 7 and 8, or 9 and 10.

GARP inhibitors include, but are not limited to, anti-GARP antibodies, such as ARGX-115 (arGEN-X) and the antibodies and their preparation methods 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/Forty Seven), CC-90002/INBRX-103 (CellGene/Inhibrx), SRF231 (Surface Oncology), IBI188 (Innovent Biologics), 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 CD47 fusion proteins, such as ALX148 (ALX Oncology; see 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), FSI-189 (Forty Seven), and anti-SIRPα fusion proteins, such as TTI-621 and TTI-662 (Trillium Therapeutics; see WO 2014/094122).

PVRIG inhibitors include, but are not limited to, anti-PVRIG antibodies, such as COM701 (CGEN-15029), and antibodies and methods of manufacturing thereof, disclosed in 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), and antibodies including the variant heavy chain region of SEQ ID NO: 5 and the variant light chain region of SEQ ID NO: 10 according to WO 2018/033798, or including antibodies including the variant heavy chain region of SEQ ID NO: 10 according to SEQ ID NO: 10, or antibodies including the variant heavy chain region of SEQ ID NO: 10 according to WO 2018/033798 ...SEQ ID NO: 10 according to WO 2018/033798, according to SEQ ID NO: 10, according to WO 2018/033798, according to SEQ ID NO: 10, according to WO 2018/033798, according to SEQ ID NO: 10, according to WO Antibodies of the heavy chain of 9 and the light chain according to SEQ ID NO: 14; WO 2018/033798 also discloses anti-TIGIT antibodies and combination therapies of anti-TIGIT and anti-PVRIG antibodies), WO2016134333, WO2018017864 (e.g., antibodies comprising a heavy chain according to SEQ ID NO: 5-7 and having at least 90% sequence homology with SEQ ID NO: 11, and/or an antibody comprising a light chain according to SEQ ID NO: 8-10 and having at least 90% sequence homology with SEQ ID NO: 12, or antibodies encoded by SEQ ID NO: 13 and/or 14 or SEQ ID NO: 24 and/or 29, or other antibodies disclosed in WO 2018/017864) and anti-PVRIG antibodies and fusion peptides, such as WO As described in 2016/134335.

CSF1R inhibitors include, but are not limited to, the anti-CSF1R antibody cabilazumab (FPA008; FivePrime; see WO 2011/140249, WO 2013/169264 and WO 2014/036357), IMC-CS4 (EiiLilly), emactuzumab (R05509554; Roche), RG7155 (see WO 2011/70024, WO 2011/107553, WO 2011/131407, WO 2013/87699, WO 2013/119716 and WO 2013/132044) and small molecule inhibitors such as BLZ945 (CAS No.: 953769-46-5) and pexidartinib (PLX3397; Selleckchem; CAS No.: 1029044-16-3).

CSF1 inhibitors include, but are not limited to, anti-CSF1 antibodies (see EP 1 223 980 and Weir et al., Bone Mineral Research 11:1474-1481, 1996), as well as antisense DNA and RNA disclosed in WO 2001/030381.

Exemplary NOX inhibitors include, but are not limited to, NOX1 inhibitors, such as small molecule ML171 (Gianni et al., 2010, ACS Chemical Biology 5(10):981-93) and NOS31 (Yamamoto et al., 2018, Chinese Journal of Biopharmaceuticals 41(3):419-426); NOX2 inhibitors, such as small molecule ceplene (histamine dihydrochloride; CAS No.: 56-92-8) and BJ-1301 (Gautam et al., 2017, Molecular Cancer Therapy 16(10): 2144-2156; CAS No.: 1287234-48-3) and the inhibitors described by Lu et al. (2017); NOX4 inhibitors, such as the small molecule inhibitor VAS2870 (Altenhöfer et al., 2012, Cell Molecular Life Sciences 69(14):2327-2343), diphenyl iodine (CAS No.: 244-54-2) and GKT137831 (CAS No.: 1218942-37-0; see Tang et al., 2018, 19(10):578-585).

TDO inhibitors include, but are not limited to, 4-(indole-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-(indole-3-yl)-pyridine derivatives (see US 2015/0225367 and WO 2015/121812), and bis-IDO/TDO inhibitors, such as WO 2015/150097, WO 2015/082499, WO 2016/026772, WO 2016/071283, WO 2016/071293, WO The small molecule dual IDO/TDO inhibitors disclosed in 2017/007700, and the small molecule inhibitor CB548 (Kim, C et al., 2018, The Annals of Oncology 29(Supplement 8): viii400-viii441).

According to this disclosure, immune checkpoint inhibitors are inhibitors of inhibitory checkpoint proteins, but preferably not inhibitors of stimulatory checkpoint proteins. As described herein, numerous inhibitors of 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, and their respective ligands, are known, and many of these are in clinical trials or have been approved. Based on these known immune checkpoint inhibitors, alternative immune checkpoint inhibitors can be developed. Specifically, inhibitors of known superior immune checkpoint proteins, or their analogues, can be used, especially chimeric, humanized, or human antibodies, as well as antibodies that compete with those described herein.

Those with general technical skills in this field should understand that other immune checkpoint targets can also be targeted by antibodies or antagonists, as long as such targeting can produce an immune response such as increased T cell proliferation, enhanced T cell activation, and/or increased production of cytokines (such as IFN-γ, IL2), for example, an antitumor immune response.

Checkpoint inhibitors can be applied by any means and pathways known in the art. The application mode and pathway will depend on the type of checkpoint inhibitor used.

Checkpoint inhibitors can be administered in any suitable pharmaceutical combination form, as described herein.

Checkpoint inhibitors can be administered in the form of nucleic acids, such as DNA or RNA molecules, which encode immune checkpoint inhibitors, for example, inhibitory nucleic acid molecules or antibodies and fragments thereof. For example, antibodies can be delivered via expression vectors, as described herein. Nucleic acid molecules can be delivered directly, for example, in the form of plasmids or mRNA molecules, or complexed with a delivery vector, for example, with liposomes, liposome complexes, or nucleic acid-liposome complexes. Checkpoint inhibitors can also be administered via oncolytic viruses comprising an expression cartridge encoding a checkpoint inhibitor. Furthermore, checkpoint inhibitors can also be delivered by administering endogenous or allogeneic cells capable of expressing checkpoint inhibitors, for example, in the form of cell-based therapy.

"Cell-based therapy" refers to the transplantation of cells expressing immune checkpoint inhibitors (such as T lymphocytes, dendritic cells, or stem cells) into an individual to treat a disease or symptom (such as cancer). In one embodiment, cell-based therapy includes genetically engineered cells. In one embodiment, these genetically engineered cells express immune checkpoint inhibitors as described herein. In one embodiment, the immune checkpoint inhibitor expressed by the genetically engineered cells is an inhibitory nucleic acid molecule, such as siRNA, shRNA, oligonucleotide, antisense DNA or RNA, adaptor, antibody, or fragment thereof, or soluble immune checkpoint protein or fusion protein. Genetically engineered cells may also express other agents that enhance T cell function. These agents are known in the art. Cell-based therapies for inhibiting immune checkpoint signaling are described in detail in WO 2018/222711, the entire contents of which are incorporated herein by reference.

As used in this invention, the term "oncolytic virus" refers to a virus capable of selectively replicating in cancer cells or hyperproliferating cells, either in vitro or in vivo, and slowing their growth or inducing their death, while having minimal or no effect on normal cells. Oncolytic viruses used for delivering immune checkpoint inhibitors include an expression cartridge that encodes an immune checkpoint inhibitor, which is an inhibitory nucleic acid molecule such as siRNA, shRNA, oligonucleotide, antisense DNA or RNA, adaptor, antibody, or fragment thereof, or a soluble immune checkpoint protein or fusion protein. Preferably, the oncolytic virus is capable of replication and the expression cartridge is controlled by a viral promoter (e.g., a synthetic early/late poxvirus promoter). Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), rhabdoviruses (such as Seneca Valley virus; SVV-001), Coxsackie virus, parvovirus, Newcastle disease virus (NDV), herpes simplex virus (HSV; such as OncoVEXGMCSF), retroviruses (such as influenza virus), measles virus, reovirus, synbivirus, poxvirus (such as the Copenhagen strain, Western Reserve strain, and Wesleyan strain described in WO2017/209053), and adenoviruses (such as Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAd1, H101, AD5/3-D24-GMCSF). The generation and use of recombinant oncolytic viruses, including soluble forms of immune checkpoint inhibitors, are described in detail in WO 2018/022831, the full text of which is incorporated herein by reference. Oncolytic viruses can be used as attenuated viruses.

In some embodiments, immune checkpoint inhibitors include antibodies selected from anti-PD-1 antibodies, anti-PD-L1 antibodies, and combinations thereof.

In some embodiments, immune checkpoint inhibitors include anti-PD-1 antibodies.

In some embodiments, anti-PD-1 antibodies include cimiprizumab (LIBTAYO, REGN2810), nivolumab (OPDIVO; BMS-936558), pembrolizumab (KEYTRUDA; MK-3475), patilizumab (CT-011), spartalizumab (PDR001), MEDI0680 (AMP-514), dostalimumab (TSR-042), cetalizumab (JNJ 63723283), toripalimab (JS001), AMP-224 (GSK-2661380), PF-06801591, tislelizumab (BGB-A317), ABBV-181, BI 754091, sintilimab (IBI308), or camrelizumab (SHR-1210).

In some embodiments, immune checkpoint inhibitors include anti-PD-L1 antibodies.

In some embodiments, anti-PD-L1 antibodies include atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267), durvalumab (MEDI4736), BMS-936559, bavencio, lodaporimab (LY3300054), CX-072 (Proclaim-CX-072), FAZ053, KN035, sugemalimab (CS1001), or MDX-1105.

As described herein, the bispecific conjugate is administered to an individual, such as a patient, together with a drug that stabilizes or increases CLDN18.2 expression. In some embodiments, the drug that stabilizes or increases CLDN18.2 expression and the bispecific conjugate are administered to the individual as a single combination. In some embodiments, the drug that stabilizes or increases CLDN18.2 expression and the bispecific conjugate are administered to the individual simultaneously (as a single combination). In some embodiments, the drug that stabilizes or increases CLDN18.2 expression and the bispecific conjugate are administered to the individual separately. In some embodiments, the drug that stabilizes or increases CLDN18.2 expression is administered to the individual before the bispecific conjugate. In some embodiments, the drug stabilizing or increasing CLDN18.2 performance is administered to the individual after the bispecific conjugate. In some embodiments, the drug stabilizing or increasing CLDN18.2 performance and the bispecific conjugate are administered to the individual on the same day. In some embodiments, the drug stabilizing or increasing CLDN18.2 performance and the bispecific conjugate are administered to the individual on different days.

The term "medical preparation" refers to any product intended for medical use. This term includes pharmaceutical compositions containing at least one active ingredient, as well as arrangements of one or more active ingredients, such as kits, which may be present together or individually, for example in individual vials, and optionally accompanied by materials containing information about their dosage, effects, etc.

The compounds and agents described in this invention can be administered in any suitable pharmaceutical combination form.

The pharmaceutical compositions described herein are preferably sterile and comprise effective amounts of the compounds and agents described herein, and may optionally include other agents further discussed herein to produce the desired response or effect.

Pharmaceutical compositions are typically provided in homogeneous dosage forms and can be prepared using known methods. Pharmaceutical compositions may, for example, be in the form of solutions or suspensions.

Pharmaceutical compositions may include salts, buffers, preservatives, carriers, thinners, and/or excipients, all of which must be pharmaceutically acceptable. The term "pharmaceutically acceptable" means a material that does not interact with the active ingredient in the pharmaceutical composition and is non-toxic.

Non-pharmaceutical acceptable salts can be used to prepare pharmaceutically acceptable salts, which are also included in this invention. Such pharmaceutically acceptable salts include, without limitation, salts 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, butyric acid, etc. Pharmaceutically acceptable salts can also be basic metal salts or basic earth metal salts, such as sodium salts, potassium salts, or calcium salts.

Suitable buffers for pharmaceutical compositions include acetate, citrate, borate, and phosphate.

Preservatives suitable for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, p-hydroxybenzoate, and thimerosal.

Injectable formulations may include medically acceptable excipients, such as lactated Ringer's solution.

The term "carrier" refers to an organic or inorganic component, which may be natural or synthetic, to which an active ingredient is bound to facilitate, enhance, or enable application. According to the present invention, "carrier" further includes one or more compatible solid or liquid fillers, thinners, or encapsulating substances suitable for administration to a patient.

Carriers suitable for hydrophilic preparations include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyethylene glycol, naphthalene hydroxide, and especially biocompatible lactic acid polymers, lactic acid/glycolic acid copolymers, or polyoxyethylene/polyoxypropylene copolymers.

The term "enhancer" as used herein is intended to refer to all substances that may be present in a pharmaceutical composition but are not active ingredients, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavorings, or colorants.

The drugs and compounds described herein can be administered via any conventional route, such as by non-enteric administration, including injection or infusion. Preferred routes of administration include intravenous, intra-arterial, subcutaneous, intradermal, or intramuscular administration.

Compositions suitable for non-enteric administration typically include sterile aqueous or non-aqueous formulations, wherein the active compound is preferably isotonic with the recipient's blood. Examples of suitable carriers and solvents include Ringer's solution and isotonic sodium chloride solution. Additionally, sterile fixed oils are commonly used as the medium for solutions or suspensions.

The drugs and combinations described herein are administered in an effective amount. An "effective amount" as referred to herein means the amount that, alone or in combination with other doses, is sufficient to achieve the desired response or effect. In the treatment of a particular disease or condition, a preferred response is the inhibition of disease progression. This includes slowing disease development, particularly interrupting or reversing disease progression. In the treatment of a disease or condition, a preferred response may also include delaying or preventing the onset of the disease.

The effective dose of the drugs or combinations described in this article will vary depending on the conditions of treatment, the severity of the disease, the patient's individual parameters (including age, physiological condition, body size, and weight), the duration of treatment, the type of concomitant therapy (if present), the specific route of administration, and other factors. Therefore, the dosage of the drugs described in this article may depend on these parameters. If the patient does not respond adequately to the initial dose, a higher dose may be used (or an even higher effective dose may be achieved through a different, more regional route of administration).

The drugs and compounds described in this disclosure can be administered to patients, such as orally, to treat or prevent a variety of diseases as described herein. Preferred patients include those with conditions that can be corrected or alleviated by administration of the drugs and compounds described in this disclosure. This includes diseases involving altered cellular expression patterns of CLDN18.2.

For example, in some embodiments, the agents described herein can be used to treat patients with cancer, such as cancer characterized by the presence of cancer cells expressing CLDN18.2.

The pharmaceutical compositions and treatments described in this invention can also be used for immunization or vaccination to prevent the diseases described herein.

The pharmaceutical conjugates described herein can be administered co-administered with supplemental immune enhancers, such as one or more adjuvants, and may include one or more immune enhancers to further improve their effectiveness, preferably achieving an additive effect of immune stimulation. The term "adjuvant" refers to a compound that can prolong, enhance, or accelerate an immune response. Various mechanisms can exist depending on the type of adjuvant. For example, compounds that allow dendritic cell maturation, such as lipopolysaccharide or CD40 ligands, constitute a suitable class of adjuvants. Generally, any agent that affects the immune system's "danger signal" type (such as LPS, GP96, dsRNA, etc.) or cytokines, such as GM-CSF, can act as an adjuvant to enhance and/or control the immune response. In this context, CpG oligodeoxynucleotides can also be used, but their side effects in certain situations need to be considered. Ideal adjuvants include cytokines such as monocytokines, 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. Other known adjuvants include aluminum hydroxide, Freund's adjuvants, or oils such as Montanide®, with Montanide® ISA51 being the preferred choice. Lipopeptides, such as Pam3Cys, are also suitable as adjuvants in the pharmaceutical compositions of this invention.

The drugs and combinations provided by this invention can be used alone or in combination with conventional treatment regimens such as surgery, radiation therapy, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated).

Cancer treatment is a field that particularly requires combination strategies, as the combined use of two, three, four, or even more cancer drugs/therapies often produces stronger synergistic effects than single therapies. Therefore, another embodiment of the present invention involves using the methods and combinations of the present invention to treat cancer via an immune or vaccine mechanism, which can be effectively combined with other drugs and/or methods targeting similar or different specific mechanisms. These combination therapies include combinations with conventional tumor treatments, multi-epitope strategies, additional immunotherapy, and therapies targeting angiogenesis or apoptosis (see, for example: Andersen et al., 2008: Cancer Treatment: Combination Use of Vaccines with Other Therapies. Cancer Immunology Immunotherapy, 57(11): 1735-1743). Administering different drugs in a specific order can inhibit the growth of cancer cells at different checkpoints, while other drugs may inhibit angiogenesis, malignant cell survival, or metastasis, potentially transforming cancer into a chronic disease. The following are some non-limiting examples of anticancer drugs and treatments that can be used in combination with this invention:

1. Chemotherapy

Chemotherapy is the standard treatment for many types of cancer. The most common chemotherapy drugs work by killing rapidly dividing cells, one of the main characteristics of cancer cells. Therefore, the combination of this invention with conventional chemotherapy drugs (such as alkylating agents, antimetabolites, anthracycline antibiotics, phytidines, topoisomerase inhibitors, and other antitumor drugs that affect cell division or DNA synthesis) can significantly improve the therapeutic effect of this invention by eliminating suppressor cells, restarting the immune system, enhancing the sensitivity of tumor cells to immune-mediated killing, or additionally activating immune system cells. Multiple studies have demonstrated the synergistic effect of combining chemotherapy drugs with vaccine-based immunotherapies in the fight against cancer (see: Quoix et al. 2011: Efficacy of first-line chemotherapy and TG4010 vaccine in advanced non-small cell lung cancer: a controlled phase IIb trial. Lancet Oncol. 12(12): 1125-33; and Liseth et al. 2010: Combination of intensive chemotherapy and anticancer vaccines in the treatment of human malignancies: hematological experience. J Biomed Biotechnol. 2010: 6920979; and Hirooka et al. 2009: Gemcitabine combined with immunotherapy is used for patients with regionally progressive pancreatic cancer who are not candidates for surgery. (Pancreas 38(3): e69-74). There are hundreds of chemotherapy drugs available that are suitable for combination therapy. Some (non-limiting) examples of chemotherapy drugs that can be used in combination with the present invention include: paraplatin, platinol (Platinol-AQ), cyclophosphamide (Cytoxan, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (VePesid), gemcitabine (Gemzar), imatinib mesylate (Gleevec), irinotecan (Camptosar), folex (Mexate, Amethopterin), taxol (Taxol, Abraxane), sorafenib (Nexavar), sunitinib (Sutent), hycamtin, oncovin (Vincasar PFS), and velban.

2. Surgery

Cancer surgery—the process of removing tumors—remains the cornerstone of cancer treatment. Surgery can be combined with other cancer treatments to eliminate any remaining tumor cells. Combining surgical procedures with subsequent immunotherapy is a promising strategy, proven in numerous cases.

3. Radiotherapy

Radiation therapy remains a crucial component of cancer treatment, with approximately 50% of cancer patients receiving it during their disease course. The primary goal of radiation therapy is to inhibit the proliferative (cell division) ability of cancer cells. Types of radiation used to treat cancer include photon radiation (X-rays and gamma rays) and particle radiation (electron, proton, and neutron beams). There are two methods for delivering radiation to the cancer cells. External beam radiation therapy (OPT) involves directing high-energy radiation (photons, protons, or particles) from outside the body to the tumor site. Internal beam radiation therapy, also known as brachytherapy, involves encapsulating a radiation source in a cannula or seed and placing it directly into the tumor. Radiation therapy techniques that can be combined with this invention include: fractionated therapy (radiation therapy is administered in fractions, such as receiving 1.5 to 3 Gy of radiation daily over several weeks), 3D conformal radiotherapy (3DCRT; delivering radiation to the gross tumor volume), modulated arc therapy (IMRT; computer-controlled modulation of the intensity of multiple radiation beams), image-guided radiotherapy (IGRT; pre-treatment imaging techniques can be used for correction), and stereotactic body radiotherapy (SBRT; delivering extremely high single doses within a few treatment fractions). For a summary of radiation therapy, please refer to the 2012 article by Baskar et al.: "Cancer and Radiation Therapy: Current Advances and Future Directions". International Journal of Medical Sciences 9(3): 193–199.

This invention is further illustrated by the following embodiments, which are not intended to limit the scope of the invention. The combination of the bispecific binder of the invention with other therapeutic agents...

In some embodiments of the compositions, pharmaceutical preparations or methods described herein, the bispecific binders described herein are used in combination with platinum compounds and fluoropyrimidine compounds or their precursors.

In some embodiments, the platinum compound is oxaliplatin. In some embodiments, the fluoropyrimidine compound or its precursor is selected from the group consisting of fluorouracil (5-FU), capecitabine, fluorouracil, tegafur, polyfluorouridine, and carmoflu. In some embodiments, the fluoropyrimidine compound or its precursor is fluorouracil (5-FU) or capecitabine.

In some embodiments of the compositions, pharmaceutical preparations, or methods described herein, the bispecific conjugates described herein are used in combination with oxaliplatin and 5-fluorouracil or their precursors. In some embodiments of the compositions, pharmaceutical preparations, or methods described herein, the bispecific conjugates described herein are used in combination with oxaliplatin and 5-fluorouracil or oxaliplatin and capecitabine. In some embodiments of the compositions, pharmaceutical preparations, or methods described herein, the bispecific conjugates described herein are used in combination with oxaliplatin and 5-fluorouracil.

In some embodiments, the combination of bispecific binders, platinum compounds, and fluoropyrimidine compounds or their precursors may further include folic acid.

In some embodiments of the compositions, pharmaceutical preparations or methods described herein, the bispecific binders described herein are used in combination with oxaliplatin, 5-fluorouracil, and folic acid.

In some embodiments of the compositions, pharmaceutical preparations or methods described herein, the bispecific binders described herein are used in combination with mFOLFOX6 chemotherapy regimens.

"Folic acid aldehyde" or "leucovorin" refers to compounds that have a synergistic effect with the chemotherapy drug 5-fluorouracil. Folate aldehyde has the following formula:

Specifically, the term refers to the compound (2S)-2-{[4-[(2-amino-5-methoxy-4-oxo-5,6,7,8-tetrahydro-1H-purin-6-yl)methylamino]benzoyl]amino}glutaric acid.

FOLFOX is a chemotherapy regimen consisting of folate (formyltetrahydrofolate), 5-fluorouracil, and oxaliplatin.

There are several different FOLFOX regimens, which differ in the dosage and administration method of the three drugs.

In some embodiments, the described bispecific conjugate is used in combination with a modified FOLFOX-6 regimen (mFOLFOX6). In some embodiments, the mFOLFOX6 regimen comprises an intravenous infusion of 85 mg/ ^m² oxaliplatin, an intravenous infusion of 400 mg/ ^m² folate, and an intravenous bolus of 400 mg/ ^m² 5-fluorouracil, followed by a continuous intravenous infusion of 2,400 mg/m² ⁵ -fluorouracil.

The mFOLFOX6 protocol can be repeated every two weeks.

In some embodiments, the combined use of bispecific binding agents, platinum compounds, fluorouracil compounds or their precursors and folate also includes immune checkpoint inhibitors, such as those selected from PD-1 inhibitors and PD-L1 inhibitors, particularly anti-PD-1 antibodies, such as nivolumab or pembrolizumab, especially pembrolizumab.

In some embodiments of the compositions, pharmaceutical preparations or methods described herein, the bispecific binders described herein are used in combination with oxaliplatin, 5-fluorouracil, folate, and nivolumab.

In some embodiments of the compositions, pharmaceutical preparations or methods described herein, the bispecific binders described herein are used in combination with oxaliplatin, 5-fluorouracil, folate, and pembrolizumab.

In some embodiments of the compositions, pharmaceutical preparations or methods described herein, the bispecific binding agents described herein are used in combination with the mFOLFOX6 chemotherapy regimen and nivolumab.

In some embodiments of the compositions, pharmaceutical preparations or methods described herein, the bispecific binders described herein are used in combination with the mFOLFOX6 chemotherapy regimen and pembrolizumab.

In some embodiments, nivolumab comprises a heavy chain and a light chain sequence, wherein: (a) the heavy chain comprises the following amino acid sequence: QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYYADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPSVFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSSVVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLTVLHQDWLNGK and (b) The light chain contains the following amino acid sequence: EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPARFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLLSSTLTLSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NO: 36).

According to institutional guidelines, published guidelines, and relevant product prescription information, nivolumab can be administered intravenously, and the dosage should be based on this protocol. In some practices, nivolumab is administered intravenously at a dose of 240 mg. In some practices, nivolumab is administered intravenously at a dose of 240 mg every 2 weeks. In some practices, nivolumab is administered intravenously at a dose of 480 mg. In some practices, nivolumab is administered intravenously at a dose of 480 mg every 4 weeks.

In some embodiments, pembrolizumab comprises a heavy chain and a light chain sequence, wherein: (a) the heavy chain comprises an amino acid sequence: QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNFNEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSSASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 37), (b) The light chain contains the amino acid sequence: EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL LIYLASYLESGVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEI KRTVAAPSVFIFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLSSTLLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC (SEQ ID NO: 38).

According to institutional guidelines, published guidelines, and relevant product prescription information, pembrolizumab can be administered intravenously, and the dosage should be based on this regimen. In some practices, pembrolizumab is administered intravenously at a dose of 200 mg. In some practices, pembrolizumab is administered intravenously at a dose of 200 mg every 3 weeks. In some practices, pembrolizumab is administered intravenously at a dose of 400 mg. In some practices, pembrolizumab is administered intravenously at a dose of 400 mg every 6 weeks.

In some embodiments, combinations of bispecific binders, platinum compounds, fluorouracil compounds or their precursors with aldehyde folic acid also include camptothecin analogs, such as irinotecan.

In some embodiments of the compositions, pharmaceutical preparations or methods described herein, the bispecific binding agent described herein is combined with oxaliplatin, 5-fluorouracil, folic acid and irinotecan.

In certain embodiments of the compositions, pharmaceutical preparations or methods described herein, the bispecific binders and mFOLFIRINOX chemotherapy regimens described herein are examples.

FOLFIRINOX is a chemotherapy regimen consisting of folate (formyltetrahydrofolate), 5-fluorouracil, oxaliplatin, and irinotecan.

In some embodiments, the bispecific conjugate described herein is used in combination with a modified FOLFIRINOX regimen (mFOLFIRINOX). In some embodiments, the mFOLFIRINOX regimen comprises: intravenous infusion of 85 mg/ ^m² oxaliplatin, intravenous infusion of 180 mg/ ^m² irinotecan, intravenous infusion of 400 mg/ ^m² methyltetrahydrofolate, and intravenous bolus of 400 mg/m² ⁵ -FU, followed by continuous intravenous infusion of 2,400 mg/m² ⁵ -FU.

The mFOLFIRINOX regimen can be repeated every two weeks.

In some embodiments of the compositions, pharmaceutical preparations or methods described herein, the bispecific binders described herein are used in combination with taxane and an antibody bound to human VEGFR2.

In some embodiments, taxane is paclitaxel. In some embodiments, the antibody that binds to human VEGFR2 is ramorumab.

In some embodiments of the compositions, pharmaceutical preparations or methods described herein, the bispecific binding agent described herein is combined with paclitaxel and ramucirumab.

In some embodiments, the cancer treated with the above combination therapy is a CLDN18.2 positive cancer.

In certain embodiments, the cancers treated with the above combination therapy, particularly combinations of bispecific binders, platinum compounds, fluorouracil compounds or their precursors, folate, and immune checkpoint inhibitors, such as the combination of bispecific binders, oxaliplatin, 5-fluorouracil, folate, and pembrolizumab, i.e., the combination of bispecific binders, mFOLFOX6 chemotherapy regimen, and pembrolizumab, are gastric cancer and/or gastroesophageal junction cancer, especially metastatic or regionally advanced unresectable gastric or gastroesophageal junction (GEJ) adenocarcinoma.

In some embodiments, the cancers treated with the above combination therapy, particularly combinations of bispecific binders, taxanes, and antibodies that bind to human VEGFR2, such as the combination of bispecific binders, taxanes, and ramorumab, are gastric cancer and/or gastroesophageal junction cancer, especially metastatic or regionally progressive unresectable gastric or gastroesophageal junction (GEJ) adenocarcinoma.

In some embodiments, the cancers treated by the above combinations, particularly combinations of bispecific binding agents, platinum compounds, fluorouracil compounds or their precursors, folate and camptothecin analogues, such as combinations of bispecific binding agents, oxaliplatin, 5-fluorouracil, folate and irinotecan (e.g., the combination of bispecific binding agents with mFOLFIRINOX chemotherapy regimens), are pancreatic cancers, especially metastatic or regionally advanced unresectable pancreatic adenocarcinomas.

In this article, "regional progression" means that the cancer has spread to nearby tissues. "Unresectable" means that the cancer cannot be removed surgically. "Metastatic" means that the cancer has spread to other parts of the body.

In some embodiments of the above combination, the bispecific binding agent comprises four polypeptide chains, wherein: (i) the first polypeptide chain contains the amino acid sequence of SEQ ID NO: 27; (ii) the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 28; (iii) the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 29.

The bispecific binding agent described herein can be administered via intravenous infusion in the arm or subcutaneously only (subcutaneous injection). Treatment can be administered in 7-day or 14-day (1 or 2-week) cycles. During each treatment cycle, intravenous infusion or subcutaneous injection can be administered once weekly or every 2 weeks. In some embodiments, the bispecific binding agent described herein is administered every 7 days within each 14-day cycle. In some embodiments, the dosage of the bispecific binding agent described herein is between 4 µg and 3600 µg, such as 4 µg, 12 µg, 36 µg, 100 µg, 280 µg, 700 µg, 1500 µg, 2400 µg or 3600 µg.

This invention is further illustrated by the following embodiments, which should not be considered as limiting the scope of the invention. Embodiment 1 : Cytotoxicity of ASP2138 in combination with gemcitabine hydrochloride and paclitaxel against pancreatic cancer cells expressing nectone 18.2 .

This study aimed to detect the antitumor cell cytotoxicity of the bispecific antibody ASP2138, a four-peptide chain (according to SEQ ID NO: 27, 28, 29, and 29) targeting human necrotizing connective tissue protein 18.2 and CD3, in a redirected T-cell cytotoxicity (RTCC) assay, and to compare the efficacy of gemcitabine hydrochloride alone and in combination with paclitaxel. Materials and Methods : Test items

ASP2138 (lot number: PDB00743-BDS; Astellas Pharmaceuticals, Tokyo, Japan) was diluted with RPMI-1640 medium (containing 5% heat-inactivated fetal bovine serum (FBS), test medium). Gemcitabine hydrochloride (Eli Lilly and Company, Indianapolis, USA) was dissolved in phosphate-buffered saline solution to a concentration of 40 mg/mL and further diluted with RPMI-1640 medium (containing 10% heat-inactivated FBS, medium). Paclitaxel (Sawai Pharmaceutical Co., Ltd., Osaka, Japan) was diluted with medium. Cell lines

MIA PaCa-2_GFP_CLDN18.2_27 (MIA PaCa-2) was established by Astellas Pharmaceuticals. This cell line was derived from the MIA PaCa-2 pancreatic cancer cell line purchased from RIKEN Bioresource Center (Ibaraki, Japan), and was established by transducing human nectone 18.2 and green fluorescent protein (GFP). MIA PaCa-2 cells were cultured at 37°C and 5% _CO2 in a medium containing 10 μg/mL blastomycin and 1 μg/mL puromycin. Chemotherapy was used for treatment.

MIA PaCa-2 cells were seeded at a concentration of 2.0 × ^10⁶ cells/10 mL in 10 cm tissue culture dishes (AGC TECHNO GLASS CO., LTD., Shizuoka, Japan) and cultured for 1 day at 37°C and 5% _CO₂ . After culturing, gemcitabine hydrochloride and paclitaxel (a chemotherapy agent) were added, and the cells were cultured for another 2 days. Gemcitabine hydrochloride was added in free form to achieve a final concentration of 0.1 µg/mL. Paclitaxel was added in free form to achieve a final concentration of 0.003 µg/mL. CLDN18.2 expression.

MIA PaCa-2 cells, whether chemotherapeutic or untreated, were stained with 1 µg of zotuximab (anti-CLDN18.2 antibody, lot number 7D900, Astellas Pharma, Inc.). Secondary antibody was APC anti-human IgG Fc antibody (BioLegend, Inc.). Dead cells were identified using a Zombie Aqua fixation survival kit (BioLegend, Inc.). CLDN18.2 expression was measured using a MACSQuant® Analyzer 10 flow cytometer (Miltenyi Biotec BV & Co. KG, Bergisch Gladbach, Germany) and analyzed using FlowJo 10.5.3 software (BD Biosciences). RTCC assay .

MIA PaCa-2 cells, human peripheral blood mononuclear cells (PBMCs; Lonza Group AG), and ASP2138 cells, whether chemotherapeutic or untreated, were seeded in assay medium in 96-well plates (AGC TECHNO GLASS CO., LTD.) and cultured for 1 day at 37°C and 5% _CO2 . MIA PaCa-2 cells were seeded at a density of 2.0 × ^10⁴ cells/100 µL/well. PBMCs were added at a density of 2.0 × ^10⁵ cells/50 µL/well (effector cell to target cell ratio of 10:1). In the first experiment, ASP2138 was added at a concentration of 50 µL/well, resulting in final concentrations of 0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, and 300 ng/mL, with each concentration repeated twice. In the second experiment, ASP2138 was added at a concentration of 50 µL/well, resulting in final concentrations of 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, and 1000 ng/mL, with each concentration repeated three times.

Cytotoxicity was assessed by flow cytometry. In the first experiment, MIA PaCa-2 and PBMCs were stained with 7-aminospermine D (7-AAD; BD Biosciences). In the second experiment, they were stained with 7-AAD and PE-anti-human CD45 antibody (BD Biosciences). GFP-positive and 7-AAD-negative (defined as viable target cells) cells were counted using a MACSQuant® Analyzer 10 flow cytometer, and analysis was performed using FlowJo software. The percentage of cytotoxicity was calculated using the following formula: 100 × [1 – (number of viable target cells per well) / (number of viable target cells in wells treated with only 0 ng/mL ASP2138)]. Results and Conclusions

This study investigated the cytotoxicity of the bispecific antibody ASP2138 against human necrin 18.2 and CD3 in combination with the chemotherapy drugs gemcitabine hydrochloride and paclitaxel. MIA PaCa-2_GFP_CLDN18.2_27 (MIA PaCa-2) is a subselected pancreatic cancer cell line that uses transduction to express human necrin 18.2 as a target cell and is treated with or without chemotherapy.

Flow cytometry revealed increased expression of human neminin 18.2 in target cells after chemotherapy treatment (Figures 2 and 4). The cytotoxicity of ASP2138 on MIA PaCa-2 cells was concentration-dependent regardless of chemotherapy treatment (Figures 3 and 5). Furthermore, the ASP2138-mediated cytotoxicity concentration-response curve of chemotherapeutic-treated MIA PaCa-2 cells shifted to the left and upward compared to the untreated group. These results indicate that chemotherapy treatment increases neminin 18.2 expression and enhances the potency and efficacy of ASP2138 in cytotoxically acting on human neminin 18.2-expressing tumor cells. Example 2 : The combination of ASP2138 with gemcitabine hydrochloride and paclitaxel demonstrated antitumor effects in a human CD3ε gene knock-in mouse model within the CLDN18.2 B16-F10 tumor model.

This study aimed to investigate the antitumor effect of ASP2138 (a bispecific antibody against clathrin 18.2 (CLDN18.2) and CD3, composed of four polypeptide chains with sequences SEQ ID NO: 27, 28, 29, and 29) combined with gemcitabine hydrochloride (gemcitabine) and paclitaxel in a human CD3ε knock-in (hCD3ε KI) mouse model exhibiting clathrin 18.2 expression in mice carrying the B16-F10 (B16F10_mCLDN18.2) tumor model. Materials and Methods Test Items

ASP2138 (lot number: PDB00743-BDS; Astellas Pharmaceuticals, Tokyo, Japan) and gemcitabine hydrochloride (Eli Lilly and Company, Indianapolis, USA) were dissolved in phosphate-buffered saline (PBS). Paclitaxel (LC Laboratory, Woburn, Massachusetts, USA) was dissolved in 50% ethanol and 50% Cremophor® to prepare a 10 mg/mL solution, which was then diluted with PBS. Cell lines

B16-F10, a mouse skin melanoma cell line, was purchased from the American Type Culture Collection (ATCC, Manzas, USA). B16F10_mCLDN18.2 was established by Astellas Pharmaceuticals by transfecting mouse CLDN18.2 into B16-F10 cells and cultured in Dulbecco modified Eagle medium containing 10% heat-inactivated fetal bovine serum and 2 mg/mL G418 at 37°C and 5% _CO2 . Animal experiments .

The experiment used 9-week-old male hCD3εKI mice purchased from Biocytogen Jiangsu Co., Ltd. in Jiangsu Province, China, and housed at Astellas Pharmaceuticals Co., Ltd. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Astellas Pharmaceuticals Ibaraki Research Center, which is AAALAC internationally accredited. Tumor vaccination .

B16F10_mCLDN18.2 cells at a concentration of 4.0 × ^10⁶ cells/mL were suspended in PBS and subcutaneously inoculated into the lateral abdomen of 9-week-old mice at a dose of 2.0 × ^10⁵ cells/50 μL. Administration and measurement were then performed.

On the third day after inoculation with B16F10_mCLDN18.2 cells, mice were assigned to groups (n=15) with similar mean tumor volume across groups, and drug administration began the following day. The first day of drug administration was defined as day 0.

The four experimental groups used in this study are as follows: Group 1: Loaded mice received intraperitoneal injections of PBS on days 0, 3, 7, and 10. Group 2: ASP2138 mice received intraperitoneal injections of ASP2138 (0.01 mg/kg) on days 0 and 7, and intraperitoneal injections of PBS on days 3 and 10. Group 3: Chemotherapy mice received intraperitoneal injections of paclitaxel (10 mg/kg) on days 0 and 7, and intraperitoneal injections of gemcitabine (40 mg/kg) on days 0, 3, 7, and 10. Group 4: Combination mice received intraperitoneal injections of ASP2138 (0.01 mg/kg) and paclitaxel (10 mg/kg) on days 0 and 7, and intraperitoneal injections of gemcitabine (40 mg/kg) on days 0, 3, 7, and 10.

Tumor diameter and weight were measured using vernier calipers and a standard scale on days -1, 3, 7, 10, and 14. The measurement on day -1 was used as the baseline for analysis on day 0. Tumor volume [ ^mm³ ] was calculated using the following formula: (major axis length of tumor [mm]) × (minor axis length of tumor [mm]) ^² × 0.5

The percentage of tumor growth inhibition (%Inh) was calculated using the following formula: %Inh = 100 × (1 – [mean tumor volume on day 14 – mean tumor volume on day 0] / [mean tumor volume on day 14 in the control group – mean tumor volume on day 0 in the control group]). Statistical analysis

Tumor volume and weight values are expressed as mean ± SEM. The mean tumor volume of groups 2 and 3 on day 14 was compared with that of group 4 using the unmatched student t-test. P < 0.05 was considered statistically significant. Data were processed using GraphPad Prism (version 8.0.2, GraphPad Software, San Diego, California, USA). Results and Conclusions

In a human CD3ε knock-in (hCD3ε KI) mouse model expressing CLDN18.2 tumor burden (B16-F10 (B16F10_mCLDN18.2)), the antitumor efficacy of the combination of the bispecific antibody ASP2138 targeting clathrin 18.2 (CLDN18.2) and CD3 with gemcitabine hydrochloride (gemcitabine) and paclitaxel (chemotherapy) was investigated. Starting from day 4 after B16F10_mCLDN18.2 vaccination, ASP2138 (0.01 mg/kg) and paclitaxel (10 mg/kg) were administered intraperitoneally on days 0 and 7, while gemcitabine (40 mg/kg) was administered intraperitoneally on days 0, 3, 7, and 10.

On day 14, ASP2138 alone and chemotherapy alone inhibited tumor growth by 74% and 64%, respectively. The combination therapy of ASP2138 and chemotherapy inhibited tumor growth by 97% on day 14, with no significant difference in body weight changes between the groups (Figure 7). The tumor volume in the combination therapy group was significantly smaller than that in the ASP2138 alone or chemotherapy alone groups (Figure 6).

This study demonstrates that in a B16F10_mCLDN18.2 tumor-loaded hCD3ε KI mouse model, the combination therapy of ASP2138 and chemotherapy improved the antitumor effect compared to ASP2138 or chemotherapy alone. Example 3 : Antitumor effect of ASP2138 combined with 5- fluorouracil and oxaliplatin in a human CD3ε gene knock-in mouse model expressing MC38 tumor load in human CLDN18.2 .

This study aimed to investigate the efficacy of ASP2138 (a bispecific antibody against clathrin 18.2 (CLDN18.2) and CD3, composed of four polypeptide chains with sequences SEQ ID NO: 27, 28, 29, and 29) in combination with 5-fluorouracil and oxaliplatin (FOLFOX) in a human CD3ε gene knock-in (hCD3ε KI) mouse model of human clathrin 18.2-expressing MC38 (MC38_hCLDN18.2) tumor burden. Materials and Methods Test Items

ASP2138 (lot number: PDB00743-BDS; Astellas Pharmaceuticals, Tokyo, Japan) and 5-fluorouracil (lot number: 22501SF; Kyowa Kirin Co., Ltd., Tokyo, Japan) and oxaliplatin (lot number: XHDFDA; Yakult Honsha Co., Ltd., Tokyo, Japan) were dissolved in phosphate-buffered saline (PBS). Cell lines

MC38, a mouse colonic adenocarcinoma cell line, was obtained from the National Cancer Institute (Bethesda, Maryland, USA). Astellas Pharmaceuticals established the MC38_hCLDN18.2 cell line by transducing human CLDN18.2 into MC38 cells and cultured them at 37°C and 5% _CO2 in Dulbecco modified Eagle medium containing 10% fetal bovine serum and 6 μg/mL blastcinon. Animals

hCD3εKI mice were purchased from Biocytogen Jiangsu Co., Ltd. (Jiangsu, China) and bred at Astellas Pharmaceuticals Co., Ltd. This study was approved by the Animal Care and Use Committee of Astellas Pharmaceuticals Co., Ltd. (Ibaraki, Japan), and the center is AAALAC internationally accredited. Tumor inoculation .

MC38_hCLDN18.2 cells at a concentration of 4.0 × ^10⁶ cells/mL were suspended in PBS and subcutaneously inoculated into the flanks of 7-week-old male mice at a dose of 2.0 × ^10⁵ cells/50 μL. Administration and measurement were performed.

On day 5 after inoculation with MC38_hCLDN18.2 cells, mice were assigned to groups (n=15) with similar mean tumor volume across groups, and drug administration began the following day. Day 0 was defined as the first day of drug administration.

The four drug administration groups used in this study are as follows: Group 1: Loaded mice received intraperitoneal injections of PBS on days 0, 4, 7, and 11. Group 2: ASP2138 mice received intraperitoneal injections of ASP2138 (0.1 mg/kg) on days 0 and 7, and intraperitoneal injections of PBS on days 4 and 11. Group 3: FOLFOX mice received intraperitoneal injections of 5-fluorouracil (25 mg/kg) and oxaliplatin (2.5 mg/kg) on days 0, 4, 7, and 11. Group 4: Combination mice received intraperitoneal injections of ASP2138 (0.1 mg/kg) on days 0 and 7, and intraperitoneal injections of 5-fluorouracil (25 mg/kg) and oxaliplatin (2.5 mg/kg) on days 0, 4, 7, and 11.

Tumor diameter and body weight were measured using calipers and weighing instruments on days -1, 4, 7, 11, and 14. The measurements taken on day -1 were used as baseline data for analysis on day 0.

The formula for calculating tumor volume [ ^mm³ ] is: (tumor major axis length [mm]) × (tumor minor axis length [mm]) ^² × 0.5

The formula for calculating the percentage of tumor growth inhibition (%Inh) is: %Inh = 100 × (1 – [mean tumor volume on day 14 of each group – mean tumor volume on day 0] / [mean tumor volume on day 14 of the control group – mean tumor volume on day 0]). Statistical analysis

Tumor volume and weight values are expressed as mean ± SEM. At day 14, mean tumor volume in groups 2 and 3 was compared separately with group 4 using the student's t-test. P < 0.05 was considered statistically significant. Data processing was performed using GraphPad Prism (version 10.1.2, GraphPad software, USA). Results and Conclusions

In a human CD3ε knock-in (hCD3ε KI) mouse model exhibiting CLDN18.2 tumor burden (MC38_hCLDN18.2), the antitumor effect of the bispecific antibody ASP2138 targeting human nectone 18.2 (hCLDN18.2) and CD3, combined with 5-fluorouracil and platinum oxide (FOLFOX), was investigated. Starting from day 6 post-MC38_hCLDN18.2 vaccination, ASP2138 (0.1 mg/kg) was administered intraperitoneally on days 0 and 7, while 5-fluorouracil (25 mg/kg) and oxaliplatin (2.5 mg/kg) were administered intraperitoneally on days 0, 4, 7, and 11. On day 14, ASP2138 or FOLFOX alone inhibited tumor growth by 37% and 39%, respectively. The combination of ASP2138 and FOLFOX inhibited tumor growth by 60% on day 14, with no significant difference in body weight changes among the groups (Figure 9). The tumor volume in the combination group was significantly smaller than that in the groups using ASP2138 or FOLFOX alone (Figure 8).

This study demonstrates that in the hCD3εKI mouse model burdened by MC38_hCLDN18.2 tumors, the combination therapy of ASP2138 and FOLFOX significantly improved the antitumor efficacy compared with ASP2138 or FOLFOX monotherapy.

In this example, 5-fluorouracil and oxaliplatin were administered to mice, and the administration of FOLFOX in this context is mentioned. Those skilled in the art will understand that, strictly speaking, the FOLFOX administration regimen should also include the administration of folate, but this was not done in this example. Those skilled in the art will recognize that the results observed in this example of the combination of 5-fluorouracil and oxaliplatin are equally applicable to the additional administration of folate. Example 4 : Antitumor effect of ASP2138 combined with 5- fluorouracil, platinum oxide, and irinotecan hydrochloride (FOLFIRINOX) in a human CD3ε gene knock-in mouse model expressing MC38 tumor burden in human CLDN18.2.

This study aimed to investigate the antitumor efficacy of ASP2138 (a bispecific antibody against clathrin 18.2 (CLDN18.2) and CD3, composed of four polypeptide chains with sequences SEQ ID NO: 27, 28, 29, and 29) in combination with 5-fluorouracil, platinum oxide, and irinotecan hydrochloride (FOLFIRINOX) in a human CD3ε gene knock-in (hCD3ε KI) mouse model expressing human clathrin 18.2 tumor burden. Materials and Methods Test Items

ASP2138 (lot number: PDB00743-BDS; Astellas Pharmaceuticals Co., Ltd., Tokyo, Japan) and 5-fluorouracil (lot number: 22501SF; Kyowa Kirin Co., Ltd., Tokyo, Japan), as well as oxaliplatin (lot number: XHDFDA; Yakult Honsha Co., Ltd., Tokyo, Japan) and irinotecan hydrochloride (lot number: IR22208B; Pfizer Japan Inc., Tokyo, Japan) were dissolved in phosphate-buffered saline (PBS). Cell lines

The mouse colonic adenocarcinoma cell line MC38 was obtained from the National Cancer Institute (Bethesda, MD, USA). The MC38_hCLDN18.2 cell line was established by Astellas Pharmaceuticals by transducing human CLDN18.2 into MC38 cells and cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum and 6 μg/mL blastcinon at 37°C and 5 % _CO2 .

hCD3εKI mice were purchased from Biocytogen Jiangsu Co., Ltd. (Jiangsu, China) and bred at Astellas Pharmaceuticals Co., Ltd. This study was approved by the Animal Care and Use Committee of Astellas Pharmaceuticals Co., Ltd. (Ibaraki, Japan), and the center is AAALAC internationally accredited. Tumor inoculation .

MC38_hCLDN18.2 cells at a concentration of 4.0 × ^10⁶ cells/mL were suspended in PBS and subcutaneously inoculated into the flanks of 5-week-old male mice at a dose of 2.0 × ^10⁵ cells/50 μL. Administration and measurement were then performed.

On day 5 after inoculation with MC38_hCLDN18.2 cells, mice were assigned to groups (n=15) with similar mean tumor volume across groups, and each mouse was given 100 μL of the test substance starting the following day. Day 0 was defined as the first day of administration.

The four groups used in this study are listed below: Group 1: Control mice received intraperitoneal injections of PBS on days 0, 4, and 7. Group 2: ASP2138 mice received intraperitoneal injections of ASP2138 (0.1 mg/kg) on days 0 and 7, and intraperitoneal injections of PBS on day 4. Group 3: FOLFIRINOX mice received intraperitoneal injections of 5-fluorouracil (25 mg/kg), oxaliplatin (2.5 mg/kg), and irinotecan hydrochloride (10 mg/kg) on days 0, 4, and 7. Group 4: Combination mice received intraperitoneal injections of ASP2138 (0.1 mg/kg) on days 0 and 7, and intraperitoneal injections of 5-fluorouracil (25 mg/kg), oxaliplatin (2.5 mg/kg), and irinotecan hydrochloride (10 mg/kg) on days 0, 4, and 7.

Tumor diameter and body weight were measured using calipers and an electronic scale on days -1, 4, 7, and 11, respectively. The measurements taken on day -1 were used for analysis as data for day 0.

The formula for calculating tumor volume [ ^mm³ ] is: (tumor major axis length [mm]) × (tumor minor axis length [mm]) ^² × 0.5

The formula for calculating the percentage of tumor growth inhibition (%Inh) is: %Inh = 100 × (1 – [mean tumor volume on day 11 of each group – mean tumor volume on day 0] / [mean tumor volume on day 11 of the control group – mean tumor volume on day 0]). Statistical analysis

Tumor volume and body weight are expressed as mean ± SEM. On day 11, the mean tumor volume of groups 2 and 3 was compared with that of group 4 using the student's t-test. P < 0.05 was considered statistically significant. Data processing was performed using GraphPad Prism (version 10.1.2, GraphPad Software, San Diego, CA, USA). Results and Conclusions

In a human CD3ε knock-in (hCD3ε KI) mouse model expressing hCLDN18.2 tumor burden, the antitumor efficacy of a combination of the bispecific antibody ASP2138 targeting CLDN18.2 and CD3 with 5-fluorouracil, oxaliplatin, and irinotecan hydrochloride (FOLFIRINOX) was investigated.

Starting on day 6 following MC38_hCLDN18.2 inoculation, ASP2138 (0.1 mg/kg) was administered intraperitoneally on days 0 and 7, followed by intraperitoneal injections of 5-fluorouracil (25 mg/kg), oxaliplatin (2.5 mg/kg), and irinotecan hydrochloride (10 mg/kg) on days 0, 4, and 7. On day 11, ASP2138 and FOLFIRINOX inhibited tumor growth by 59% and 38%, respectively. The combination of ASP2138 and FOLFIRINOX inhibited tumor growth by 77% on day 11, with no significant difference in body weight change between groups (Figure 11). The tumor volume in the combination therapy group was significantly smaller than that in the ASP2138 or FOLFIRINOX groups (Figure 10).

In an hCD3ε KI mouse model burdened with MC38_hCLDN18.2 tumors, this study demonstrated that the combination therapy of ASP2138 and FOLFIRINOX significantly increased antitumor efficacy compared to ASP2138 or FOLFIRINOX monotherapy.

In this example, a combination of 5-fluorouracil, oxaliplatin, and irinotecan hydrochloride was administered to mice, and the administration of FOLFIRINOX is mentioned in this context. Those skilled in the art will understand that, strictly speaking, the FOLFIRINOX administration regimen should also include the administration of folate aldehydes, but this was not done in this example. Those skilled in the art will recognize that the results observed in this example with the combination of 5-fluorouracil, oxaliplatin, and irinotecan hydrochloride are equally applicable to the case of separate administration of folate aldehydes.

This article describes the following sequences: Sequence No. 1: MAVTACQGLG FVVSLIGIAG IIAATCMDQW STQDLYNNPV TAVFNYQGLW RSCVRESSGF 60 TECRGYFTLL GLPAMLQAVR ALMIVGIVLG AIGLLVSIFA LKCIRIGSME DSAKANMTLT 120 SGIMFIVSGL CAIAGVSVFA NMLVTNFWMS TANMYTGMGG MVQTVQTRYT FGAALFVGWV 180 AGGLTLIGGV MMCIACRGLA PEETNYKAVS YHASGHSVAY KPGGFKASTG FGSNTKNKKI 240 YDGGARTEDE VQSYPSKHDY V 261 Sequence No. 2: GKPGSGKPGS GKPGSGKPGS 20 Sequence No. 3: KTHTCPPCP 9 Sequence No. 4: GGGGSGGGGS KTHTCPPCP 19 Serial Number 5: GGGGSGGGGS 10 Serial Number 6: EPKSCDKTHT CPPCP 15 Serial Number 7: ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 60 GLYSLSSVVT VPSSSLGTQT YICNVNHKPS DTKVDKKVEP KSCDKTHTCP PCPAPPVAGP 120 SVFLFPPKPK DTLMISRTPE VTCVVVDVKH EDPEVKFNWY VDGVEVHNAK TKPREEEYNS 180 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM 240 TKNQVSLTCD VSGFYPSDIA VEWESDGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWE 300 QGDVFSCSVM HEALHNHYTQ KSLSLSPGK 329 Sequence No. 8:APPVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVKHEDP EVKFNWYVDG VEVHNAKTKP 60 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL 120 PPSREQMTKN QVKLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT 180 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 216 Sequence No. 9:RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD 60 SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 107 Serial Number 10:SYWIN 5 Serial Number 11:NIYPSDSYTN YNQKFQG 17 Serial Number 12:SWRGNSFDY 9 Serial Number 13:KSSQSLLNSG NQKNYLT 17 Serial Number 14:WASTRES 7 Serial Number 15:QNDYSYPFT 9 Serial Number 16:EVQLVQSGAE VKKPGESLRI SCKASGYTFT SYWINWVRQM PGKGLEWMGN IYPSDSYTNY 60 NQKFQGHVTI SVDKSISTAY LQWSSLKASD TAMYYCTRSW RGNSFDYWGQ GTLVTVSS 118 Serial Number 17:DIVMTQSPDS LAVSLGERAT INCKSSQSLL NSGNQKNYLT WYQQKPGQPP KLLIYWASTR 60 ESGVPDRFTG SGSGTDFTLT ISSLQAEDVA VYYCQNDYSY PFTFGSGTKL EIK 113 Serial Number 18:TYAMN 5 Serial Number 19:HGNFGDSYVS WFAY 14 Serial Number 20:GSSTGAVTTS NYAN 14 Serial Number 21:GTNKRAP 7 Serial Number 22:ALWYSNHWV 9 Serial Number 23:RIRSKANNYA TYYADSVKG 19 Serial Number 24:QAVVTQEPSL TVSPGGTVTL TCGSSTGAVT TSNYANWVQQ KPGKSPRGLI GGTNKRAPGV 60 PARFSGSLLG GKAALTISGA QPEDEADYYC ALWYSNHWVF Serial number 25: EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKANNYAT 60 YYADSVKGRF TISRDDSKNT LYLQMNSLRA EDTAVYYCVR HGNFGDSYVS WFAYWGQGTL 120 VTVSS Serial number 26: QAVVTQEPSL TVSPGGTVTL TCGSSTGAVT TSNYANWVQQ KPGKSPRGLI GGTNKRAPGV 60 PARFSGSLLG GKAALTISGA QPEDEADYYC ALWYSNHWVF GGGTKLTVLG KPGSGKPGSG 120 KPGSGKPGSE VQLVESGGGL VQPGGSLRLS CAASGFTFST YAMNWVRQAP GKGLEWVGRI 180 RSKANNYATY YADSVKGRFT ISRDDSKNTL YLQMNSLRAE DTAVYYCVRH GNFGDSYVSW 240 FAYWGQGTLV TVSS 254 Serial Number 27: EVQLVQSGAE VKKPGESLRI SCKASGYTFT SYWINWVRQM PGKGLEWMGN IYPSDSYTNY 60 NQKFQGHVTI SVDKSISTAY LQWSSLKASD TAMYYCTRSW RGNSFDYWGQ GTLVTVSSAS 120 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 180 YSLSSVVTVP SSSLGTQTYI CNVNHKPSDT KVDKKVEPKS CDKTHTCPPC PAPPVAGPSV 240 FLFPPKPKDT LMISRTPEVT CVVVDVKHED PEVKFNWYVD GVEVHNAKTK PREEEYNSTY 300 RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK 360 NQVSLTCDVS GFYPSDIAVE WESDGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWEQG 420 DVFSCSVMHE ALHNHYTQKS LSLSPGK 447 Serial Number 28:EVQLVQSGAE VKKPGESLRI SCKASGYTFT SYWINWVRQM PGKGLEWMGN IYPSDSYTNY 60 NQKFQGHVTI SVDKSISTAY LQWSSLKASD TAMYYCTRSW RGNSFDYWGQ GTLVTVSSAS 120 TKGPSVFPLA 180 GGKAALTISG AQPEDEADYY CALWYSNHWV FGGGTKLTVL GKPGSGKPGS GKPGSGKPGS 360 EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKANNYAT 420 YYADSVKGRF TISRDDSKNT LYLQMNSLRA EDTAVYYCVR HGNFGDSYVS WFAYWGQGTL 480 VTVSSGGGGS GGGGSKTHTC PPCPAPPVAG PSVFLFPPKP KDTLMISRTP EVTCVVVDVK 540 HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA 600 LPAPIEKTIS KAKGQPREPQ VYTLPPSREQ MTKNQVKLTC LVKGFYPSDI AVEWESNGQP 660 ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 720 Serial Number 29:DIVMTQSPDS LAVSLGERAT INCKSSQSLL NSGNQKNYLT WYQQKPGQPP KLLIYWASTR 180 LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC 220

Figure 1 shows the “Fab2-scFv” form of the bispecific binder described herein, which includes a VH fused to one side of the heterodimer Fc by recombination (the first polypeptide chain described herein), a VH linked to the scFv and then to the other side of the heterodimer Fc by recombination (the second polypeptide chain described herein), and LCs (the third and fourth polypeptide chains described herein) that form Fab domains with the VH of the first polypeptide chain and additional VH of the second polypeptide chain.

Figure 2 : Histograms showing necrin 18.2 expression in pancreatic cancer cells with and without gemcitabine hydrochloride and paclitaxel treatment ( first experiment ) . The histograms show necrin 18.2 expression on the surface of MIA PaCa-2_GFP_CLDN18.2_27 cells. Unfilled: treated with 0.1 µg/mL gemcitabine hydrochloride and 0.003 µg/mL paclitaxel for 2 days; Filled: untreated.

Figure 3 : Cytotoxicity of ASP2138 in pancreatic cancer cells treated with or without gemcitabine hydrochloride and paclitaxel co-cultured with PBMCs ( first experiment ). MIA PaCa-2 cells, after treatment with or without gemcitabine hydrochloride and paclitaxel, were co-cultured with human peripheral blood mononuclear cells (PBMCs) at an effector cell to target cell ratio of 10:1 and ASP2138 (concentrations of 0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, and 300 ng/mL) for 1 day. The number of viable target cells was measured by flow cytometry and cytotoxicity (%) was calculated. Each data point represents the mean of repeated measurements in a single experiment. Data points treated with gemcitabine hydrochloride, paclitaxel, and ASP2138 at 1 ng/mL or 30 ng/mL are single measurements. Each line represents a fitted nonlinear regression curve. MIA PaCa-2: MIA PaCa-2_GFP_CLDN18.2_27; Chemotherapy treatment: treated with 0.1 µg/mL gemcitabine hydrochloride and 0.003 µg/mL paclitaxel for 2 days.

Figure 4 : Histogram showing the expression of neminin 18.2 in pancreatic cancer cells with and without gemcitabine hydrochloride and paclitaxel treatment ( Second Experiment ) . The histogram shows the expression of neminin 18.2 on the surface of MIA PaCa-2_GFP_CLDN18.2_27 cells. Not filled in: treated with 0.1 µg/mL gemcitabine hydrochloride and 0.003 µg/mL paclitaxel for 2 days; Filled in: no treatment.

Figure 5 : Cytotoxicity of ASP2138 in pancreatic cancer cells with and without gemcitabine hydrochloride and paclitaxel treatment in co-culture with PBMCs ( Second Experiment ). MIA PaCa-2 cells were co-cultured with human peripheral blood mononuclear cells (PBMCs) at an effector cell to target cell ratio of 10:1 and ASP2138 (concentrations of 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300 and 1000 ng/mL) for 1 day with and without gemcitabine hydrochloride and paclitaxel treatment. The number of live target cells was measured by flow cytometry, and cytotoxicity (%) was calculated. Each data point represents the mean of the triplet test in a single experiment. Each line represents the fitted nonlinear regression curve. MIA PaCa-2: MIA PaCa-2_GFP_CLDN18.2_27; Chemotherapy: treated for 2 days with 0.1 µg/mL gemcitabine hydrochloride and 0.003 µg/mL paclitaxel.

Figure 6 : Antitumor effect of ASP2138 in combination with gemcitabine hydrochloride and paclitaxel in B16-F10 tumor-bearing and human CD3ε knock-in mouse models expressing CLDN18.2 . On day -4, 2.0 × ^10⁵ B16F10_mCLDN18.2 cells were subcutaneously seeded into the flank of mice. hCD3εKI mice received intraperitoneal injections of ASP2138 (0.01 mg/kg) and paclitaxel (10 mg/kg) on days 0 and 7, and gemcitabine hydrochloride (40 mg/kg) on days 0, 3, 7, and 10. Above: Tumor volume at each time point for each group is plotted as mean ± SEM (n=15). Below: Scatter plot showing tumor volume for each mouse on day 14. Short horizontal lines and error lines represent mean ± SEM (n=15). Statistical analysis was performed on the values at day 14. **: P < 0.01 compared to the values in the combination treatment group on day 14 (unpaired student t-test). B16F10_mCLDN18.2: B16-F10 representing mouse CLDN18.2; hCD3εKI: human CD3ε knock-in; Chemotherapy: gemcitabine hydrochloride and paclitaxel.

Figure 7 : Body weight changes in mice with B16-F10 tumor and human CD3ε knock-in models of CLDN18.2 expression after ASP2138 and / or chemotherapy. Mice received intraperitoneal injections of ASP2138 (0.01 mg/kg) and paclitaxel (10 mg/kg) on days 0 and 7, and gemcitabine hydrochloride (40 mg/kg) on days 0, 3, 7, and 10. Each point represents mean ± SEM (n=15). CLDN18.2: Neminin 18.2; Chemotherapy: Gemcitabine hydrochloride and paclitaxel

Figure 8 : Antitumor effect of the ASP2138 combined with FOLFOX in MC38 tumors expressed in human CLDN18.2 and human CD3ε knock-in mouse models. 2.0 × ^10⁵ MC38_hCLDN18.2 cells were subcutaneously seeded into the flank of mice on day -6. hCD3εKI mice received intraperitoneal injections of ASP2138 (0.1 mg/kg) on days 0 and 7, and were treated with 5-fluorouracil (25 mg/kg) and oxaliplatin (2.5 mg/kg) on days 0, 4, 7, and 11. Top: Tumor volume for each group at each time point is plotted as mean ± SEM (n=15). The following figure: A scatter plot showing the individual tumor volumes of each group on day 14. The short horizontal line and error line represent the mean ± SEM (n=15). Statistical analysis was performed on the values on day 14. **: P < 0.01 compared to the values in the combination therapy group on day 14 (Student's t-test). MC38_hCLDN18.2: MC38 expressed as human necrin 18.2; hCD3εKI: human CD3ε gene knock-in; FOLFOX: 5-fluorouracil and oxaliplatin.

Figure 9 : Body weight changes in MC38 tumors and human CD3ε knock-in mouse models exemplified by human CLDN18.2 , after treatment with ASP2138 and / or FOLFOX . Mice received intraperitoneal injections of ASP2138 (0.1 mg/kg) on days 0 and 7, and 5-fluorouracil (25 mg/kg) and oxaliplatin (2.5 mg/kg) on days 0, 4, 7, and 11. Each point represents mean ± SEM (n=15). CLDN18.2: Neminin 18.2; FOLFOX: 5-fluorouracil and oxaliplatin

Figure 10 : MC38 tumors expressed by human CLDN18.2 and human CD3ε knock-in mouse models. The antitumor effect of the ASP2138 combined with FOLFIRINOX was observed on day -6, when 2.0 × ^10⁵ MC38_hCLDN18.2 cells were subcutaneously injected into the flank of mice. hCD3εKI mice received intraperitoneal injections of ASP2138 (0.1 mg/kg) on days 0 and 7, and received 5-fluorouracil (25 mg/kg), oxaliplatin (2.5 mg/kg), and irinotecan hydrochloride (10 mg/kg) on days 0, 4, and 7. Top: Mean ± SEM of tumor volume at each time point for each group (n=15). The following figure: A scatter plot showing the individual tumor volume of each group on day 11. The short horizontal line and error line represent the mean ± SEM (n=15). Statistical analysis was performed on the values on day 11. **: Compared with the combination therapy group on day 11, P < 0.01 (Student's t-test). MC38_hCLDN18.2: MC38 representing human nectone 18.2; hCD3εKI: human CD3ε gene knock-in; FOLFIRINOX: 5-fluorouracil, oxaliplatin, and irinotecan hydrochloride.

Figure 11 : Body weight changes in human CLDN18.2 -expressing MC38 tumor and human CD3ε knock-in mouse models after ASP2138 and / or FOLFIRINOX treatment . Mice received intraperitoneal injections of ASP2138 (0.1 mg/kg) on days 0 and 7, and 5-fluorouracil (25 mg/kg), oxaliplatin (2.5 mg/kg), and irinotecan hydrochloride (10 mg/kg) on days 0, 4, and 7. Each point represents mean ± SEM (n=15). CLDN18.2: Neminin 18.2; FOLFIRINOX: 5-fluorouracil, oxaliplatin, and irinotecan hydrochloride.

TW202541837A_113147162_SEQL.xml

Claims (165)

A composition or pharmaceutical preparation comprising: (a) a bispecific binding agent comprising a first binding domain for human CLDN18.2, a second binding domain for human CLDN18.2, and a third binding domain for human CD3, wherein the binding agent comprises four polypeptide chains, wherein (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 80% sequence identity with 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 having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 28; and (iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 28. (iv) an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 29; and (b) an agent that stabilizes or enhances the performance of CLDN18.2. As in claim 1, the combination or pharmaceutical preparation wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 27. As in the combination or pharmaceutical preparation of claim 1 or 2, wherein the second polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 28. Combinations or pharmaceutical preparations of any of claims 1 to 3, wherein the third polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 29. Combinations or pharmaceutical preparations of any of claims 1 to 4, wherein the fourth polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 29. The combination or pharmaceutical preparation of any one of claims 1 to 5, wherein: (i) the first polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 27; (ii) the second polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 28; (iii) the third polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 29. A composition or pharmaceutical preparation comprising: (a) a bispecific binding agent comprising a first binding domain for human CLDN18.2, a second binding domain for human CLDN18.2, and a third binding domain for human CD3, wherein the binding agent comprises four polypeptide chains, wherein (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27; (ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28; (iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and (b) a medicament for stabilizing or increasing the expression of CLDN18.2. As in claim 7, the combination or pharmaceutical preparation wherein the first polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 27. Such as the combination or pharmaceutical preparation of claim 7 or 8, wherein the second polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 28. Combinations or pharmaceutical preparations of any of claims 7 to 9, wherein the third polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 29. Combinations or pharmaceutical preparations of any of claims 7 to 10, wherein the fourth polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 29. The combination or pharmaceutical preparation of any of claims 7 to 11, wherein: (i) the first polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 27; (ii) the second polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 28; (iii) the third polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 29. A composition or pharmaceutical preparation comprising: (a) a bispecific binding agent comprising a first binding domain for human CLDN18.2, a second binding domain for human CLDN18.2, and a third binding domain for human CD3, wherein the binding agent is encoded by one or more nucleic acid molecules comprising: (i) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 27; (ii) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 28; and (iii) a third nucleic acid sequence encoding a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 29; and (b) a medicament for stabilizing or increasing the expression of CLDN18.2. As in the combination or pharmaceutical preparation of claim 13, wherein the one or more nucleic acid molecules are a group of nucleic acids. The combination or pharmaceutical preparation of claims 13 or 14, wherein the bispecific binding agent comprises a tetrapeptide chain, wherein (i) a first polypeptide chain is encoded by the first nucleic acid sequence; (ii) a second polypeptide chain is encoded by the second nucleic acid sequence; (iii) a third polypeptide chain is encoded by the third nucleic acid sequence; and (iv) a fourth polypeptide chain is encoded by the third nucleic acid sequence. The composition or pharmaceutical preparation of claim 15, wherein the first polypeptide chain includes the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 27 includes the deletion of lysine at position 447 of SEQ ID NO: 27. Combinations or pharmaceutical preparations of claims 15 or 16, wherein the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 28 includes the deletion of lysine at position 720 of SEQ ID NO: 28. Compositions or pharmaceutical preparations of any of claims 15 to 17, wherein the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 29. Combinations or pharmaceutical preparations of any of claims 15 to 18, wherein the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 29. The combination or pharmaceutical preparation of any of claims 15 to 19, wherein (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 27 comprises the deletion of lysine at position 447 of SEQ ID NO: 27; (ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 28 comprises the deletion of lysine at position 720 of SEQ ID NO: 28; (iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29. Combinations or pharmaceutical preparations of any of claims 15 to 20, wherein (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 27 includes the deletion of lysine at position 447 of SEQ ID NO: 27; (ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 28 includes the deletion of lysine at position 720 of SEQ ID NO: 28; (iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29. Combinations or pharmaceutical preparations of any of claims 1 to 21, wherein the first polypeptide chain interacts with the second polypeptide chain and the third polypeptide chain. Combinations or pharmaceutical preparations of any of claims 1 to 12 and 15 to 22, wherein the second polypeptide chain interacts with the fourth polypeptide chain. Compositions or pharmaceutical preparations of any of claims 1 to 12 and 15 to 23, wherein 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. The combination or pharmaceutical preparation of any of claims 1 to 24, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus: (i) a variable region (VH) of a heavy chain derived from an immunoglobulin binding to human CLDN18.2 (VH(CLDN18.2)), (ii) a first constant region (CH1) of a heavy chain derived from an immunoglobulin or a functional variant thereof, (iii) a second constant region (CH2) of a heavy chain derived from an immunoglobulin or a functional variant thereof, and (iv) a third constant region (CH3) of a heavy chain derived from an immunoglobulin or a functional variant thereof. The combination or pharmaceutical preparation of any of claims 1 to 25, wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus: (i) a variable region (VH) of the heavy chain derived from an immunoglobulin binding to human CLDN18.2 (VH(CLDN18.2)), (ii) a first constant region (CH1) of the heavy chain derived from an immunoglobulin or a functional variant thereof, (iii) a variable region (VL) of the light chain derived from an immunoglobulin binding to human CD3 (VL(CD3)), (iv) a variable region (VH) of the heavy chain derived from an immunoglobulin binding to human CD3 (VH(CD3)), (v) a constant region 2 (CH2) of the heavy chain derived from an immunoglobulin or a functional variant thereof, and (vi) a constant region 3 (CH3) of the heavy chain derived from an immunoglobulin or a functional variant thereof. Compositions or pharmaceutical preparations of any of claims 1 to 26, wherein the third polypeptide chain comprises, from the N-terminus to the C-terminus: (i) a variable light chain region (VL) derived from an immunoglobulin binding to human CLDN18.2 (VL(CLDN18.2)), and (ii) a constant light chain region (CL) derived from an immunoglobulin or a functional variant thereof. Compositions or pharmaceutical preparations of any of claims 1 to 12 and 15 to 27, wherein the fourth polypeptide chain comprises, from the N-terminus to the C-terminus: (i) a variable light chain region (VL) derived from an immunoglobulin binding to human CLDN18.2 (VL(CLDN18.2)), and (ii) a constant light chain region (CL) derived from an immunoglobulin or a functional variant thereof. As in claims 27 or 28, the composition or pharmaceutical preparation wherein the VH (CLDN18.2) on the first polypeptide chain and the VL (CLDN18.2) on the third polypeptide chain interact to form a binding domain that binds to human CLDN18.2. As in claims 28 or 29, the combination or pharmaceutical preparation wherein the VH (CLDN18.2) on the second polypeptide chain and the VL (CLDN18.2) on the fourth polypeptide chain interact to form a binding domain that binds to human CLDN18.2. Compositions or pharmaceutical preparations of any of claims 26 to 30, wherein the VH(CD3) and the VL(CD3) interact to form a binding domain that binds to human CD3. The combination or pharmaceutical preparation of any of claims 25 to 31, wherein the VH (CLDN18.2) includes CDR1 containing the amino acid sequence SYWIN (SEQ ID NO: 10), CDR2 containing the amino acid sequence NIYPSDSYTNYNQKFQG (SEQ ID NO: 11), and CDR3 containing the amino acid sequence SWRGNSFDY (SEQ ID NO: 12). The combination or pharmaceutical preparation of any of claims 27 to 32, wherein the VL (CLDN18.2) comprises CDR1 containing the amino acid sequence KSSQSLLNSGNQKNYLT (SEQ ID NO: 13), CDR2 containing the amino acid sequence WASTRES (SEQ ID NO: 14), and CDR3 containing the amino acid sequence QNDYSYPFT (SEQ ID NO: 15). The combination or pharmaceutical preparation of any of claims 26 to 33, wherein the VH(CD3) includes CDR1 containing the amino acid sequence TYAMN (SEQ ID NO: 18), CDR2 containing the amino acid sequence RIRSKANNYATYYADSVKG (SEQ ID NO: 23), and CDR3 containing the amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO: 19). The composition or pharmaceutical preparation of any of claims 26 to 34, wherein the VL (CD3) includes CDR1 containing the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 20), CDR2 containing the amino acid sequence GTNKRAP (SEQ ID NO: 21), and CDR3 containing the amino acid sequence ALWYSNHWV (SEQ ID NO: 22). Compositions or pharmaceutical preparations of any of claims 26 to 35, wherein 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. Compositions or pharmaceutical preparations of any of claims 27 to 36, wherein CH1 on the first polypeptide chain interacts with CL on the third polypeptide chain. Compositions or pharmaceutical preparations of any of claims 28 to 37, wherein CH1 on the second polypeptide chain interacts with CL on the fourth polypeptide chain. Compositions or pharmaceutical preparations of any of claims 25 to 38, wherein the immunoglobulin is IgG1. As in claim 39, the composition or pharmaceutical preparation wherein the IgG1 is human IgG1. Compositions or pharmaceutical preparations of any of claims 25 to 40, wherein the VH (CLDN18.2) comprises SEQ ID NO: 16 or is composed of the amino acid sequence represented thereto. Compositions or pharmaceutical preparations of any of claims 27 to 41, wherein the VL (CLDN18.2) comprises SEQ ID NO: 17 or is composed of the amino acid sequence represented thereto. Compositions or pharmaceutical preparations of any of claims 26 to 42, wherein the VL(CD3) comprises SEQ ID NO: 24 or is composed of the amino acid sequence represented thereto. Compositions or pharmaceutical preparations of any of claims 26 to 43, wherein the VH(CD3) comprises SEQ ID NO: 25 or is composed of the amino acid sequence represented thereto. The combination or pharmaceutical preparation of any of claims 27 to 44, wherein the VH (CLDN18.2) comprises SEQ ID NO: 16 or is composed of the amino acid sequence represented thereto, the VL (CLDN18.2) comprises SEQ ID NO: 17 or is composed of the amino acid sequence represented thereto, the VH (CD3) comprises SEQ ID NO: 25 or is composed of the amino acid sequence represented thereto, and the VL (CD3) comprises SEQ ID NO: 24 or is composed of the amino acid sequence represented thereto. Compositions or pharmaceutical preparations of any of claims 25 to 45, wherein VH(CLDN18.2), VL(CLDN18.2), VH(CD3) and/or VL(CD3) are humanized. Compositions or pharmaceutical preparations of any of claims 25 to 46, wherein CH1 on the first polypeptide chain is linked to CH2 by a peptide linker L1. As in claim 47, the combination or pharmaceutical preparation wherein the peptide linker L1 comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 6) or a functional variant thereof. The composition or pharmaceutical preparation of any of claims 26 to 48, wherein the VL(CD3) is linked to CH1 by a peptide linker L2. As in claim 49, the combination or pharmaceutical preparation wherein the peptide linker L2 comprises an amino acid sequence ( _G4S ) _x or a functional variant thereof, wherein x is 2, 3, 4, 5 or 6. Compositions or pharmaceutical preparations such as those in claims 49 or 50, wherein the peptide linker L2 comprises an amino acid sequence ( _G4S ) ₂ (SEQ ID NO: 5) or a functional variant thereof. The composition or pharmaceutical preparation of any of claims 26 to 51, wherein the VL(CD3) and the VH(CD3) are linked to each other by a peptide linker L3. As in claim 52, the combination or pharmaceutical preparation wherein the peptide linker L3 comprises an amino acid sequence (GKPGS) _x or a functional variant thereof, wherein x is 2, 3, 4, 5 or 6. Combinations or pharmaceutical preparations such as claims 52 or 53, wherein the peptide linker L3 comprises an amino acid sequence (GKPGS) ₄ (SEQ ID NO: 2) or a functional variant thereof. Compositions or pharmaceutical preparations of any of claims 26 to 54, wherein the VH(CD3) is linked to CH2 via a peptide linker L4. As in claim 55, the composition or pharmaceutical preparation wherein the peptide linker L4 comprises an amino acid sequence ( _G4S ) _x or a functional variant thereof, wherein x is 2, 3, 4, 5 or 6. Compositions or pharmaceutical preparations such as those in claims 55 or 56, wherein the peptide linker L4 comprises an amino acid sequence ( _G4S ) ₂ (SEQ ID NO: 5) or a functional variant thereof. Combinations or pharmaceutical preparations of any of claims 1 to 57, wherein the first polypeptide chain comprises an amino acid sequence represented by SEQ ID NO: 7. Combinations or pharmaceutical preparations of any of claims 1 to 58, wherein the second polypeptide chain comprises an amino acid sequence represented by SEQ ID NO: 8. Combinations or pharmaceutical preparations of any of claims 1 to 59, wherein the third and/or fourth polypeptide chain comprises an amino acid sequence represented by SEQ ID NO: 9. Compositions or pharmaceutical preparations of any of claims 1 to 60, wherein the bispecific binder binds to the extracellular portion of human CLDN18.2. Combinations or pharmaceutical preparations of any of claims 1 to 61, wherein the bispecific binder, upon binding to CD3 on T cells, leads to the proliferation and/or activation of T cells. Combinations or pharmaceutical preparations of any of claims 1 to 62, wherein the bispecific binder induces T cell-mediated cytotoxicity against cancer cells expressing CLDN18.2. Combinations or pharmaceutical preparations of any of the claims 1 to 63, wherein the preparation that stabilizes or increases the performance of CLDN18.2 includes chemotherapy preparations. Combinations or pharmaceutical preparations of any of claims 1 to 64, wherein the stabilizing or enhancing CLDN18.2 performance includes cytotoxic and/or cell growth inhibitors. Combinations or pharmaceutical preparations of any of claims 1 to 65, wherein the preparations that stabilize or enhance CLDN18.2 performance include the group consisting of preparations selected from anthracycline antibiotics, nucleoside analogs, platinum compounds, camptothecin analogs and taxanes, their prodrugs, their salts, and combinations thereof. Combinations or pharmaceutical preparations of any of claims 1 to 66, wherein the preparations that stabilize or enhance CLDN18.2 performance include nucleoside analogs, their prodrugs, or their salts. Combinations or pharmaceutical preparations of any of claims 1 to 67, wherein the preparations that stabilize or enhance CLDN18.2 performance include taxanes, their prodrugs, or their salts. Combinations or pharmaceutical preparations of any of the claims 1 to 68, wherein the preparations that stabilize or increase the performance of CLDN18.2 include (i) gemcitabine, its prodrug, or salts thereof, (ii) paclitaxel, its prodrug, or salts thereof, or (iii) combinations of gemcitabine, its prodrug, or salts thereof, and paclitaxel, its prodrug, or salts thereof. Combinations or pharmaceutical preparations of any of claims 1 to 66 include: (i) the bispecific binding agent; (ii) oxaliplatin; (iii) 5-fluorouracil or a precursor thereof; and optionally (iv) folic acid. Combinations or pharmaceutical preparations as claimed in claim 70 further include pembrolizumab. The composition or pharmaceutical preparation of claim 70 further includes irinotecan. Combinations or pharmaceutical preparations of any of claims 1 to 66, including: (i) the bispecific binding agent; (ii) paclitaxel; and (iii) ramorumab. If any of the combinations or pharmaceutical preparations in claims 1 to 73 are pharmaceutical compositions. As in claim 74, the composition or pharmaceutical formulation further includes one or more pharmaceutically acceptable carriers, diluents and/or excipients. If any of the combinations or pharmaceutical preparations in requests 1 to 73 are in a set. As in claim 76, the combination or pharmaceutical preparation wherein the bispecific binder and the preparation that stabilizes or enhances CLDN18.2 performance are stored separately in vials. The combination or pharmaceutical preparation of claims 76 or 77 further includes the bispecific binding agent and instructions for use of the preparation for the treatment or prevention of cancer that stabilizes or enhances CLDN18.2 performance. Combinations or pharmaceutical preparations of any of claims 1 to 78, intended for medical use. Such as composition or pharmaceutical preparation in claim 79, wherein the pharmaceutical use includes the treatment or prevention of disease or condition. As in claim 80, the composition or pharmaceutical preparation wherein the treatment or prevention of a disease or condition includes the treatment or prevention of cancer. Compositions or pharmaceutical preparations of any of claims 78 to 81, wherein the cancer involves cancer cells expressing CLDN18.2. Combinations or pharmaceutical preparations of any of claims 78 to 82, wherein the cancer is selected from the group consisting of gastric cancer (especially gastric adenocarcinoma), esophageal cancer, gastroesophageal junction cancer (GEJ) (especially gastroesophageal junction adenocarcinoma), pancreatic cancer (especially pancreatic cancer), lung cancer (such as 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 its metastases, Crugenburg tumor, peritoneal metastases and/or lymph node metastases. Combinations or pharmaceutical preparations of any of the claims 1 to 83, intended for administration to humans. A method for treating or preventing cancer in an individual, comprising administering to the individual: (a) a bispecific binding agent comprising a first binding domain to human CLDN18.2, a second binding domain to human CLDN18.2, and a third binding domain to human CD3, wherein the binding agent comprises four polypeptide chains, wherein (i) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 27; (ii) the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 28; and (iii) the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 28. (iv) an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 29; and (b) an agent that stabilizes or enhances the performance of CLDN18.2. The method of claim 85, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 27. The method of claim 85 or 86, wherein the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 28. The method of any one of claims 85 to 87, wherein the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 29. The method of any one of claims 85 to 88, wherein the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 29. The method of any one of claims 85 to 89, wherein (i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 80% sequence identity with 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 having at least 80% sequence identity with 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 having at least 80% sequence identity with 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 with the amino acid sequence of SEQ ID NO: 29. A method for treating or preventing cancer in an individual, comprising administering to an individual: (a) a bispecific binding agent comprising a first binding domain of human CLDN18.2, a second binding domain of human CLDN18.2, and a third binding domain of human CD3, wherein the binding agent comprises four polypeptide chains, wherein (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27; (ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28; (iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and (b) a drug that stabilizes or increases the expression of CLDN18.2. The method of claim 91, wherein the first polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 27. The method of claim 91 or 92, wherein the second polypeptide chain is composed of the amino acid sequence of SEQ ID NO: 28. The method of any one of claims 91 to 93, wherein the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29. The method of any one of claims 91 to 94, wherein the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29. The method of any one of claims 91 to 95, wherein: (i) the first polypeptide chain consists of the amino acid sequence of SEQ ID NO: 27; (ii) the second polypeptide chain consists of the amino acid sequence of SEQ ID NO: 28; (iii) the third polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain consists of the amino acid sequence of SEQ ID NO: 29. A method for treating or preventing cancer in an individual, comprising administering to an individual: (a) a bispecific binding agent comprising a first binding domain of human CLDN18.2, a second binding domain of human CLDN18.2, and a third binding domain of human CD3, wherein the binding agent is encoded by one or more nucleic acid molecules comprising: (i) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 27; (ii) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 28; and (iii) a third nucleic acid sequence encoding a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 29; and (b) a drug that stabilizes or increases the expression of CLDN18.2. The method of claim 97, wherein the one or more nucleic acid molecules are a group of nucleic acids. The method of claim 97 or 98, wherein the bispecific binding agent comprises a tetrapeptide chain, wherein (i) a first polypeptide chain is encoded by the first nucleic acid sequence; (ii) a second polypeptide chain is encoded by the second nucleic acid sequence; (iii) a third polypeptide chain is encoded by the third nucleic acid sequence; and (iv) a fourth polypeptide chain is encoded by the third nucleic acid sequence. The method of claim 99, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 27 comprises the deletion of lysine at position 447 of SEQ ID NO: 27. The method of claim 99 or 100, wherein the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 28 comprises the deletion of lysine at position 720 of SEQ ID NO: 28. The method of any of claims 99 to 101, wherein the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 29. The method of any of claims 99 to 102, wherein the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 29. The method of any one of claims 99 to 103, wherein (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 27 comprises the deletion of lysine at position 447 of SEQ ID NO: 27; (ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 28 comprises the deletion of lysine at position 720 of SEQ ID NO: 28; (iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29. The method of any one of claims 99 to 104, wherein (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 27 includes the deletion of lysine at position 447 of SEQ ID NO: 27; (ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 or a C-terminal truncated variant thereof, wherein the C-terminal truncated variant of SEQ ID NO: 28 includes the deletion of lysine at position 720 of SEQ ID NO: 28; (iii) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; and (iv) the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29. The method of any of claims 85 to 105, wherein the first polypeptide chain interacts with the second polypeptide chain and the third polypeptide chain. The method of any of claims 85 to 96 and 99 to 106, wherein the second polypeptide chain interacts with the fourth polypeptide chain. The method of any of claims 85 to 96 and 99 to 107, wherein 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. The method of any of claims 85 to 108, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus: (i) a variable region (VH) of a heavy chain derived from an immunoglobulin binding to human CLDN18.2 (VH(CLDN18.2)), (ii) a first constant region (CH1) of a heavy chain derived from an immunoglobulin or a functional variant thereof, (iii) a second constant region (CH2) of a heavy chain derived from an immunoglobulin or a functional variant thereof, and (iv) a third constant region (CH3) of a heavy chain derived from an immunoglobulin or a functional variant thereof. The method of any of claims 85 to 109, wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus: (i) a heavy chain variable region (VH) derived from an immunoglobulin binding to human CLDN18.2 (VH(CLDN18.2)), (ii) a first constant region (CH1) of the heavy chain derived from an immunoglobulin or a functional variant thereof, (iii) a light chain variable region (VL) derived from an immunoglobulin binding to human CD3 (VL(CD3)), (iv) a heavy chain variable region (VH) derived from an immunoglobulin binding to human CD3 (VH(CD3)), (v) a second constant region (CH2) of the heavy chain derived from an immunoglobulin or a functional variant thereof, and (vi) a third constant region (CH3) of the heavy chain derived from an immunoglobulin or a functional variant thereof. The method of any one of claims 85 to 110, wherein the third polypeptide chain comprises, from the N-terminus to the C-terminus: (i) a light chain variable region (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. The method of any one of claims 85 to 96 and 99 to 111, wherein the fourth polypeptide chain comprises, from the N-terminus to the C-terminus: (i) a light chain variable region (VL)(VL(CLDN18.2)) derived from an immunoglobulin that binds to human CLDN18.2, and (ii) a light chain constant region (CL) derived from an immunoglobulin or a functional variant thereof. The method of claim 111 or 112, wherein the VH (CLDN18.2) on the first polypeptide chain and the VL (CLDN18.2) on the third polypeptide chain interact to form a binding domain that binds to human CLDN18.2. The method of claim 112 or 113, wherein the VH (CLDN18.2) on the second polypeptide chain and the VL (CLDN18.2) on the fourth polypeptide chain interact to form a binding domain that binds to human CLDN18.2. The method of any of claims 110 to 114, wherein the VH(CD3) and the VL(CD3) interact to form a binding domain that binds to human CD3. The method of any one of claims 109 to 115, wherein the VH (CLDN18.2) includes CDR1 containing the amino acid sequence SYWIN (SEQ ID NO: 10), CDR2 containing the amino acid sequence NIYPSDSYTNYNQKFQG (SEQ ID NO: 11), and CDR3 containing the amino acid sequence SWRGNSFDY (SEQ ID NO: 12). The method of any one of claims 111 to 116, wherein the VL (CLDN18.2) comprises CDR1 containing the amino acid sequence KSSQSLLNSGNQKNYLT (SEQ ID NO: 13), CDR2 containing the amino acid sequence WASTRES (SEQ ID NO: 14), and CDR3 containing the amino acid sequence QNDYSYPFT (SEQ ID NO: 15). The method of any one of claims 110 to 117, wherein the VH(CD3) comprises CDR1 containing the amino acid sequence TYAMN (SEQ ID NO: 18), CDR2 containing the amino acid sequence RIRSKANNYATYYADSVKG (SEQ ID NO: 23), and CDR3 containing the amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO: 19). The method of any one of claims 110 to 118, wherein the VL (CD3) comprises CDR1 containing the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 20), CDR2 containing the amino acid sequence GTNKRAP (SEQ ID NO: 21), and CDR3 containing the amino acid sequence ALWYSNHWV (SEQ ID NO: 22). The method of any of claims 110 to 119, wherein 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. The method of any of claims 111 to 120, wherein CH1 on the first polypeptide chain interacts with CL on the third polypeptide chain. The method of any of claims 112 to 121, wherein CH1 on the second polypeptide chain interacts with CL on the fourth polypeptide chain. The method of any of claims 109 to 122, wherein the immunoglobulin is IgG1. The method of claim 123, wherein the IgG1 is human IgG1. The method of any of claims 109 to 124, wherein the VH (CLDN18.2) comprises SEQ ID NO: 16 or is composed of the amino acid sequence represented thereto. The method of any of claims 111 to 125, wherein the VL (CLDN18.2) comprises SEQ ID NO: 17 or is composed of the amino acid sequence represented thereto. The method of any of claims 110 to 126, wherein the VL(CD3) comprises SEQ ID NO: 24 or is composed of the amino acid sequence represented thereto. The method of any of claims 110 to 127, wherein the VH(CD3) comprises SEQ ID NO: 25 or is composed of the amino acid sequence represented thereto. The method of any of claims 111 to 128, wherein the VH (CLDN18.2) comprises SEQ ID NO: 16 or is composed of the amino acid sequence represented thereto, the VL (CLDN18.2) comprises SEQ ID NO: 17 or is composed of the amino acid sequence represented thereto, the VH (CD3) comprises SEQ ID NO: 25 or is composed of the amino acid sequence represented thereto, and the VL (CD3) comprises SEQ ID NO: 24 or is composed of the amino acid sequence represented thereto. The method of any of the claims 109 to 129, wherein VH(CLDN18.2), VL(CLDN18.2), VH(CD3) and/or VL(CD3) are humanized. The method of any of claims 109 to 130, wherein CH1 on the first polypeptide chain is linked to CH2 by a peptide linker L1. The method of claim 131, wherein the peptide linker L1 comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 6) or a functional variant thereof. The method of any of claims 110 to 132, wherein the VL(CD3) is linked to CH1 by a peptide linker L2. The method of claim 133, wherein the peptide linker L2 comprises an amino acid sequence ( _G4S ) _x or a functional variant thereof, wherein x is 2, 3, 4, 5 or 6. The method of claim 133 or 134, wherein the peptide linker L2 comprises an amino acid sequence ( _G4S ) ₂ (SEQ ID NO: 5) or a functional variant thereof. The method of any of claims 110 to 135, wherein the VL(CD3) and the VH(CD3) are linked to each other by a peptide linker L3. The method of claim 136, wherein the peptide linker L3 includes an amino acid sequence (GKPGS) _x or a functional variant thereof, wherein x is 2, 3, 4, 5 or 6. The method of claim 136 or 137, wherein the peptide linker L3 comprises an amino acid sequence (GKPGS) ₄ (SEQ ID NO: 2) or a functional variant thereof. The method of any of claims 110 to 138, wherein the VH(CD3) is linked to CH2 via a peptide linker L4. The method of claim 139, wherein the peptide linker L4 comprises an amino acid sequence ( _G4S ) _x or a functional variant thereof, wherein x is 2, 3, 4, 5 or 6. The method of claims 139 or 140, wherein the peptide linker L4 comprises an amino acid sequence ( _G4S ) ₂ (SEQ ID NO: 5) or a functional variant thereof. The method of any of claims 85 to 141, wherein the first polypeptide chain comprises an amino acid sequence represented by SEQ ID NO: 7. The method of any of claims 85 to 142, wherein the second polypeptide chain comprises an amino acid sequence represented by SEQ ID NO: 8. The method of any of claims 85 to 143, wherein the third and/or the fourth polypeptide chain comprises an amino acid sequence represented by SEQ ID NO: 9. The method of any of claims 85 to 144, wherein the bispecific binder binds to the extracellular portion of human CLDN18.2. The method of any of claims 85 to 145, wherein the bispecific binder, upon binding to CD3 on T cells, leads to the proliferation and/or activation of T cells. The method of any of claims 85 to 146, wherein the bispecific binder induces T cell-mediated cytotoxicity against cancer cells expressing CLDN18.2. The method of any of claims 85 to 147, wherein the agent that stabilizes or increases the performance of CLDN18.2 includes chemotherapy agents. The method of any of claims 85 to 148, wherein the agent stabilizing or increasing the performance of CLDN18.2 includes cytotoxic and/or cell growth inhibitors. The method of any of claims 85 to 149, wherein the agent that stabilizes or increases the performance of CLDN18.2 comprises the group consisting of agents selected from anthracycline antibiotics, nucleoside analogs, platinum compounds, camptothecin analogs and taxanes, their prodrugs, their salts, and combinations thereof. The method of any of claims 85 to 150, wherein the agent that stabilizes or increases the performance of CLDN18.2 includes nucleoside analogues, their prodrugs, or their salts. The method of any of claims 85 to 151, wherein the agent that stabilizes or increases the performance of CLDN18.2 includes paclitaxel, its prodrug, or its salt. The method of any of claims 85 to 152, wherein the agent that stabilizes or increases the performance of CLDN18.2 includes (i) gemcitabine, its prodrug, or a salt thereof, (ii) paclitaxel, its prodrug, or a salt thereof, or (iii) a combination of gemcitabine, its prodrug, or a salt thereof, and paclitaxel, its prodrug, or a salt thereof. The method of any of claims 85 to 150 includes (i) the bispecific binding agent; (ii) oxaliplatin; (iii) 5-fluorouracil or a precursor thereof; and optionally (iv) folic acid. The method of claim 154 further includes pembrolizumab. The method of claim 154 further includes irinotecan. The method of any of claims 85 to 150 includes: (i) the bispecific binding agent; (ii) paclitaxel; and (iii) ramorumab. The method of any of claims 85 to 157, wherein the bispecific binder and the agent that stabilizes or enhances the performance of CLDN18.2 are present in the same pharmaceutical composition or separate pharmaceutical compositions. The method of claim 158, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients. The method of any of claims 85 to 159, wherein the cancer involves cancer cells expressing CLDN18.2. The method of any of claims 85 to 160, wherein the cancer is selected from the group consisting of gastric cancer (especially gastric adenocarcinoma), esophageal cancer, gastroesophageal junction cancer (GEJ) (especially gastroesophageal junction adenocarcinoma), pancreatic cancer (especially pancreatic cancer), lung cancer (such as 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 its metastases, Crugenburg tumor, peritoneal metastases and/or lymph node metastases. The method described in claim 155 or 157, wherein the cancer is gastric cancer and/or gastroesophageal junction cancer. The method of claim 156, wherein the cancer is pancreatic cancer. The method of any of the requests 85 to 163, wherein the individual is a human. Compositions or pharmaceutical preparations of any of claims 1 to 84, and methods of using any of claims 85 to 164.