Claudin18 antibodies and methods of treating cancer

Abstract

To provide antigen-binding proteins which bind to Claudin-18.2 (CLDN18.2).SOLUTION: The present disclosure provides antigen-binding proteins which bind to human Claudin-18.2 (CLDN18.2) proteins, wherein, a. the antigen-binding proteins bind to an extracellular loop 1 (EL1) of the extracellular domain (ECD) of CLDN18.2 and does not bind to an extracellular loop 2 (EL2) of the ECD of CLDN18.2, or b. does not bind to Claudin 18.1 (CLDN18.1) and binds to CLDN18.2 endogenously expressed by HLPT4 cells with an affinity greater than 1.5 times that of a reference antibody, or c. a combination thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates generally to antibodies specific for claudin 18.2 and their use to treat cancer.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 875,416, filed July 17, 2019, the entire contents of which are incorporated herein by reference in their entirety. [Background technology]

[0003] Due to their ability to specifically target different antigens on cells, bacteria, viruses, or toxins, antibodies constitute powerful therapeutic agents characterized by limited side effects. In 1986, the first therapeutic monoclonal antibody, Orthoclone OKT3, was introduced to the market. Since then, this class of biopharmaceutical products has grown significantly. As of the end of 2014, 47 monoclonal antibody products have been approved in the United States or Europe for the treatment of a variety of diseases, including cancer and inflammatory, cardiovascular, respiratory, and infectious diseases.

[0004] More than a dozen monoclonal antibodies are currently approved by the U.S. Food and Drug Administration to treat cancer. Among these drugs are alemtuzumab (Campath®), indicated for chronic lymphocytic leukemia (CLL), and trastuzumab (Herceptin®), used to treat breast cancer. Some antibodies are classified as chemotherapy drugs, including, for example, brentuximab vedotin (Adcetris®) and ado-trastuzumab emtansine (Kadcyla®). Other antibody products, such as blinatumomab (Blincyto), are designed to recognize and bind to two different antigens. Despite the commercial availability of such antibody products, current cancer incidence and cancer deaths remain high. Cancer incidence is reported to be more than 450 per 100,000 men and women per year, and cancer deaths are just over 170 per 100,000 men and women per year. Summary of the Invention

[0005] Provided herein are antigen binding proteins that bind to claudin-18 (CLDN18). In various aspects, the antigen binding proteins of the disclosure bind to human CLDN18, and optionally to murine CLDN18. In various aspects, the antigen binding proteins bind to the extracellular domain (ECD) of CLDN18. In various embodiments, the disclosure provides antigen binding proteins for CLDN18.2. In various examples, the antigen binding proteins bind to extracellular loop 1 (EL1) of the ECD of CLDN18.2. In various aspects, the antigen binding proteins bind to EL1 and do not bind to extracellular loop 2 (EL2) of the ECD of CLDN18.2. In various embodiments, the antigen binding proteins that bind to CLDN18.2 bind to an epitope within the amino acid sequence of residues 28-74 of CLDN18.2 (SEQ ID NO:1). In various embodiments, the antigen binds to the amino acid sequence of GLWRSCVRESSGFTECRFYFTL (SEQ ID NO: 4), QGLWRSCVRESSGFTECRGYFTLK (SEQ ID NO: 5), DQWSTQDLYNNPVTAVFNYQGLWRSC (SEQ ID NO: 6) or CRGYFTLLFLPAMLQAVR (SEQ ID NO: 7) of CLDN18.2.

[0006] In various examples, the antigen binding protein binds to CLDN18.2 and does not bind to any other member of the claudin family. In various aspects, the antigen binding protein binds to CLDN18.2 endogenously expressed by human cancer cells, e.g., HUPT4 pancreatic cells. In various examples, the antigen binding protein of the present disclosure inhibits tumor growth in a subject, e.g., a human, without any other moiety binding to the antigen binding protein. In various examples, the antigen binding protein that is not conjugated to a foreign moiety (e.g., not conjugated to any chemotherapeutic agent, drug, or toxic moiety) inhibits tumor growth in a subject, e.g., a human.

[0007] In various embodiments, the antigen binding protein binds to CLDN18.2 expressed by human cancer cells. In various embodiments, the antigen binding protein inhibits the binding interaction between human CLDN18.2 and a reference anti-CLDN18.2 antibody. Without being bound to a particular theory, the inhibitory effect of the antigen binding proteins provided herein renders such entities useful in methods of reducing tumor growth and treating subjects with tumors or cancer. As further discussed herein, in various embodiments, the antigen binding protein is an antibody, an antigen-binding antibody fragment thereof, or an antibody protein product.

[0008] The present disclosure also provides antigen binding proteins that comprise at least three, four, five, or all of the amino acid sequences of a particular group of amino acid sequences. In various embodiments, the antigen binding proteins comprise at least three, four, five, or six complementarity determining region (CDR) amino acid sequences of the CLDN18.2 antibody disclosed herein.

[0009] The present disclosure further provides antigen binding proteins comprising the amino acid sequences detailed herein. In various embodiments, the antigen binding proteins comprise the amino acid sequences of the SEQ ID NOs listed in Table A, Table B, Table C, Table D, Table 6, Table 8, Table 9, Table 10, Table 11, or combinations thereof, as further described herein.

[0010] The present disclosure provides bispecific antigen binding proteins that bind CLDN18.2 and a second antigen. The bispecific antigen binding proteins may include any one of the antigen binding proteins described herein. The second antigen may be a cell surface protein, optionally a protein whose binding modulates an immune response. The bispecific antigen binding proteins may take any structure, e.g., diabodies, TandAbs (tandem diabodies), BiTEs (bispecific T cell engagers), etc. Exemplary bispecific antigen binding proteins include the sequences shown in Table 11.

[0011] The present disclosure also provides a conjugate comprising an antigen-binding protein or a bispecific antigen-binding protein and a heterologous moiety (e.g., a cytotoxic drug). The conjugate may comprise a cleavable or non-cleavable linker. The conjugate may have a variable number of heterologous moieties (agents) conjugated to the antigen-binding protein or bispecific antigen-binding protein described herein, preferably 1-8 agents per protein or 3-8 agents per protein. The conjugate may be a site-specific conjugate. The conjugate may be a homogeneous or heterogeneous conjugate.

[0012] Related polypeptides, nucleic acids, vectors, host cells, and conjugates are further provided herein. Kits and pharmaceutical compositions comprising such entities are further contemplated.

[0013] Also provided are methods of making the antigen binding proteins, in various embodiments, the methods comprise culturing a host cell comprising a nucleic acid encoding an antigen binding protein or polypeptide described herein to express the antigen binding protein or polypeptide.

[0014] Further provided herein is a method of treating a subject having cancer. In various embodiments, the method comprises administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to treat the cancer in the subject.

[0015] Also provided is a method for treating a subject with a cancer that expresses CLDN18.2, comprising administering to the subject a pharmaceutical composition as described herein.In various embodiments, the cancer that expresses CLDN18.2 expresses CLDN18.2.Furthermore, a method for inhibiting tumor growth in a subject is contemplated, comprising administering to the subject a pharmaceutical composition as described herein.

[0016] A method of reducing tumor size in a subject or preventing recurrence of cancer in a subject, comprising administering to the subject a pharmaceutical composition described herein.

[0017] Also provided herein is a method of treating cancer in a subject diagnosed with a low overexpressor of CLDN18, such as CLDN18.2, comprising administering to the subject a pharmaceutical composition described herein.

[0018] In various embodiments, administration induces apoptosis in tumor cells, e.g., cells expressing CLDN18, particularly CLDN18.2. In various embodiments, administration induces antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), tumor necrosis and cell death or depletion, and / or disruption of tumor cell adhesion, each of which results in tumor regression or slowing of tumor growth. [Brief description of the drawings]

[0019] [Figure 1] 1 depicts a graph of CLDN18.2 expression in a panel of cancer types. [Diagram 2] 1 depicts a graph of CLDN18.2 expression in additional cancer types. [Diagram 3] 1 depicts a graph of CLDN18.1 and CLDN18.2 expression in cancer cell lines as determined by RNASeq. [Figure 4] The anti-CLDN18.2 antibodies described herein demonstrate the ability to detect CLDN18.2 on the surface of cancer cell lines that endogenously express CLDN18.2. [Diagram 5] A represents a graph of tumor volume (mm3) of tumors in mice bearing upper gastrointestinal (SNU601) tumors as a function of time (days) after treatment with chimeric anti-CLDN18.2 antibodies. B represents a graph of the mean change in tumor volume (mm3) at day 30 in the same treatment groups. [Figure 6]A represents a graph of tumor volume (mm3) of tumors in mice bearing pancreatic (HUPT4) tumors as a function of time (days) after treatment with chimeric anti-CLDN18.2 antibodies. B represents a graph of the mean change in tumor volume (mm3) at day 28 in the same treatment groups. [Figure 7A] 1 depicts a graph of tumor volume (mm3) of tumors in mice bearing pancreatic (HUPT4) tumors as a function of time (days) after treatment with humanized anti-CLDN18.2 antibody. [Figure 7B] A graph of the mean change in tumor volume (mm3) at day 42 in the same treatment groups is presented. [Figure 8A] 1 shows the difference in tumor volume in mice bearing pancreatic (HUPT4) tumors following different dosing schedules as a function of time (days) after treatment with humanized anti-CLDN18.2 antibody. [Figure 8B] A graph of the mean change in tumor volume (mm3) at day 42 in the same treatment groups is presented. [Figure 9A] 1 depicts a graph of tumor volume (mm3) of tumors in mice bearing pancreatic (HUPT4) tumors as a function of time (days) after treatment with humanized anti-CLDN18.2 antibody drug conjugates (ADCs). [Figure 9B] 1 depicts a graph of tumor volume (mm3) in mice treated with a single dose of anti-CLDN18.2 ADC or control antibody. [Figure 10] A depicts a graph of tumor volume (mm3) of tumors in mice bearing pancreatic (PATU8988S) tumors as a function of time (days) after treatment with different doses of humanized anti-CLDN18.2 ADC. B depicts a graph of the mean change in tumor volume (mm3) at day 33 in the same treatment groups. [Figure 11] 1 depicts a graph of tumor volume (mm3) of tumors in mice bearing SNU601 gastric cancer as a function of time (days) after treatment with different doses of humanized anti-CLDN18.2 ADC. [Figure 12]A represents a graph of tumor volume (mm3) of tumors in mice bearing pancreatic (HUPT4) tumors as a function of time (days) after treatment with different doses of humanized anti-CLDN18.2 antibody drug conjugates (ADCs). B represents a graph of tumor volume (mm3) at day 32 in the same treatment groups. [Figure 13-1] 1 shows the amino acid sequences of the heavy and light chain variable regions of the CLDN18.2 antibody described herein. [Figure 13-2] Continued from Figure 13-1. [Figure 13-3] Continued from Figure 13-2. [Figure 13-4] Continued from Figure 13-3. [Figure 13-5] Continued from Figure 13-4. [Figure 14] A represents a graph of tumor volume (mm3) of tumors in mice bearing pancreatic patient-derived (PDX) tumors expressing CLDN18.2 as a function of time (days) after treatment with different doses of humanized anti-CLDN18.2 antibody drug conjugates (ADCs). B represents a graph of tumor volume (mm3) at day 27 in the same treatment groups. C represents immunohistochemistry (IHC) showing CLDN18.2 expression in PDX models. [Figure 15] A represents a graph of tumor volume (mm3) of tumors in mice bearing pancreatic patient-derived (PDX) tumors that do not express CLDN18.2 as a function of time (days) after treatment with different doses of humanized anti-CLDN18.2 antibody drug conjugates (ADCs). B represents a graph of tumor volume (mm3) at day 18 in the same treatment groups. C represents immunohistochemistry (IHC) showing the lack of CLDN18.2 expression in PDX models. [Figure 16A] 1 depicts a graph of tumor volume (mm3) of tumors in mice bearing melanoma (M202) tumors that do not express CLDN18.2 as a function of time (days) after treatment with different doses of humanized anti-CLDN18.2 antibody drug conjugate (ADC). [Figure 16B] A graph of tumor volume (mm3) at day 23 in the same treatment groups is presented. [Figure 17] The sequences for antibodies 307-4h and 358-h3 as well as additional sequence variants are shown. These antibodies were generated to provide additional information on potential manufacturable sequence burden. [Figure 18] It represents the humanized antibody binding activity against native positive cells (cells with endogenous expression of CLDN18.2) or artificially overexpressing cells (cells engineered to overexpress CLDN18.2) as analyzed by flow cytometry. [Figure 19] 1 shows the binding activity of humanized antibodies against human CLDN18.2, human CLDN18.1, mouse CLDN18.2, canine CLDN18, and rat CLDN18. [Figure 20] 1 shows in vitro characterization of antibody internalization using CLDN18.2 humanized antibody in HUPT4 cells. [Figure 21] A represents the binding affinity (KD) of humanized CLDN18.2 antibodies to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. B represents CLDN18.2 expression levels to various cancer cell lines. pNK-307-h1F06-4 refers to 307-h4 antibody; pNK-358-h2c10-3 refers to 358-h3 antibody; #307-6h refers to 307-6h antibody; #358-5h refers to 358-5h antibody. [Figure 22] A depicts a graph of tumor volume (mm3) in mice bearing pancreatic (HUPT4) tumors as a function of time (days) after treatment with humanized anti-CLDN18.2 antibodies (307-4h, 307-6h, 358-3h, and 358-5h). B depicts a graph of tumor volume (mm3) at day 24 in the same treatment groups. [Figure 23A-1] The sequences of the CLDN18.2-CD3 bispecific T cell engager (BiTE) (Figures 23A and 23C) and the CLDN18.2-CD16 TandAb (Figures 23B and 23C) are shown. [Figure 23A-2] Continued from Figure 23A-1. [Figure 23B-1] The sequences of the CLDN18.2-CD3 bispecific T cell engager (BiTE) (Figures 23A and 23C) and the CLDN18.2-CD16 TandAb (Figures 23B and 23C) are shown. [Figure 23B-2] Continued from Figure 23B-1. [Figure 23C-1] The sequences of the CLDN18.2-CD3 bispecific T cell engager (BiTE) (Figures 23A and 23C) and the CLDN18.2-CD16 TandAb (Figures 23B and 23C) are shown. [Figure 23C-2] Continued from Figure 23C-1. [Figure 24A] Figure 1 shows the binding activity of CLDN18.2-CD3 BiTE to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 24B] Figure 1 shows the binding activity of CLDN18.2-CD3 BiTE to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 24C] Figure 1 shows the binding activity of CLDN18.2-CD3 BiTE to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 24D] Figure 1 shows the binding activity of CLDN18.2-CD3 BiTE to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 24E] Figure 1 shows the binding activity of CLDN18.2-CD3 BiTE to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 25A]Figure 1 shows the binding activity of CLDN18.2-CD16 TandAb to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 25B] Figure 1 shows the binding activity of CLDN18.2-CD16 TandAb to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 25C] Figure 1 shows the binding activity of CLDN18.2-CD16 TandAb to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 25D] Figure 1 shows the binding activity of CLDN18.2-CD16 TandAb to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 25E] Figure 1 shows the binding activity of CLDN18.2-CD16 TandAb to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 26A] Figure 1 shows the binding activity of CLDN18.2-CD16 TandAb to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 26B] Figure 1 shows the binding activity of CLDN18.2-CD16 TandAb to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 26C]Figure 1 shows the binding activity of CLDN18.2-CD16 TandAb to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 26D] Figure 1 shows the binding activity of CLDN18.2-CD16 TandAb to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 26E] Figure 1 shows the binding activity of CLDN18.2-CD16 TandAb to cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. Binding activity was analyzed by flow cytometry. [Figure 27A] 1 depicts the binding activity of CLDN19.2-CD3 BiTE to Jurkat cells as analyzed by flow cytometry. [Figure 27B] Shown is surface staining of Jurkat cells with BiTE. [Figure 28] 1 depicts a T cell activation assay using Jurkat cells with an NFAT-RE reporter and CLDN18.2-CD3 BiTE using cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. [Figure 29] 1 depicts a T cell activation assay using Jurkat cells with an NFAT-RE reporter and CLDN18.2-CD3 BiTE using cells that endogenously express CLDN18.2 or cells engineered to overexpress CLDN18.2. [Figure 30A] Shown is a representative picture of a CLDN18.2-CD3 BiTE cytotoxicity assay after 5 days of treatment. [Figure 30B] Shown is a representative picture of a CLDN18.2-CD3 BiTE cytotoxicity assay after 5 days of treatment. [Figure 30C]Shown is a representative picture of a CLDN18.2-CD3 BiTE cytotoxicity assay after 5 days of treatment. [Figure 30D] Shown is a representative picture of a CLDN18.2-CD3 BiTE cytotoxicity assay after 5 days of treatment. [Figure 30E] Shown is a representative picture of a CLDN18.2-CD3 BiTE cytotoxicity assay after 5 days of treatment. [Figure 30F] Shown is a representative picture of a CLDN18.2-CD3 BiTE cytotoxicity assay after 5 days of treatment. [Diagram 31] Figure 1 shows lactate dehydrogenase (LDH) activity of CLDN18.2-CD3 BiTEs after 5 days of treatment. [Diagram 32] 1 depicts a graph of tumor volume (mm3) of tumors in mice bearing pancreatic (HUPT4) tumors and injected with human PBMCs as a function of time (days) after treatment with different doses of anti-CLDN18.2 BiTEs. [Figure 33A] 1 depicts a graph of tumor volume (mm3) of tumors in humanized BLT mice bearing pancreatic (HUPT4) tumors as a function of time (days) after treatment with different doses of humanized anti-CLDN18.2 BiTE. [Figure 33B] A graph of tumor volume (mm3) at day 28 in the same treatment groups is presented. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure describes antigen binding proteins for CLDN18, e.g., specific for CLDN18.2, for treating cancers that express CLDN18.2. Anti-CLDN18.2 antibodies are believed to be effective through direct inhibition of the function of CLDN18.2 at cell-cell junctions and through immune recruitment.

[0021] Claudine Family

[0022] Tight junctions, also known as occluding junctions or zonula occludens, are spine structures located between two adjacent cells that regulate paracellular permeability and maintain cell polarity in epithelial and endothelial cell sheets. Claudin (CLDN) family genes code for membrane proteins that are key components of tight junctions. CLDN proteins contain four transmembrane (TM) helices (TM1, TM2, TM3, and TM4) and two extracellular loops (EL1 and EL2). The extracellular loops of CLDN proteins from adjacent cells interact with each other to seal the cell sheets and regulate paracellular transport between the luminal and basolateral spaces.

[0023] CLDN proteins play a role in various human diseases and pathologies. For example, mutations in the CLDN1 gene have been shown to result in progressive skin scaling along with bile duct obstruction. Mutations in the CLDN16 gene cause magnesium wasting disorders. CLDN19 mutations lead to ocular pathologies such as macular coloboma and myopia, while CLDN14 mutations can lead to nonsyndromic recessive hearing loss. CLDN3 and CLDN4 are known to be surface receptors for the enterotoxin of Clostridium perfringens in the gastrointestinal tract, and CLDN1, CLDN6, and CLDN9 are co-receptors for Hepatitis C virus (HCV) entry. Several CLDN proteins have been shown to be aberrantly expressed in cancer. For example, CLDN1 is downregulated in breast and colon cancer, while CLDN3 and CLDN4 are strongly upregulated in multiple cancers.

[0024] Claudin-6 (CLDN6) is a member of the CLDN family. The gene encoding the human CLDN6 protein is located on the p arm of human chromosome 16 at 16p13.3 and is conserved in chimpanzees, rhesus monkeys, dogs, cows, mice, rats, zebrafish, and frogs. CLDN6 is normally expressed in humans as a 220 amino acid precursor protein, the first 21 amino acids of which constitute a signal peptide.

[0025] Claudin-18 (CLDN18) is a tight junction protein that is largely absent in non-germline adult tissues. CLDN18.2 is elevated in stem cells compared to non-stem cells. The human CLDN18 gene has two alternative first exons, giving rise to two protein isoforms (CLDN18.1 and CLDN18.2) that differ in the N-terminal 69 amino acids, including the first extracellular loop (Niimi et al., Mol Cell Biol 2001;21:7380-90). The two variants differ by 8 / 51 amino acids in the first extracellular domain, and the sequences of their second extracellular loops are identical. CLDN18.2 is present only in normal gastric tissues. CLDN18.2 is overexpressed in pancreatic, esophageal and lung cancers. The high tumor-normal expression differential and its putative role in tumor biology make CLDN18.2 a very attractive therapeutic target. The amino acid sequence of the CLDN18.2 precursor protein is publicly available at the National Center for Biotechnology Information (NCBI) website as NCBI reference sequence NP_001002026 and is provided herein as SEQ ID NO: 1. Extracellular loop 1 spans amino acids 28-80 of SEQ ID NO: 1 and EL2 is amino acids 144-174 of SEQ ID NO: 1. Antibodies that bind to claudin-18.2 are described in U.S. Patent No. 10,137,195.

[0026] Antigen-binding proteins

[0027] Provided herein is an antigen binding protein that binds to Claudin-18 (CLDN18). In various embodiments, the antigen binding protein binds to the isoform CLDN18.2. The antigen binding protein of the present disclosure may take any one of the many forms of antigen binding proteins known in the art. In various embodiments, the antigen binding protein of the present disclosure takes the form of an antibody, or an antigen-binding antibody fragment, or an antibody protein product.

[0028] In various embodiments of the present disclosure, the antigen-binding protein comprises, consists essentially of, or consists of an antibody. As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin format, including heavy and light chains, and including variable and constant regions. For example, an antibody can be an IgG, which is a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair having one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In the IgG format, the variable region is usually about 100-110 or more amino acids, includes three complementarity determining regions (CDRs), and is primarily responsible for antigen recognition and differs substantially among other antibodies that bind to different antigens. The constant region enables the antibody to recruit cells and molecules of the immune system. The variable region is composed of the N-terminal regions of each light and heavy chain, whereas the constant region is composed of the C-terminal portions of each of the heavy and light chains. (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4 th ed.Elsevier Science Ltd. / Garland Publishing, (1999)).

[0029] The general structure and properties of antibody CDRs have been described in the art. Briefly, in an antibody scaffold, the CDRs are embedded within the framework of heavy and light chain variable regions, which constitute the regions largely responsible for antigen binding and antigen recognition. A variable region typically comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883) within a framework region (designated framework regions 1-4, FR1, FR2, FR3 and FR4 by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra). In related embodiments, framework residues are altered. The heavy chain framework regions that may be altered are within the regions designated H-FR1, H-FR2, H-FR3 and H-FR4, which surround the heavy chain CDR residues, and the light chain framework region residues that may be altered are within the regions designated L-FR1, L-FR2, L-FR3 and L-FR4, which surround the light chain CDR residues. Amino acids within the framework regions may be replaced with any suitable amino acid, for example, as specified in the human framework or human consensus framework.

[0030] The antibody may include any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. All such classes or isotypes of antibodies are included in the embodiments of the present disclosure. The light chain constant region may be, for example, a kappa or lambda type light chain constant region, for example, a human kappa or lambda type light chain constant region. The heavy chain constant region may be, for example, an alpha, delta, epsilon, gamma, or mu type heavy chain constant region, for example, a human alpha, delta, epsilon, gamma, or mu type heavy chain constant region. Thus, in various embodiments, the antibody is of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4. In various aspects, the antibody comprises a constant region that comprises one or more amino acid modifications compared to the natural counterpart to improve half-life / stability or to make the antibody more suitable for expression / manufacturability. In various examples, the antibody comprises a constant region in which the C-terminal Lys residue present in the natural counterpart has been removed or truncated.

[0031] The antibody may be a monoclonal antibody. In some embodiments, the antibody comprises a sequence substantially similar to a naturally occurring antibody produced by a mammal, such as a mouse, rabbit, goat, horse, chicken, hamster, human, etc. In this regard, the antibody may be considered as a mammalian antibody, such as a mouse antibody, a rabbit antibody, a goat antibody, a horse antibody, a chicken antibody, a hamster antibody, a human antibody, etc. In certain aspects, the antigen binding protein is an antibody, such as a human antibody. In certain aspects, the antigen binding protein is a chimeric antibody or a humanized antibody. The term "chimeric antibody" refers to an antibody that contains domains from two or more different antibodies. A chimeric antibody may, for example, contain a constant domain from one species and a variable domain from a second species, or more commonly, may contain stretches of amino acid sequences from at least two species. A chimeric antibody may also contain domains of two or more different antibodies within the same species. The term "humanized" when used in reference to an antibody refers to an antibody that has at least a CDR region from a non-human source that has been engineered to have a structure and immunological function more similar to a pure human antibody than the original source antibody. For example, humanization can include grafting CDRs from a non-human antibody, such as a mouse antibody, onto a human antibody. Humanization can also include selecting amino acid substitutions to make the non-human sequence more similar to a human sequence. Information including sequence information of heavy and light chain constant regions of human antibodies is publicly available through the Uniprot database and other databases familiar to those skilled in the art of antibody engineering and generation. For example, the IgG2 constant region is available from the Uniprot database as Uniprot number P01859 (incorporated herein by reference).

[0032] Antibodies can be cleaved into fragments by enzymes such as, for example, papain and pepsin. Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce an F(ab') fragment. 2In various aspects of the disclosure, the antigen-binding proteins of the disclosure are antigen-binding fragments of antibodies (also known as antigen-binding antibody fragments, antigen-binding fragments, or antigen-binding portions). In various examples, antigen-binding antibody fragments include Fab fragments or F(ab') fragments. 2 It is a fragment.

[0033] The structure of antibodies has been exploited to generate an increasingly wide range of alternative antibody formats, spanning a molecular weight range of at least about 12-150 kDa, with valencies (n) ranging from monomers (n=1), to dimers (n=2), trimers (n=3), tetramers (n=4), and possibly higher polymers; such alternative antibody formats are referred to herein as "antibody protein products." Antibody protein products include those based on the complete antibody structure and those that mimic antibody fragments that retain complete antigen-binding ability, such as scFv, Fab, and VHH / VH (discussed below). The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, consisting exclusively of the variable (V) region. To stabilize the molecule, soluble and flexible amino acid peptide linkers are used to link the V region to scFv (single chain variable fragment) fragments, or constant (C) domains are added to the V region to generate Fab fragments [fragment, antigen binding]. Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells. Other antibody protein products include dimeric and multimeric antibody formats such as disulfide bond stabilized scFv (ds-scFv), single chain Fab (scFab), as well as diabodies, triabodies and tetrabodies, which contain different formats consisting of scFv linked to oligomerization domains, or minibodies (miniAbs). The smallest fragments are the VHH / VH of camelid heavy chain Abs and single domain Abs (sdAbs). The building blocks most frequently used to create new antibody formats are single chain variable (V) domain antibody fragments (scFv), which contain the V domains from heavy and light chains (VH and VL domains) linked by a peptide linker of about 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto the Fc domain. Peptibodies are well described in the art, see, e.g., Shimamoto et al., mAbs 4(5):586-591 (2012).

[0034] Other antibody protein products include single chain antibodies (SCAs), diabodies, triabodies, tetrabodies, bispecific or trispecific antibodies, etc. Bispecific antibodies can be divided into five major classes: BsIgG, adduct IgG, bispecific antibody (BsAb) fragments, bispecific fusion proteins, and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2)Part A:97-106(2015).

[0035] In various aspects, the antigen binding proteins of the disclosure comprise, consist essentially of, or consist of any one of these antibody protein products. In various aspects, the antigen binding proteins of the disclosure comprise, consist essentially of, or consist of any one of the following: scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibodies, multimeric antibodies (e.g., diabodies, triabodies, tetrabodies), miniAbs, peptibody VHH / VH of camelid heavy chain antibodies, sdAbs, diabodies, triabodies, tetrabodies, bispecific or trispecific antibodies, BsIgG, adjunct IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates.

[0036] In various examples, the antigen binding proteins of the present disclosure are antibody protein products in monomeric form, or in polymeric, oligomeric, or multimeric form. In certain embodiments where an antibody comprises two or more different antigen binding region fragments, the antibody is considered to be bispecific, trispecific, or multispecific, or bivalent, trivalent, or multivalent, depending on the number of different epitopes recognized and bound by the antibody.

[0037] In various embodiments, the anti-CLDN18.2 antibody or antibody variant thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0038] In various aspects, the antigen binding proteins of the present disclosure are linked to a therapeutic agent. As described below, the therapeutic agent can be any known in the art, including but not limited to chemotherapeutic agents, cytokines and growth factors, cytotoxic agents, etc. See "Conjugates" below.

[0039] bisingularity form

[0040] In exemplary aspects, the antigen binding protein is bispecific and thus capable of binding to two different and distinct antigens, hi exemplary embodiments, the antigen binding protein is bispecific and binds to CLDN18.2 and a second antigen.

[0041] In an exemplary embodiment, the second antigen is a cell surface protein expressed by a T cell. In an exemplary embodiment, the cell surface protein is a component of the T cell receptor (TCR), e.g., CD3. In an exemplary embodiment, the second antigen is a costimulatory molecule that assists in T cell activation, e.g., CD40 or 4-1BB (CD137). In an exemplary embodiment, the second antigen is an Fc receptor. In various embodiments, the Fc receptor is an Fc gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor. In an exemplary embodiment, the Fc receptor is CD64 (Fc-gamma RI), CD32 (Fc-gamma RIIA), CD16A (Fc-gamma RIIIA), CD16b (Fc-gamma RIIIb), FcεRI, CD23 (Fc-epsilon RII), CD89 (Fc-epsilon RI), Fcα / μR, or FcRn. In an exemplary embodiment, the Fc receptor is CD16A. In an exemplary example, the second antigen is an immune checkpoint molecule, e.g., a protein involved in an immune checkpoint pathway. Immune checkpoint pathways and molecules or proteins that function therein are known in the art. See, e.g., Pardoll, Nat Rev Genet 12(4):252-264(2012). In an exemplary example, the immune checkpoint molecule is A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA, or SIGLEC7. Optionally, the immune checkpoint molecule is PD-1, LAG3, TIM3, or CTLA4.

[0042] More than 50 formats of bispecific antigen binding proteins are known in the art, some of which are described in Kontermann and Brinkmann, Drug Discovery Today 20(7):838-847(2015); Zhang et al., Exp Hematol Oncol 6:12(2017); Spiess et al., Mol Immunol.; 67(2 Pt A):95-106(2015). In exemplary aspects, the bispecific antigen binding proteins of the present disclosure are generated via chemical engineering, genetic engineering, or quadroma technology.

[0043] In exemplary embodiments, the bispecific antigen binding proteins are constructed with some or all of the constant domains of an antibody. In exemplary embodiments, the bispecific antigen binding proteins of the present disclosure include an Fc polypeptide and retain Fc-mediated effector functions. In various examples, the bispecific antigen binding proteins are bispecific monoclonal antibodies formed, for example, by chemical cross-linking of two monoclonal antibodies (mab) or by knob-and-hold technology. In exemplary embodiments, the bispecific antigen binding proteins are generated via the "knobs-into-holes" technology, where H-chain heterodimerization is pushed by introducing different mutations into the two CH3 domains resulting in an asymmetric antibody. A "knob" mutation is generated in one HC and a "hole" mutation is generated in the other HC to promote heterodimerization. In exemplary embodiments, the bispecific antigen binding proteins are bispecific antibodies generated by quadroma technology, which is based on somatic cell fusion of two different hybridoma cells to generate monoclonal antibodies with the desired specificity. Zhang et al., supra, 2017. In exemplary embodiments, the bispecific antigen binding proteins include crossMab, ortho-Fab IgG, DVD-Ig, two-in-one IgG, IgG-scFv and scFv. 2-Fc (Kontermann and Brinkmann, supra, 2015). In various aspects, the bispecific antigen-binding protein is an Ig-scFv fusion in which new antigen-binding sites are added to full-length IgG resulting in a fusion protein with tetravalency for two different antigens, e.g., IgG C-terminal scFv fusion and IgG N-terminal scFv fusion. In an exemplary example, the bispecific antigen-binding protein is a dual-variable-domain-IgG (DVD-IgG), in which the LC and HC variable regions of an IgG specific for one antigen are fused via a linker to the N-terminal LC and HC variable regions of an IgG specific for a second antigen to form a DVD-IgG. In an exemplary aspect, the bispecific antigen-binding protein is a diabody-Fc fusion involving the replacement of the Fab fragment of IgG with a bispecific diabody.

[0044] In alternative examples, the bispecific antigen binding proteins of the present disclosure do not comprise an Fc polypeptide. In exemplary embodiments, the bispecific antigen binding proteins comprise the variable domains of each parent monoclonal antibody, and a linker is cloned and joined to form a single chain bispecific antibody. In exemplary embodiments, the bispecific antigen binding proteins are tandem scFv, diabody formats, single chain diabodies, tandem diabodies (TandAbs), dual affinity retargeting molecules (DARTs), dock and lock (DNLs), and nanobodies (Fan et al., J Hematol Oncol. 2015;8:130). In various embodiments, the bispecific antigen binding proteins are bispecific F(mab 1 ) 2, scFv, bispecific diabody (BsDb), single chain bispecific diabody (scBsDb), single chain bispecific tandem variable domain (scBsTaFv), dock-and-lock trivalent Fab (DNL-(Fab)3), single domain antibody (sdAb), or bispecific single domain antibody (BssdAb). In an exemplary embodiment, the bispecific antigen binding protein is a tandem scFv comprising two scFv fragments linked by an extra peptide linker, e.g., a glycine-serine repeat motif. Optionally, the tandem scFv has the structure: VL A -Linker1-VH A -Linker2-VH B -Linker3-VL B (VL and VH are derived from single chain antibody fragments; A and B represent parent monoclonal antibodies A and B). In an exemplary embodiment, the bispecific antigen binding protein is a TandAb that contains two pairs of VL and VH domains connected in a single polypeptide chain (Reusch et al., MAbs. 2015;7(3):584-604). The two polypeptide products dimerize in a head-to-tail manner upon expression to form a homodimer with high molecular weight (about 105 kDa). In an exemplary embodiment, the bispecific antigen binding protein is one that is generated using the crossMab technology described in PNAS 108(27):11187-92 (2011). CrossMab is generated by a method that does not have any chemical linker or connector and that forces the correct light chain association in a bispecific heterodimeric IgG antibody. In exemplary embodiments, the CrossMab is a bivalent (1+1), trivalent (2+1) or tetravalent (2+2) bispecific crossMab, or a non-Fc tandem antigen-binding fragment (Fab) based crossMab. Fab , crossMab VH-VL , or crossMab CH1-CL It is.

[0045] In exemplary aspects, the bispecific antigen binding protein comprises a single domain antibody or a nanobody comprising a single monomeric variable antibody domain. Optionally, the variable domain is based on a heavy chain variable domain. In alternative aspects, the variable domain is based on a light chain variable domain.

[0046] In exemplary aspects, the bispecific antigen binding protein is a bispecific T cell engager or BiTE®. BiTEs are bivalent small molecules that contain only the variable regions of an antibody in the form of an scFv connected by a flexible peptide linker. In exemplary aspects, the bispecific antigen binding protein comprises an scFV that contains the LC and HC variable regions of the CLDN18.2 antibody of the present disclosure and the LC and HC variable regions of a second antibody specific for a second antigen. In some embodiments, the BiTE contains the LC and HC variable regions of a second antibody specific for CD3. In some embodiments, the CD3 is CD3E.

[0047] In an exemplary embodiment, the bispecific antigen-binding protein is a dual affinity retargeting (DART), which is different from BiTE®, in that the covalent bond between the two chains of DART restricts the freedom of the antigen-binding site. Therefore, DART is structurally compact and can form stable contact between target and effector cells. DART comprises two engineered Fv fragments whose own VH is exchanged with the VH of the other. The interchanged Fv domains advantageously free the variant fragments from the conformational constraints imposed by the short connecting peptide.

[0048] In an exemplary embodiment, the bispecific antigen binding protein is an HSABody comprising two scFvs fused to a modified HSA. HSABody is described in Mol Cancer Ther. 2012;11(3):582-93.

[0049] Thus, in an exemplary embodiment, the bispecific antigen-binding protein comprises an antigen-binding fragment of any of the CLDN18.2 antibodies of the present disclosure. In an exemplary embodiment, the antigen-binding fragment is a Fab. In an exemplary embodiment, the bispecific antigen-binding protein comprises a F(ab) of any of the CLDN18.2 antibodies of the present disclosure. 2 '. In an exemplary embodiment, the bispecific antigen binding protein comprises an scFv comprising the LC and HC variable regions of any of the CLDN18.2 antibodies of the present disclosure. In an exemplary embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 514 or 515. In various embodiments, the antigen binding fragment is based on the heavy chain variable region, and in other embodiments, the antigen binding fragment is based on the light chain variable region. In an exemplary embodiment, the antigen binding fragment comprises at least a portion of both the HC variable region and the LC variable region. In an exemplary embodiment, the bispecific antigen binding protein comprises at least one, if not both, of the LC or HC variable region of a CLDN18.2 antibody of the present disclosure, and at least one, if not both, of the LC and HC variable regions of a second antibody specific for a second antigen. In an exemplary example, the bispecific antigen binding protein comprises an scFV comprising the LC and HC variable regions of a CLDN18.2 antibody of the present disclosure, and the LC and HC variable regions of a second antibody specific for a second antigen.

[0050] CLDN18 and epitopes

[0051] The antigen binding protein of the present disclosure binds to CLDN18.2. In various embodiments, CLDN18.2 has the amino acid sequence: MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV (SEQ ID NO: 1) It is a human CLDN18.2 isoform having the structure:

[0052] In various aspects, the antigen binding protein of the present disclosure binds to an epitope within the amino acid sequence of CLDN18.2. In various aspects, the CLDN18.2 is human CLDN18.2, and the antigen binding protein of the present disclosure binds to an epitope within the amino acid sequence of human CLDN18.2, e.g., SEQ ID NO: 1. "Epitope" refers to a region of or within CLDN18.2 that is bound by an antigen binding protein. In some embodiments, the epitope is a linear epitope. "Linear epitope" refers to a region of or within CLDN18.2 that is bound by an antigen binding protein, which region is composed of consecutive amino acids of the amino acid sequence of CLDN18.2. The amino acids of a linear epitope are adjacent to each other in the primary structure of CLDN18.2. Thus, a linear epitope is a fragment or part of the amino acid sequence of an antigen, i.e., CLDN18.2. In various other embodiments, epitope refers to a conformational or structural epitope. "Conformational epitope" or "structural epitope" refers to an epitope that is composed of amino acids that are located close to each other only when CLDN18.2 is in its properly folded state. Unlike linear epitopes, the amino acids of a conformational or structural epitope are not adjacent to each other in the primary structure (i.e., amino acid sequence) of CLDN18.2. A conformational or structural epitope is not composed of consecutive amino acids of the amino acid sequence of an antigen (CLDN18.2).

[0053] In various embodiments, the epitope is located within the extracellular domain (ECD) of CLDN18.2, e.g., human CLDN18.2. In various embodiments, the antigen binding protein binds to extracellular loop 1 (EL1) of the ECD of CLDN18.2 having the amino acid sequence of residues 28-80 of SEQ ID NO:1. In various embodiments, the epitope to which the antigen binding protein binds is within GLWRSCVRESSGFTECRFYFTL (SEQ ID NO:4), QGLWRSCVRESSGFTECRGYFTLK (SEQ ID NO:5), DQWSTQDLYNNPVTAVFNYQGLWRSC (SEQ ID NO:6) or CRGYFTLLFLPAMLQAVR (SEQ ID NO:7) of CLDN18.2. In various examples, the antigen binding protein of the present disclosure binds to EL1 but not to extracellular loop 2 (EL2) of CLDN18.2. In various aspects, the epitope(s) bound by an antigen binding protein of the disclosure is distinct from the epitope bound by an anti-CLDN18.2 antibody comprising a light chain variable region comprising the sequence of SEQ ID NO:58 and a heavy chain variable region comprising the sequence of SEQ ID NO:59, or comprising a light chain variable region comprising the sequence of SEQ ID NO:60 and a heavy chain variable sequence encoded by SEQ ID NO:61, or comprising a light chain variable region comprising the sequence of SEQ ID NO:62 and a heavy chain variable sequence encoded by SEQ ID NO:63.

[0054] In various embodiments, the antigen binding protein binds to human CLDN18.2 and non-human CLDN18.2. In various examples, the non-human CLDN18.2 is chimpanzee, rhesus monkey, dog, cow, mouse, rat, zebrafish, or frog CLDN18.2. In various examples, the antigen binding protein binds to human CLDN18.2 and mouse CLDN18.2.

[0055] Affinity and Avidity

[0056] The antigen binding proteins provided herein bind to CLDN18.2 in a non-covalent and reversible manner. In various embodiments, the binding strength of an antigen binding protein to CLDN18.2 can be described in terms of its affinity, which is an indication of the strength of the interaction between the binding site of the antigen binding protein and the epitope. In various aspects, the antigen binding proteins provided herein have a high affinity for CLDN18.2 and thus bind to a greater amount of CLDN18.2 in a shorter period of time than low affinity antigen binding proteins. In various aspects, the antigen binding proteins bind to CLDN18.2 in a shorter period of time than low affinity antigen binding proteins. 5 Mol -1 , at least 10 6 Mol -1 , at least 10 7 Mol -1 , at least 10 8 Mol -1 , at least 10 9 Mol -1 , or at least 10 10 Mol -1 Or at least 10 10 Mol -1 At least 10 10 Mol -1 The equilibrium association constant K A As will be appreciated by those skilled in the art, K A can be affected by factors including pH, temperature and buffer composition.

[0057] In various embodiments, the binding strength of an antigen binding protein to CLDN18.2 can be described in terms of its sensitivity. D is the k between the antigen-binding protein and CLDN18.2 off / k on The equilibrium dissociation constant is the ratio of K D and K A is inversely correlated. D The value is related to the concentration of the antigen-binding protein (the amount of antigen-binding protein needed for a particular experiment) and therefore the K DThe lower the value (the lower the concentration), the higher the affinity of the antigen binding protein. In various embodiments, the binding strength of an antigen binding protein to CLDN18.2 is expressed as K D In various embodiments, the K of the antigen binding proteins provided herein can be described in terms of D is about 10 -1 , about 10 -2 , about 10 -3 , about 10 -4 , about 10 -5 , about 10 -6 In various embodiments, the K of the antigen binding proteins provided herein is D is a micromolar, nanomolar, picomolar or femtomolar concentration. In various embodiments, the K D is about 10 -4 ~10 -6 or 10 -7 ~10 -9 or 10 -10 ~10 -12 or 10 -13 ~10 -15 or 10 -9 ~10 -12 or 10 -9 ~10 -15 In various embodiments, the K of the antigen binding proteins provided herein is within the range D is approximately 1.0 x 10 -12 M ~ approx. 1.0×10 -8 In various embodiments, the K of the antigen binding protein is within the range of D is approximately 1.0 x 10 -11 M ~ approx. 1.0×10 -9 It is within the range of M.

[0058] In various embodiments, the affinity of antigen-binding proteins is measured or ranked using flow cytometry or fluorescence-activated cell sorting (FACS)-based assays. Flow cytometry-based binding assays are known in the art. See, for example, Cedeno-Arias et al., Sci Pharm 79(3):569-581(2011); Rathanaswami et al., Analytical Biochem 373:52-60(2008); and Geuijen et al., J Immunol Methods 302(1-2):68-77(2005). In various embodiments, the affinity of antigen-binding proteins can be measured or ranked using the competitive assays described in Trikha et al., Int J Cancer 110:326-335(2004) and Tam et al., Circulation 98(11):1085-1091(1998) and below. See the section entitled "Competition Assays" below. In Trikh et al., cells expressing the antigen were used in a radioassay. 125 The binding of I-labeled antigen-binding proteins (e.g., antibodies) is measured using suspended cells. In various embodiments, the relative affinity of CLDN18.2 antibodies is determined via a FACS-based assay in which different concentrations of fluorophore-conjugated CLDN18.2 antibodies are incubated with cells expressing CLDN18.2 and the emitted fluorescence, which is a direct indicator of antibody-antigen binding, is determined. A curve is generated plotting the fluorescence for each dose or concentration. The maximum is the lowest concentration at which the fluorescence reaches a plateau or maximum, which is when binding saturation occurs. Half of the maximum is considered to be the EC50 or IC50, and the antibody with the lowest EC50 / IC50 is considered to have the highest affinity compared to other antibodies tested in the same manner. Such an assay is described in Example 5.

[0059] In various embodiments, IC 50 The K value of an antigen-binding protein as determined by competitive binding inhibition assays.D In various examples, as discussed below, the competitive assay is a FACS-based assay performed with a reference antibody, a fluorophore-conjugated secondary antibody, and cells expressing CLDN18.2. In various embodiments, the cells are genetically engineered to overexpress CLDN18.2. In some embodiments, the cells are HEK293T cells transduced with a viral vector to express CLDN18.2. In alternative embodiments, the cells endogenously express CLDN18.2. Before the FACS-based assay is performed, in some embodiments, the cells that endogenously express CLDN18.2 are pre-determined as low CLDN18.2 expressing cells or high CLDN18.2 expressing cells. In some embodiments, the cells are cancer or tumor cells. In various embodiments, the cells are cells from a cell line, such as an ovarian cell line, an endometrial cell line, a bladder cell line, a lung cell line, a gastrointestinal (GI) cell line, a liver cell line, a lung cell line, etc. In various aspects, the cells that endogenously express CLDN18.2 are selected from the group consisting of HUPT4 pancreatic cells, UMUC-4 bladder cells, MKN7, KATO III, SNU601, NUGC4, NUGC3, SNU620 SNU520, and OE19 upper GI cells. In various aspects, the antigen binding protein inhibits the binding interaction between human CLDN18.2 expressed by the cells and a reference antibody, the reference antibody being known to bind CLDN18.2 but not an antigen binding protein of the present disclosure. In various examples, the antigen binding protein of the present disclosure competes with the reference antibody for binding to human CLDN18.2, thereby decreasing the amount of human CLDN18.2 bound to the reference antibody as determined by an in vitro competitive binding assay. In various aspects, the antigen binding protein of the present disclosure inhibits the binding interaction between human CLDN18.2 and the reference antibody, the inhibition being measured by an IC 50 In various embodiments, the antigen binding protein has an IC of less than about 2500 nM for inhibiting the binding interaction between human CLDN18.2 and the reference antibody. 50In various embodiments, the antigen binding protein exhibits an IC of less than about 2000 nM, less than about 1500 nM, less than about 1000 nM, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 100 nm. 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM. 50 In various examples, the antigen binding proteins of the present disclosure compete for binding to CLDN18.2 against a reference antibody known to bind to CLDN18.2 (wherein the reference antibody is different from any of the antigen binding proteins of the present disclosure). See further description under Competition Assays.

[0060] Avidity provides an indication of the overall strength of the antibody-antigen complex. It depends on three main parameters: the affinity of the antigen-binding protein to the epitope, the valency of both the antigen-binding protein and CLDN18.2, as well as the structural arrangement of the interacting moieties. The higher the valency (number of antigen-binding sites) of the antigen-binding protein, the higher the amount of antigen (CLDN18.2) it can bind. In various embodiments, the antigen-binding protein has a strong avidity for CLDN18.2. In various embodiments, the antigen-binding protein is multivalent. In various embodiments, the antigen-binding protein is bivalent. In various examples, the antigen-binding protein is monovalent.

[0061] Cross-reactivity

[0062] In various embodiments, the antigen binding protein of the present disclosure binds to CLDN18.2 and does not bind to any other member of the CLDN family, e.g., does not cross-react with any other member of the CLDN family. In various examples, the antigen binding protein of the present disclosure is specific for CLDN18.2. In various embodiments, the antigen binding protein of the present disclosure has a selectivity for CLDN18.2 that is at least 10-fold, 5-fold, 4-fold, 3-fold, 2-fold higher than the selectivity of the antigen binding protein for CLDN18.1, or another CLDN protein. In various embodiments, the antigen binding protein of the present disclosure has a selectivity for CLDN18.2 that is at least 10-fold, 5-fold, 4-fold, 3-fold, 2-fold higher than the selectivity of the antigen binding protein for CLDN18.1, or any other CLDN protein, respectively. Selectivity is measured by the K exhibited by the antigen binding protein for CLDN18.2, or a CLDN family member. D may be based on K D can be determined by techniques known in the art, e.g., surface plasmon resonance, FACS-based affinity assays.

[0063] Competition assay

[0064] In various embodiments, the antigen binding protein inhibits the binding interaction between human CLDN18.2 and a reference antibody, the reference antibody being known to bind CLDN18.2 but not an antigen binding protein of the present disclosure. In various examples, the antigen binding protein of the present disclosure competes with the reference antibody for binding to human CLDN18.2, thereby decreasing the amount of human CLDN18.2 bound to the reference antibody as determined by an in vitro competitive binding assay. In various embodiments, the reference antibody binds to an epitope within the amino acid sequence of the extracellular domain of human CLDN18.2, optionally within EL2 or EL1. In various aspects, the reference antibody comprises a light chain variable sequence encoded by SEQ ID NO:58 and a heavy chain variable sequence encoded by SEQ ID NO:59, or comprises a light chain variable region comprising the sequence of SEQ ID NO:60 and a heavy chain variable sequence encoded by SEQ ID NO:61, or comprises a light chain variable region comprising the sequence of SEQ ID NO:62 and a heavy chain variable sequence encoded by SEQ ID NO:63. In various aspects, the antigen binding protein of the disclosure inhibits the binding interaction between human CLDN18.2 and a reference antibody, and the inhibition is 50 In various embodiments, the antigen binding protein has an IC of less than about 2500 nM for inhibiting the binding interaction between human CLDN18.2 and the reference antibody. 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 2000 nM, less than about 1500 nM, less than about 1000 nM, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 100 nm. 50 In various embodiments, the antigen binding protein exhibits an IC of less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM. 50 Shows.

[0065] In various examples, the antigen binding protein of the present disclosure competes with a reference antibody for binding to human CLDN18.2, thereby decreasing the amount of human CLDN18.2 bound to the reference antibody as determined by an in vitro competitive binding assay. In various aspects, the in vitro competitive binding assay is a FACS-based assay in which the fluorescence of a fluorophore-conjugated secondary antibody that binds to the Fc of the reference antibody is measured in the absence or presence of a particular amount of an antigen binding protein of the present disclosure. Such a FACS-based assay is described in the Examples. In various aspects, the FACS-based assay is performed with a reference antibody, a fluorophore-conjugated secondary antibody, and cells expressing CLDN18.2. In various aspects, the cells are genetically engineered to overexpress CLDN18.2. In some aspects, the cells are HEK293T cells transduced with a viral vector to express CLDN18.2. In alternative aspects, the cells express CLDN18.2 endogenously. Before the FACS-based assay is performed, in some embodiments, the cells that endogenously express CLDN18.2 are pre-determined as low CLDN18.2 expressing cells or high CLDN18.2 expressing cells. In some embodiments, the cells are cancer or tumor cells. In various embodiments, the cells are cells from a cell line, such as an ovarian cell line, an endometrial cell line, a bladder cell line, a lung cell line, a gastrointestinal (GI) cell line, a liver cell line, a lung cell line, etc. In various embodiments, the cells that endogenously express CLDN18.2 are selected from the group consisting of HUPT4 pancreatic cells, UMUC-4 bladder cells, MKN7, KATO III, SNU601, NUGC4, NUGC3, SNU620 SNU520, and OE19 upper GI cells. In various examples, the antigen binding proteins of the present disclosure bind with high affinity to CLDN18.2 endogenously expressed by one or more of the cells that endogenously express CLDN18.2. In various aspects, the antigen binding protein has an IC of less than about 3000 nM as determined in a FACS-based competitive binding inhibition assay using one or more of HUPT4, UMUC-4, MKN7, KATO III, SNU601, NUGC4, NUGC3, SNU620 SNU520 and OE19 cells as described herein. 50In various aspects, the antigen binding protein exhibits an IC of less than about 2500 nM, less than about 2000 nM, less than about 1750 nM, less than about 1500 nM, less than about 1250 nM, less than about 1000 nM, less than about 750 nM, or less than about 500 nM as determined in a FACS-based competitive binding inhibition assay using one or more of HUPT4, UMUC-4, MKN7, KATO III, SNU601, NUGC4, NUGC3, SNU620 SNU520, and OE19 cells. 50 In various aspects, the antigen binding protein exhibits an IC of less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM as determined in a FACS-based competitive binding inhibition assay using one or more of HUPT4, UMUC-4, MKN7, KATO III, SNU601, NUGC4, NUGC3, SNU620 SNU520, and OE19 cells. 50 Shows.

[0066] Other binding assays are known in the art, such as competitive binding assays or competition assays that test the ability of an antibody to compete with a second antibody for binding to an antigen or to its epitope. See, for example, Trikha et al., Int J Cancer 110:326-335 (2004); Tam et al., Circulation 98(11):1085-1091 (1998). US Patent Application Publication No. US20140178905, Chand et al., Biologicals 46:168-171 (2017); Liu et al., Anal Biochem 525:89-91 (2017); and Goolia et al., J Vet Diagn Invest 29(2):250-253 (2017). Other methods for comparing two antibodies are also known in the art, including, for example, surface plasmon resonance (SPR). SPR can be used to determine the binding constant of an antibody and a second antibody, and the two binding constants can be compared.

[0067] Antibody Generation and Related Methods

[0068] Suitable methods for producing antigen-binding proteins (e.g., antibodies, antigen-binding antibody fragments, and antibody protein products) are known in the art. For example, standard hybridoma methods for producing antibodies are described in, e.g., Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5 th Ed., Garland Publishing, New York, NY (2001). Various methods for preparing the CLDN18.2 monoclonal antibodies of the present disclosure are provided herein in the Examples.

[0069] Depending on the host species, various adjuvants can be used to increase the immune response and result in greater antibody production by the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels, such as aluminum hydroxide, and surfactants, such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants.

[0070] Other methods for antibody generation are summarized in Table 1. [Table 1]

[0071] Methods for testing the ability of an antibody to bind to an epitope of CLDN18.2, regardless of how the antibody is generated, are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266, and the section above regarding competitive assays).

[0072] Arrays / Structures

[0073] As used herein, the term "heavy chain (HC) complementarity determining region (CDR) 1 amino acid sequence" refers to a heavy chain (HC) CDR 2 amino acid sequence as set forth in Table A, or a sequence selected from the group consisting of SEQ ID NOs: 64, 88, 69, 89, 72, 75, 91, 94, 95, 97, 99, 101, 103, 106, 109; or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (b) a heavy chain (HC) CDR 2 amino acid sequence as set forth in Table A, or a sequence selected from the group consisting of SEQ ID NOs: 64, 88, 69, 89, 72, 75, 91, 94, 95, 97, 99, 101, 103, 106, 109; or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (c) a CDR2 amino acid sequence or a sequence selected from the group consisting of SEQ ID NOs: 65, 67, 70, 90, 73, 76, 92, 73, 96, 98, 100, 102, 104, 107, 110; or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (d) the light chain (LC) CDR1 amino acid sequences shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 78, 81, 82, 86, 114, 120, 123, 126; or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (e) the light chain (LC) CDR2 amino acid sequences shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 78, 81, 82, 86, 114, 120, 123, 126; or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; CDR2 amino acid sequences or sequences selected from the group consisting of SEQ ID NOs: 79, 112, 79, 84, 116, 118, 119, 129, 121, 124, 127; or variant sequences thereof differing by one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity;(f) an LC CDR3 amino acid sequence as set forth in Table A or a sequence selected from the group consisting of SEQ ID NOs: 80, 83, 113, 85, 87, 115, 117, 122, 125, 128; or a variant sequence thereof differing by one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (g) an antigen binding protein comprising a combination of any two or more of (a)-(f); [Table 2]

[0074] In various embodiments, the antigen binding protein comprises at least one or two of the LC CDR1, LC CDR2, and LC CDR3 amino acid sequences shown in Table A and the HC CDR amino acid sequences shown in Table A. In various embodiments, the antigen binding protein comprises at least one or two of the HC CDR1, HC CDR2, and HC CDR3 amino acid sequences shown in Table A and the LC CDR amino acid sequences shown in Table A.

[0075] In various embodiments, the antigen binding protein comprises at least three, four, or five of the amino acid sequences specified by SEQ ID NOs in a single row of Table A. In various embodiments, the antigen binding protein comprises each of the LC CDR amino acid sequences specified by a SEQ ID NO in a single row of Table A and at least one or two of the HC CDR amino acid sequences specified by a SEQ ID NO in a single row of Table A. In various embodiments, the antigen binding protein comprises each of the HC CDR amino acid sequences specified by a SEQ ID NO in a single row of Table A and at least one or two of the LC CDR amino acid sequences specified by a SEQ ID NO in a single row of Table A. In various embodiments, the antigen binding protein comprises all six of the CDR amino acid sequences specified by a SEQ ID NO in a single row of Table A. In various embodiments, the antigen binding protein is selected from the group consisting of: (a) SEQ ID NOs: 78, 79, 80, 64, 65, 66; (b) SEQ ID NOs: 81, 112, 80, 88, 65, 66; (c) SEQ ID NOs: 81, 79, 80, 64, 67, 68; (d) SEQ ID NOs: 82, 79, 83, 39, 70, 71; (e) SEQ ID NOs: 81, 79, 113, 89, 90, 77; (f) SEQ ID NOs: 81, 84, 85, 72, 73, 74; (g) SEQ ID NOs: 86, 79, 87, 75, 76, 77; (h) SEQ ID NOs: 114, 79, 115, 91, 92, 93; (i) SEQ ID NOs: 81, 116, 117 , 94, 73, 74; (j) SEQ ID NOs: 81, 118, 117, 95, 96, 74; (k) SEQ ID NOs: 81, 119, 117, 97, 98, 74; (l) SEQ ID NOs: 81, 118, 85, 99, 100, 74; (m) SEQ ID NOs: 81, 129, 117, 101, 102, 74; (n) SEQ ID NOs: 120, 121, 122, 103, 104, 105; (o) SEQ ID NOs: 123, 124, 125, 106, 107, 108; and (p) SEQ ID NOs: 126, 127, 128, 109, 110, 111.

[0076] Also contemplated are HCDR and / or LCDR sequences disclosed herein that include amino acid sequences that contain one or more amino acid changes (e.g., substitutions, insertions or deletions) compared to any of the HCDR or LCDR amino acid sequences identified in Table A. Preferred substitutions include substitutions for an amino acid at a corresponding position in another HCDR1, HCDR2, HCDR3 or LCDR1, LCDR2 or LCDR3 of Table A. Alternatively, the HCDR1, HCDR2, HCDR3 and / or LCDR1, LCDR2 or LCDR3 sequences may comprise the consensus amino acid sequence of HCDR1, HCDR2, HCDR3 or LCDR1, LCDR2 or LCDR3 described herein.

[0077] In various examples, the amino acid sequences of Table A are separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acid(s). In various examples, there are about 10 to about 20 amino acids between the LC CDR1 and LC CDR2 sequences and about 25 to about 40 amino acids between the LC CDR2 and LC CDR3 sequences. In various examples, there are about 14 to about 16 amino acids between the LC CDR1 and LC CDR2 sequences and about 30 to about 35 amino acids between the LC CDR2 and LC CDR3 sequences. In various examples, there are about 10 to about 20 amino acids between the HC CDR1 and HC CDR2 sequences and about 25 to about 40 amino acids between the HC CDR2 and HC CDR3 sequences. In various examples, there are about 14 to about 16 amino acids between the HC CDR1 and HC CDR2 sequences and about 30 to about 35 amino acids between the HC CDR2 and HC CDR3 sequences.

[0078] In various embodiments, the antigen binding protein comprises: (a) a heavy chain variable region amino acid sequence as set forth in Table B, or a sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 2, 34, 36, 38, and 40, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; and (b) a light chain variable region amino acid sequence as set forth in Table B, or a sequence selected from the group consisting of SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or (c) both (a) and (b). [Table 3]

[0079] In various embodiments, the antigen binding protein comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 10 and 11; (b) SEQ ID NOs: 12 and 13; (c) SEQ ID NOs: 14 and 15; (d) SEQ ID NOs: 16 and 17; (e) SEQ ID NOs: 18 and 19; (f) SEQ ID NOs: 20 and 21; (g) SEQ ID NOs: 22 and 23; (h) SEQ ID NOs: 24 and 25; (i) SEQ ID NOs: 26 and 27; (j) SEQ ID NOs: 28 and 29; (k) SEQ ID NOs: 30 and 31; (l) SEQ ID NOs: 32 and 33; (m) SEQ ID NOs: 34 and 35; (n) SEQ ID NOs: 36 and 37; (o) SEQ ID NOs: 38 and 39; (p) SEQ ID NOs: 40 and 41.

[0080] Also contemplated are VH and / or VL sequences disclosed herein that include amino acid sequences that contain one or more amino acid changes (e.g., substitutions, insertions, or deletions) compared to any VH or VL amino acid sequence identified in Table B. Preferred substitutions include substitutions for an amino acid at a corresponding position in another VH or VL of Table B. Alternatively, a VH or VL sequence may include a consensus amino acid sequence of a VH or VL described herein. For example, a CDR or a VH or VL may be replaced with an amino acid sequence identified as different from the parent sequence shown in Figure 13. The amino acid substitutions in the humanized sequence are highlighted. Consensus sequences of humanized VH or VL sequences described herein are contemplated herein.

[0081] In various aspects, the antigen binding protein does not comprise the pair of amino acid sequences encoded by the sequences of SEQ ID NOs: 58 and 59. In various aspects, the antigen binding protein does not comprise the pair of amino acid sequences of SEQ ID NOs: 60 and 61. In various aspects, the antigen binding protein does not comprise the pair of amino acid sequences of SEQ ID NOs: 62 and 63.

[0082] In various embodiments, the antigen binding protein comprises an amino acid sequence similar to the amino acid sequence set forth above, but the antigen binding protein substantially retains its biological function, e.g., its ability to bind to human CLDN18.2, reduce tumor growth, and treat cancer.

[0083] In various embodiments, the antigen binding protein comprises an amino acid sequence that differs from the above amino acid sequence(s) by only 1, 2, 3, 4, 5, 6, or more amino acids. In various embodiments, the antigen binding protein comprises a variant sequence of the above sequence, which variant sequence differs from the above sequence by only 1 or 2 amino acids. In various embodiments, the antigen binding protein comprises one or more amino acid substitutions that occur outside the CDRs, for example, one or more amino acid substitutions occur within the framework region(s) of the heavy or light chain. In various embodiments, the antigen binding protein comprises one or more amino acid substitutions, but the antigen binding protein retains the amino acid sequence of the six CDRs. In various embodiments, the antigen binding protein comprises an amino acid sequence with only 1, 2, 3, 4, 5, 6, or more conservative amino acid substitutions from the above amino acid sequence(s). As used herein, the term "conservative amino acid substitution" refers to the replacement of one amino acid with another amino acid having similar properties, such as size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, including exchanges within one of the following five groups: I. Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar negatively charged residues and their amides and esters: Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid; III. Polar positively charged residues: His, Arg, Lys; Ornithine (Orn) IV. Large aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine V. Large aromatic residues: Phe, Tyr, Trp, acetylphenylalanine

[0084] In various embodiments, conservative amino acid substitutions are exchanges within one of the following groups of amino acids: [Table 4]

[0085] In various embodiments, the antigen binding protein comprises an amino acid sequence having greater than or about 30%, greater than or about 50%, or greater than or about 70% sequence identity with the amino acid sequence described above. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or greater than 90% sequence identity with the amino acid sequence described above. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90% or greater than 90% sequence identity along the entire length of the amino acid sequence described above. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity along the entire length of the amino acid sequence described above.

[0086] In various embodiments, the antigen binding protein comprises a variant sequence of the above sequences, the variant sequence having at least or about 70% sequence identity to the above sequences. In various embodiments, the antigen binding protein comprises a variant sequence of the above sequences, the variant sequence having at least or about 80% sequence identity to the above sequences. In various embodiments, the antigen binding protein comprises a variant sequence of the above sequences, the variant sequence having at least or about 90% sequence identity to the above sequences. In various embodiments, the antigen binding protein comprises a variant sequence of the above sequences, the variant sequence having at least or about 95% sequence identity to the above sequences.

[0087] In various embodiments, the antigen binding protein comprises one, two, three, four, or five of the SEQ ID NOs in a single row of Table A, and at least one variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 64-129. In various embodiments, the antigen binding protein comprises one or more variant sequences having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 64-129. In various embodiments, the antigen binding protein comprises at least one variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 64-129, and ...5%) sequence identity to any of SEQ ID NOs: 64-129, and at least one variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 95%) sequence identity to any of SEQ ID NOs: 64-129, and at least one variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 95%) sequence identity to any of SEQ ID NOs: 64-129, and at least one variant sequence Nos. 81, 84, 85, 72, 73, 74; (g) SEQ ID NOs: 86, 79, 87, 75, 76, 77; (h) SEQ ID NOs: 114, 79, 115, 91, 92, 93; (i) SEQ ID NOs: 81, 116, 117, 94, 73, 74; (j) SEQ ID NOs: 81, 118, 117, 95, 96, 74; (k) SEQ ID NOs: 81, 119, 117, 97, 98, 74; ( (l) one, two, three, four, or five of a set of sequences selected from SEQ ID NOs: 81, 118, 85, 99, 100, 74; (m) SEQ ID NOs: 81, 129, 117, 101, 102, 74; (n) SEQ ID NOs: 120, 121, 122, 103, 104, 105; (o) SEQ ID NOs: 123, 124, 125, 106, 107, 108; and (p) SEQ ID NOs: 126, 127, 128, 109, 110, 111, wherein the antigen binding protein further comprises at least one variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to at least one of the sequences of the set of sequences.

[0088] In various embodiments, the antigen binding protein comprises a pair of variant sequences having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 10-41. In various examples, the antigen binding protein comprises pairs of variant sequences having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to: (a) SEQ ID NOs: 10 and 11; (b) SEQ ID NOs: 12 and 13; (c) SEQ ID NOs: 14 and 15; (d) SEQ ID NOs: 16 and 17; (e) SEQ ID NOs: 18 and 19; (f) SEQ ID NOs: 20 and 21; (g) SEQ ID NOs: 22 and 23; (h) SEQ ID NOs: 24 and 25; (i) SEQ ID NOs: 26 and 27; (j) SEQ ID NOs: 28 and 29; (k) SEQ ID NOs: 30 and 31; (l) SEQ ID NOs: 32 and 33; (m) SEQ ID NOs: 34 and 35; (n) SEQ ID NOs: 36 and 37; (o) SEQ ID NOs: 38 and 39; and (p) SEQ ID NOs: 40 and 41. In various embodiments, the antigen binding protein comprises a pair of sequences: one of the sequences of Table B and another sequence that is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 10-41. In various embodiments, the antigen binding protein comprises one sequence selected from the pair of sequences: (a) SEQ ID NOs: 10 and 11; (b) SEQ ID NOs: 12 and 13; (c) SEQ ID NOs: 14 and 15; (d) SEQ ID NOs: 16 and 17; (e) SEQ ID NOs: 18 and 19; (f) SEQ ID NOs: 20 and 21; (g) SEQ ID NOs: 22 and 23; (h) SEQ ID NOs: 24 and 25; (i) SEQ ID NOs: 26 and 27; (j) SEQ ID NOs: 28 and 29; (k) SEQ ID NOs: 30 and 31; (l) SEQ ID NOs: 32 and 33; (m) SEQ ID NOs: 34 and 35; (n) SEQ ID NOs: 36 and 37; (o) SEQ ID NOs: 38 and 39; (p) SEQ ID NOs: 40 and 41, and another sequence which is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to the sequence of (a)-(p).For example, in various embodiments, the antigen binding protein comprises the sequence of SEQ ID NO: 10, and the antigen binding protein further comprises a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to SEQ ID NO: 11.

[0089] In various examples, the antigen binding protein comprises an amino acid sequence of the above amino acid sequence having one or more amino acid substitutions to reduce or eliminate reactive amino acids to reduce or prevent unwanted side chain reactions. For example, the antigen binding protein comprises an amino acid sequence of the above amino acid sequence having one or more of (i) a Trp residue substituted with His, Tyr, or Phe; (ii) an Asn residue substituted with Gln, Ser, Ala, or Asp; (iii) an Asp residue immediately preceding a Pro residue substituted with Ala, Ser, or Glu; (iv) an Asn residue substituted with Gln, Ser, or Ala; and / or (v) a Cys residue substituted with Tyr, Ser, or Ala. In various embodiments, the antigen binding protein comprises an amino acid sequence of the above amino acid sequence having an amino acid substitution that is predicted to have a higher binding affinity, higher stability, or other positive attribute based on SHM events or based on statistical analysis of a large number of other similar antibody sequences.

[0090] Humanized antibodies

[0091] In various embodiments, the antigen binding protein is a humanized version of an antigen binding protein described in Table A or Table B.

[0092] In various embodiments, the antigen binding protein is a humanized version of the antibody shown in Table B having one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) amino acid substitutions in the heavy or light chain variable region at the positions shown in Figure 13, or a consensus sequence thereof.

[0093] In various embodiments, the antigen binding protein comprises: (a) a heavy chain variable region amino acid sequence as set forth in Table C, or a sequence selected from the group consisting of 42, 46, 49, 52, 55, 56, 57, and 131, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70%, or about 80%, or about 85%, or about 90%, or about 95% sequence identity; or (b) a light chain variable region amino acid sequence as set forth in Table C, or a sequence selected from the group consisting of 43-45, 47-48, 50-51, 53-54, 149, and 150, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70%, or about 80%, or about 85%, or about 90%, or about 95% sequence identity; or (c) both (a) and (b). [Table 5]

[0094] In various embodiments, the humanized antigen binding protein comprises a pair of amino acid sequences shown in Table D. [Table 6]

[0095] In various embodiments, the antigen binding protein comprises a pair of variant sequences, each having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to a SEQ ID NO: listed in Table C. In various embodiments, the antigen binding protein comprises a pair of sequences: one sequence selected from the SEQ ID NOs listed in Table C and another sequence that is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to a sequence having a SEQ ID NO: listed in Table C.

[0096] In various embodiments, the antigen binding protein comprises a pair of sequences: one sequence selected from the SEQ ID NOs listed in Table D, and another sequence that is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to the sequence having a SEQ ID NO listed in Table D. For example, in various aspects, the antigen binding protein comprises the sequence of SEQ ID NO: 42, and the antigen binding protein further comprises a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity to SEQ ID NO: 43.

[0097] Nonfucosylated antibodies

[0098] Many secreted proteins undergo post-translational glycosylation, a process in which sugar moieties (e.g., glycans, saccharides) are covalently attached to specific amino acids of proteins. In eukaryotic cells, two types of glycosylation reactions occur: (1) N-linked glycosylation, in which the glycan is attached to an asparagine at the recognition sequence Asn-X-Thr / Ser (where "X" is any amino acid except proline), and (2) O-linked glycosylation, in which the glycan is attached to a serine or threonine. Regardless of the glycosylation type (N-linked or O-linked), microheterogeneity of protein glycoforms occurs due to the wide range of glycan structures associated with each site (O or N).

[0099] All N-glycans share the same core sugar sequence: Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr(Man 3 GlcNAc 2Asn) and are classified into one of three types: (A) high mannose (HM) or oligomannose (OM) type, consisting of two N-acetylglucosamine (GalNAc) sites and multiple (e.g., 5, 6, 7, 8, or 9) mannose (Man) residues; (B) complex type, containing more than two GlcNAc sites and any number of other sugar types; or (C) hybrid type, containing a Man residue in one strand of a branch and a GlcNAc at the base of a complex branch. Figure 1A (Stanley et al., Chapter 8: N-Glycans, Essentials of Glycobiology, 2 nd ed., Cold Spring Harbor Laboratory Press; 2009) shows three types of N-glycans:

[0100] N-linked glycans typically include one or more monosaccharides of galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (GLc), N-acetylglucoasamine (ClcNAc), glucoasamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N0 acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-doxynononic acid (Kdn), fucose (Fuc), glucuronic acid (GLcA), iduronic acid (IdoA), galacturonic acid (GalA), and mannuronic acid (ManA). Commonly used symbols for such saccharides are shown in FIG. 29A.

[0101] N-linked glycosylation begins in the endoplasmic reticulum (ER), where a complex series of reactions results in the attachment of a core glycan structure essentially composed of two GlcNAc and three Man residues. The glycan complex formed in the ER is modified by the action of enzymes in the Golgi apparatus. If the saccharide is relatively inaccessible to the enzymes, it typically remains in its original HM form. If the enzymes can access the saccharide, then many of the Man residues are cleaved and the saccharide is further modified, resulting in complex-type N-glycan structures. For example, mannosidase-1 located in the cis-Golgi can cleave or hydrolyze HM glycans, while fucosyltransferase FUT-8 located in the medial-Golgi fucosylates the glycans (Hanrue Imai- Nishiya (2007), BMC Biotechnology, 7:84).

[0102] Thus, the sugar composition and structural configuration of the glycan structures vary depending, inter alia, on the glycosylation machinery in the ER and Golgi apparatus, the accessibility of the glycan structures to the enzymes of that machinery, the order of action of each enzyme, and the stage at which the protein is released from the glycosylation machinery.

[0103] In an exemplary embodiment of the disclosure, the antigen binding protein comprises an Fc polypeptide. The term "Fc polypeptide" as used herein includes native and mutant forms of a polypeptide derived from the Fc region of an antibody. In an exemplary aspect, the Fc polypeptide of the antigen binding protein of the disclosure comprises a glycan. In various examples, the glycan lacks fucose or is nonfucosylated. In an exemplary aspect, the antigen binding protein comprises a nonfucosylated glycan. As used herein, the term "nonfucosylated glycan" or "nonfucoglycan" or "nonfucosylated glycoform" or "nonfuco" refers to a glycoform that lacks core fucose, e.g., α1,6-linked fucose on the GlcNAc residue that is involved in an amide bond with the Asn of the N-glycosylation site. Nonfucosylated glycoforms include, but are not limited to, A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. Further non-fucosylated glycans include, for example, A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], FO-N[H3N3]. See, e.g., Reusch and Tejada, Glycobiology 25(12):1325-1334 (2015).

[0104] The present disclosure also provides compositions, e.g., pharmaceutical compositions, comprising antigen binding proteins comprising Fc polypeptides comprising non-fucosylated glycans. In exemplary embodiments, at least or about 25% of the antigen binding proteins present in the composition are antigen binding proteins comprising Fc polypeptides comprising non-fucosylated glycans. In exemplary embodiments, at least or about 25% of the antigen binding proteins present in the composition are non-fucosylated. Optionally, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the antigen binding proteins present in the composition are non-fucosylated. Methods for producing compositions comprising antigen binding proteins of specific glycoprofiles are known in the art. In exemplary embodiments, the antigen binding proteins are recombinantly produced in cells genetically modified to alter the activity of enzymes of the de novo or salvage pathways. These two pathways of fucose metabolism are depicted in Figure 29B. In an exemplary embodiment, the cells are genetically modified to alter the activity of any one or more of fucosyl-transferases (FUTs, e.g., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucose kinase, GDP-fucose pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX). In an exemplary embodiment, the cells are genetically modified to knock out the gene encoding FX. See, e.g., International Patent Publication No. WO2017 / 079165A1; Kanda et al., J Biotechnol 130, 2007, 300-310, Yamane-Ohunuki et al., Biotechnol Bioeng 87, 2004, 614-622, Malphettes et al., Biotechnol Bioeng 106, 2010, 774-783.

[0105] nucleic acid

[0106] The present disclosure further provides a nucleic acid comprising a nucleotide sequence encoding an antigen binding protein of the present disclosure. "Nucleic acid," as used herein, includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, or modified forms thereof, which may be single-stranded or double-stranded, may be synthesized or obtained (e.g., isolated and / or purified) from a natural source, may contain natural, non-natural, or altered nucleotides, and may contain natural, non-natural, or altered internucleotide linkages, e.g., phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between the nucleotides of unmodified oligonucleotides. A nucleic acid may comprise any nucleotide sequence that encodes any of the antigen binding proteins of the present disclosure. In various embodiments, the nucleic acid comprises: (a) a heavy chain (HC) complementarity determining region (CDR) 1 amino acid sequence set forth in Table A, or a sequence selected from the group consisting of SEQ ID NOs: 64, 88, 69, 89, 72, 75, 91, 94, 95, 97, 99, 101, 103, 106, 109; or a variant sequence thereof differing by one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (b) a HC CDR2 amino acid sequence set forth in Table A, or a sequence selected from the group consisting of SEQ ID NOs: 64, 88, 69, 89, 72, 75, 91, 94, 95, 97, 99, 101, 103, 106, 109; or a variant sequence thereof differing by one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; or variant sequences thereof differing by one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (c) HC CDR3 amino acid sequences as set forth in Table A or sequences selected from the group consisting of SEQ ID NOs: 66, 68, 71, 77, 74, 77, 93, 74, 105, 108, 111; or variant sequences thereof differing by one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity;(d) a light chain (LC) CDR1 amino acid sequence shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 78, 81, 82, 86, 114, 120, 123, 126; or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (e) a LC CDR2 amino acid sequence shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 79, 112, 79, 84, 116, 118, 119, 129, 121, 124, 127; or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (f) a LC CDR3 amino acid sequence shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 79, 112, 79, 84, 116, 118, 119, 129, 121, 124, 127; or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; CDR3 amino acid sequences or sequences selected from the group consisting of SEQ ID NOs: 80, 83, 113, 85, 87, 115, 117, 122, 125, 128 or variant sequences thereof differing by one or two amino acids or having at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity;or (g) a nucleotide sequence encoding an antigen binding protein comprising a combination of any two or more of (a)-(f). In various aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising at least one or two of the LC CDR1, LC CDR2, and LC CDR3 amino acid sequences shown in Table A and the HC CDR amino acid sequences shown in Table A. In various aspects, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising at least one or two of the HC CDR1, HC CDR2, and HC CDR3 amino acid sequences shown in Table A and the LC CDR amino acid sequences shown in Table A. In various embodiments, the nucleic acid comprises at least: (a) at least three, four, or five of the amino acid sequences specified by SEQ ID NOs in a single row of Table A; (b) at least one or two of the LC CDR amino acid sequences specified by SEQ ID NOs in a single row of Table A and the HC CDR amino acid sequences specified by SEQ ID NOs in a single row of Table A; (c) at least one or two of the LC CDR amino acid sequences specified by SEQ ID NOs in a single row of Table A and the HC CDR amino acid sequences specified by SEQ ID NOs in a single row of Table A; (d) all six of the CDR amino acid sequences specified by SEQ ID NOs in a single row of Table A; and / or (e) (a) SEQ ID NOs: 78, 79, 80, 64, 65, 66; (b) SEQ ID NOs: 81, 112, 80, 88, 65, 66; (c) SEQ ID NOs: 81, 79, 80, 64, 67, 68; (d) SEQ ID NOs: 82, 79, 83, 39, 70, 71; (e) SEQ ID NOs: 81, 79, 113, 89, 90, 77; (f) SEQ ID NOs: 81, 84, 85, 72, 73, 74;(g) SEQ ID NOs:86, 79, 87, 75, 76, 77;(h) SEQ ID NOs:114, 79, 115, 91, 92, 93;(i) SEQ ID NOs:81, 116, 117, 94, 73, 74;(j) SEQ ID NOs:81, 118, 117, 95, 96, 74;(k) SEQ ID NOs:81, 119, 117, 97, 98, 74;(l) SEQ ID NOs:81, 118, 85, 99, 100, 74;(m) SEQ ID NOs:81, 129, 117, 101, 102, 74;(n) SEQ ID NOs:120, 121, 122, 103, 104, 105;(o) SEQ ID NO: 123, 124, 125, 106, 107, 108; and (p) SEQ ID NO: 126, 127, 128, 109, 110, 111.

[0107] In various embodiments, the nucleic acid comprises: (a) a heavy chain variable region amino acid sequence set forth in Table B, or a sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; or (b) a light chain variable region amino acid sequence set forth in Table B. a nucleotide sequence encoding an antigen binding protein comprising: (a) a region amino acid sequence or a sequence selected from the group consisting of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 or 41, or a variant sequence thereof differing by one or two amino acids or having at least or about 70% (e.g., at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%) sequence identity; or (c) a nucleotide sequence encoding an antigen binding protein comprising both (a) and (b). In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 10 and 11; (b) SEQ ID NOs: 12 and 13; (c) SEQ ID NOs: 14 and 15; (d) SEQ ID NOs: 16 and 17; (e) SEQ ID NOs: 18 and 19; (f) SEQ ID NOs: 20 and 21; (g) SEQ ID NOs: 22 and 23; (h) SEQ ID NOs: 24 and 25; (i) SEQ ID NOs: 26 and 27; (j) SEQ ID NOs: 28 and 29; (k) SEQ ID NOs: 30 and 31; (l) SEQ ID NOs: 32 and 33; (m) SEQ ID NOs: 34 and 35; (n) SEQ ID NOs: 36 and 37; (o) SEQ ID NOs: 38 and 39; (p) SEQ ID NOs: 40 and 41. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein comprising a pair of amino acid sequences selected from the group consisting of the pairs listed in Table D. In various aspects, the nucleic acid comprises a nucleotide sequence comprising a sequence encoding the amino acids set forth in any one or more of SEQ ID NOs: 42-57. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions, while in other embodiments, the nucleic acid contains one or more insertions, deletions, inversions, and / or substitutions.

[0108] In some aspects, the nucleic acid of the present disclosure is recombinant.As used herein, the term "recombinant" refers to (i) a molecule that is constructed outside living cells by combining natural or synthetic nucleic acid segments with a nucleic acid molecule that can replicate in living cells, or (ii) a molecule that results from replication of the molecule described in (i) above.For purposes herein, replication can be in vitro replication or in vivo replication.

[0109] In some embodiments, the nucleic acid is constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Sambrook et al., supra; and Ausubel et al., supra. For example, the nucleic acid can be chemically synthesized using natural nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to improve the biological stability of the molecule or to improve the physical stability of the duplex formed by hybridization. Examples of modified nucleotides that can be used to generate the nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosyl eosin, inosine, N 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenines, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosyl eosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocin, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present disclosure can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0110] vector

[0111] The nucleic acids of the present disclosure in some embodiments are incorporated into a vector. In this regard, the present disclosure provides a vector comprising any of the nucleic acids of the present disclosure. In various embodiments, the vector is a recombinant expression vector. For purposes herein, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that allows expression of an mRNA, protein, polypeptide, or peptide by a host cell when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vectors of the present disclosure are not naturally occurring as a whole. However, portions of the vector may be naturally occurring. The vectors of the present disclosure may be single-stranded or double-stranded, may be partially synthetic or obtained from natural sources, and may contain any type of nucleotide, including but not limited to DNA and RNA, which may contain natural, non-natural, or altered nucleotides. The vectors may contain natural or non-natural nucleotide bonds, or both types of bonds. In some embodiments, the altered nucleotides or non-natural internucleotide bonds do not prevent transcription or replication of the vector.

[0112] The vector of the present disclosure may be any suitable vector and may be used to transduce, transform or transfect any suitable host. Suitable vectors include those designed for propagation and propagation or for expression or both, e.g., plasmids and viruses. The vector may be a plasmid-based expression vector. In various aspects, the vector is selected from the group consisting of pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, e.g., λGTIO, λGTl1, λZapII (Stratagene), λEMBL4, and λNMl149, may also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). In some embodiments, the vector is a viral vector, e.g., a retroviral vector. In various embodiments, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes simplex viral (HSV) vector, a vesicular stomatitis viral (VSV) vector, a vaccinia viral vector, or a lentiviral vector. See, e.g., Howarth et al., Cell Biol. Toxicol. 26(1):1-20 (2010). In various embodiments, the vector is a baculoviral vector that infects arthropods, e.g., insects. In various embodiments, the baculovirus vector is Autographa californica polyhedrosis virus (AcMNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV).See, e.g., Khan, Adv Pharm Bull 3(2):257-263 (2013); Miller, Bioessays 11(4):91-96 (1989); Atkinson et al., Pestic Sci 28:215-224 (1990).

[0113] The vectors of the present disclosure can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., supra, and Ausubel et al., supra. Circular or linear expression vector constructs can be prepared to contain a replication system functional in eukaryotic organisms or eukaryotic host cells. Replication systems can be derived, for example, from CoIE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0114] In some embodiments, vectors include regulatory sequences, such as transcriptional and translational initiation and termination codons, specific for the type of host (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced, as appropriate, considering whether the vector is DNA- or RNA-based.

[0115] Vectors may contain one or more marker genes to allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy. Suitable marker genes for expression vectors of the present disclosure include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0116] The vector may comprise a native or non-native promoter operably linked to a nucleotide sequence encoding a polypeptide (including a functional portion or functional variant thereof) or a nucleotide sequence complementary to or hybridizing to the nucleotide sequence encoding the polypeptide. The selection of a promoter (e.g., strong, weak, inducible, tissue-specific and development-specific) is within the ordinary skill of the art. Similarly, combining a nucleotide sequence with a promoter is also within the ordinary skill of the art. The promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat of the murine stem cell virus.

[0117] host cell

[0118] Provided herein is a host cell comprising the nucleic acid or vector of the present disclosure. As used herein, the term "host cell" refers to any type of cell that can contain the vector of the present disclosure and produce the expression product encoded by the nucleic acid (e.g., mRNA, protein). In some embodiments, the host cell is an adherent cell or a suspension cell, i.e., a cell that grows in suspension. In various embodiments, the host cell is a cultured cell or a primary cell, i.e., directly isolated from an organism, e.g., a human. The host cell can be of any cell type, can be derived from any type of tissue, and can be at any developmental stage.

[0119] In various aspects, the antigen binding protein is a glycosylated protein and the host cell is a glycosylation-competent cell. In various aspects, the glycosylation-competent cell is a eukaryotic cell, including but not limited to a yeast cell, a filamentous fungal cell, a protozoan cell, an algae cell, an insect cell, or a mammalian cell. Such host cells are described in the art. See, for example, Frenzel, et al., Front Immunol 4:217 (2013). In various aspects, the eukaryotic cell is a mammalian cell. In various aspects, the mammalian cell is a non-human mammalian cell. In some embodiments, the cells are selected from the group consisting of Chinese hamster ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical carcinoma cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonal carcinoma cells P19, mouse embryonic fibroblast cells NIH 3T3, mouse fibroblast L929, mouse neuroblastoma N2a, human breast cancer MCF-7, retinoblastoma Y79, human retinoblastoma SO-Rb50, human hepatoma Hep G2, mouse B myeloma J558L, or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0120] For purposes of amplifying or replicating the vector, the host cell is in some embodiments a prokaryotic cell, such as a bacterial cell.

[0121] Also provided by the present disclosure is a population of cells comprising at least one host cell as described herein. In some aspects, the population of cells is a heterogeneous population comprising host cells comprising the vectors described in addition to at least one other cell that does not comprise any vector. Alternatively, in some aspects, the population of cells is a substantially homogenous population, where the population comprises primarily (e.g., consists essentially of) host cells comprising the vector. In some aspects, the population is a clonal population of cells, where all cells of the population comprise the vector, as all cells of the population are clones of a single host cell comprising the vector. In various embodiments of the present disclosure, the population of cells is a clonal population comprising host cells comprising the vectors as described herein.

[0122] Manufacturing method

[0123] Also provided herein is a method of producing an antigen binding protein that binds to CLDN18.2. In various embodiments, the method comprises culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding an antigen binding protein described herein in a cell culture medium, and harvesting the antigen binding protein from the cell culture medium. The host cell can be any of the host cells described herein. In various aspects, the host cell is selected from the group consisting of CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In various aspects, the step of culturing the host cell comprises culturing the host cell in a growth medium to support the growth and proliferation of the host cell. In various aspects, the growth medium improves cell density, culture viability, and productivity in a timely manner. In various aspects, the growth medium comprises amino acids, vitamins, inorganic salts, glucose, and serum as sources of growth factors, hormones, and accessory factors. In various aspects, the growth medium is a fully chemically defined medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factors. In addition to nutrients, the growth medium also serves to maintain pH and osmolality. Several growth media are commercially available and described in the art, see, e.g., Arora, "Cell Culture Media: A Review" MATER METHODS 3:175 (2013).

[0124] In various embodiments, the method comprises culturing the host cells in a feed medium. In various embodiments, the method comprises culturing in a fed-batch mode in a feed medium. Methods for recombinant protein production are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production" MAbs 2(5):466-477 (2010).

[0125] The method of producing an antigen-binding protein may include one or more steps for purifying the protein from the cell culture or its supernatant, and preferably recovering the purified protein. In various embodiments, the method includes one or more chromatography steps, such as affinity chromatography (e.g., Protein A affinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography. In various embodiments, the method includes purifying the protein using Protein A affinity chromatography resin.

[0126] In various embodiments, the method further comprises formulating the purified protein, etc., thereby obtaining a formulation comprising the purified protein. Such steps are described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0127] In various aspects, the antigen binding protein linked to the polypeptide and the antigen binding protein are part of a fusion protein. Thus, the present disclosure further provides a method of producing a fusion protein comprising an antigen binding protein that binds to CLDN18.2. In various embodiments, the method comprises culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding a fusion protein described herein in a cell culture medium, and harvesting the fusion protein from the cell culture medium.

[0128] Conjugates

[0129] The present disclosure also provides antigen binding proteins bound, linked or conjugated to a second moiety (e.g., a heterologous moiety, a conjugate moiety). Thus, the present disclosure provides a conjugate comprising an antigen binding protein and a heterologous moiety. As used herein, the term "heterologous moiety" is synonymous with "conjugate moiety" and refers to any molecule (chemical or biochemical, natural or non-encoded) that is distinct from the antigen binding protein of the present disclosure. Various heterologous moieties include, but are not limited to, polymers, carbohydrates, lipids, nucleic acids, oligonucleotides, DNA or RNA, amino acids, peptides, polypeptides, proteins, therapeutic agents, (e.g., cytotoxic agents, cytokines), or diagnostic agents.

[0130] In some embodiments, the heterogeneous moiety is a polymer. The polymer may be branched or unbranched. The polymer may be of any molecular weight. The polymer in some embodiments has an average molecular weight between about 2 kDa and about 100 kDa (the term "about" indicates that in preparations of water-soluble polymers, some molecules may weigh more or less than the stated molecular weight). The average molecular weight of the polymer is, in some aspects, between about 5 kDa and about 50 kDa, about 12 kDa and about 40 kDa, or about 20 kDa and about 35 kDa.

[0131] In some embodiments, the polymer is modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled. In some embodiments, the polymer is water-soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. In some embodiments, the polymer is pharma- ceutically acceptable, for example, when the composition is used for therapeutic applications. Also, in some aspects, the polymer is a mixture of polymers, such as a copolymer, a block copolymer.

[0132] In some embodiments, the polymer is polyamide, polycarbonate, polyalkylene glycol, polyalkylene oxide, polyalkylene and its derivatives including polyalkylene terephthalate, polymers of acrylic and methacrylic acid esters including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate), polyvinyl alcohol, polyvinyl ethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), and polyvinyl pyrrolidinyl. The polyvinyl polymers including polydons, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkylcelluloses, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitrocellulose, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethylcellulose, cellulose triacetate, and cellulose sulfate sodium salt, polyethylenes including polypropylene, poly(ethylene glycol), poly(ethylene oxide), and poly(ethylene terephthalate), and polystyrene.

[0133] A particularly preferred water-soluble polymer for use herein is polyethylene glycol (PEG). As used herein, polyethylene glycol is meant to include any of the forms of PEG that can be used to derivatize other proteins, such as mono-(C1-C10)alkoxy- or aryloxy-polyethylene glycol. PEG is a linear or branched neutral polyether available in a wide range of molecular weights, and is soluble in water and most organic solvents.

[0134] In some embodiments, the heterologous moiety is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan).

[0135] In some embodiments, the heterogeneous moiety is a lipid, which in some embodiments is a fatty acid, an eicosanoid, a prostaglandin, a leukotriene, a thromboxane, an N-acylethanolamine), a glycerolipid (e.g., mono-, di-, or tri-substituted glycerol), a glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), a sphingolipid (e.g., sphingosine, ceramide), a sterol lipid (e.g., steroids, cholesterol), a prenol lipid, a glycolipid, or a polyketide, an oil, a wax, a cholesterol, a sterol, a fat-soluble vitamin, a monoglyceride, a diglyceride, a triglyceride, or a phospholipid.

[0136] In some embodiments, the heterogeneous moiety is a therapeutic agent. The therapeutic agent can be any of those known in the art. Examples of therapeutic agents contemplated herein include natural enzymes, proteins derived from natural sources, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic agents, immunoglobins, beta-adrenergic blockers, calcium channel blockers, coronary dilators, cardiac glycosides, antiarrhythmic agents, cardiac sympathomimetics, angiotensin-converting enzyme (ACE) inhibitors, diuretics, vasoactive agents, cholesterol and triglyceride reducing agents, bile acid suppressants, fibrates, 3-hydroxy-3-methylgluteryl (HMG)-CoA reductase inhibitors, niacin derivatives, antiadrenergic agents, alpha-adrenergic blockers, centrally acting antiadrenergic agents, vasodilators, potassium-sparing agents, thiazides and related agents, angiotensin I I receptor antagonists, peripheral vasodilators, antiandrogens, estrogens, antibiotics, retinoids, insulin and analogs, alpha-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas, thiazolidinediones, androgens, progestogens, bone metabolism regulators, anterior pituitary hormones, hypothalamic hormones, posterior pituitary hormones, gonadotropins, gonadotropin releasing hormone antagonists, ovulation stimulants, selective estrogen receptor modulators, antithyroid agents, thyroid hormones, bulking agents, laxatives, antiperistaltic agents, microbiota modifiers, intestinal adsorbents, intestinal anti-infective agents, anti-anorexic agents, anti-cachexia agents, anti-bulimic agents, appetite suppressants, anti-obesity agents, antacids, upper gastrointestinal agents, anticholinergic agents, aminosalicylic acid derivatives, biological response modifiers, corticosteroids, antispasmodics, 5-HT 4 Partial agonists, antihistamines, cannabinoids, dopamine antagonists, serotonin antagonists, cytoprotectants, histamine H2-receptor antagonists, mucosal protectants, proton pump inhibitors, H. Pylori eradication therapy, erythropoiesis stimulators, hematopoietic agents, anemia agents, heparin, antifibrinolytic agents, hemostatic agents, blood coagulation factors, adenosine diphosphate inhibitors, glycoprotein receptor inhibitors, fibrinogen-platelet binding inhibitors, thromboxane A 2inhibitors, plasminogen activators, antithrombotic agents, glucocorticoids, mineralocorticoids, corticosteroids, selective immunosuppressants, antifungals, prophylactic therapy, AIDS-related infections, drugs involved in cytomegalovirus, non-nucleoside reverse transcriptase inhibitors, nucleoside analog reverse transcriptase inhibitors, protease inhibitors, anemia, Kaposi's sarcoma, aminoglycosides, carbapenems, cephalosporins, glycopeptides, lincosamides, macrolides, oxazolidinones, penicillins, streptogramins, sulfonamides, trimethoprim and derivatives, tetracyclines, anthelmintics, amoebic agents, biguanides, cinchona alkaloids, folate antagonists, quinoline derivatives, Pneumocystis carinii therapy, hydrazides, imidazoles, triazoles, nitroimidazoles, cyclic amines, neuraminidase inhibitors, nucleosides, phosphate binders, cholinesterase inhibitors, adjunctive therapy, barbiturates and derivatives, benzodiazepines, gamma aminobutyric acid derivatives, hydantoin derivatives, iminostilbene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids, antimigraine preparations, biological response modifiers, carbamates, tricyclic derivatives, depolarizing agents, non-depolarizing agents, neuromuscular paralytic agents, CNS stimulants, dopamine agonists, monoamine oxidase inhibitors, COMT inhibitors, alkylsulfonates, ethylenimines, imidazotetrazines, nitrogen mustard analogues, nitrosoureas, platinum-containing compounds, antimetabolites, purine Analogs, pyrimidine analogs, urea derivatives, anthracyclines, actinomycins, camptothecin derivatives, epipodophyllotoxins, taxanes, vinca alkaloids and analogs, antiandrogens, antiestrogens, nonsteroidal aromatase inhibitors, protein kinase inhibitors antineoplastic agents, azaspirodecanedione derivatives, anxiolytics, stimulants, monoamine reuptake inhibitors, selective serotonin reuptake inhibitors, antidepressants, benzisoxazole derivatives, butyrophenone derivatives, dibenzodiazepine derivatives, dibenzothiazepine derivatives, diphenylbutylpiperidine derivatives, phenothiazines, thienobenzodiazepine derivatives, thioxanthene derivatives, allergen extracts, nonsteroidal agents, leukotriene receptor antagonists, xanthines,Endothelin receptor antagonists, prostaglandins, pulmonary surfactants, mucolytics, antimitotics, uricosurics, xanthine oxidase inhibitors, phosphodiesterase inhibitors, methaamine salts, nitrofuran derivatives, quinolones, smooth muscle relaxants, parasympathomimetics, halogenated hydrocarbons, esters of aminobenzoic acid, amides (e.g., lidocaine, articaine hydrochloride, bupivacaine hydrochloride), antipyretics, hypnotics and sedatives, cyclopyrrolones, pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids, These include, but are not limited to, iods, para-aminophenol derivatives, alcohol dehydrogenase inhibitors, heparin antagonists, adsorbents, emetics, opoid antagonists, cholinesterase reactivators, nicotine replacement therapy, vitamin A analogs and antagonists, vitamin B analogs and antagonists, vitamin C analogs and antagonists, vitamin D analogs and antagonists, vitamin E analogs and antagonists, vitamin K analogs and antagonists.

[0137] The antigen binding proteins of the present disclosure may be conjugated to one or more cytokines and growth factors that are effective in inhibiting tumor metastasis, where the cytokine or growth factor has been shown to have an anti-proliferative effect on at least one cell population. Such cytokines, lymphokines, growth factors, or other hematopoietic factors include, but are not limited to, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.Additional growth factors for use herein include angiogenin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein-16, bone morphogenetic protein-17, bone morphogenetic protein-18, bone morphogenetic protein-19, bone morphogenetic protein-20, bone morphogenetic protein-21, bone morphogenetic protein-22, bone morphogenetic protein-23, bone morphogenetic protein-24, bone morphogenetic protein-25, bone morphogenetic protein-26, bone morphogenetic protein-27, bone morphogenetic protein-28, bone morphogenetic protein-29, bone morphogenetic protein-30, bone morphogenetic protein-31, bone morphogenetic protein-32, bone morphogenetic protein-33, bone morphogenetic protein-34, bone morphogenetic protein-35, bone morphogenetic protein-36, bone morphogenetic protein-37, bone morphogenetic protein-38, bone morphogenetic protein-39, bone morphogenetic protein-40, bone morphogenetic protein-41, bone morphogenetic protein-42, bone morphogenetic protein-43, bone morphogenetic protein-44, bone morphogenetic protein-45, bone morphogenetic protein-46, bone morphogenetic protein-47, bone morphogenetic protein-48, bone morphogenetic protein-49, bone morphogenetic protein-50, bone morphogenetic protein-5 morphogenetic protein-15, bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor α, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2α, cytokine-induced neutrophil chemotactic factor 2β, beta endothelial growth factor, endothelin 1, epithelial-derived neutrophil attractant factor, glial cell line-derived neurotrophic factor receptor α1, glial cell line-derived neurotrophic factor receptor α2, growth-related protein, growth-related protein α, growth Included are growth-related protein beta, growth-related protein gamma, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor alpha, nerve growth factor nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor alpha, transforming growth factor beta, transforming growth factor beta 1, transforming growth factor beta 1.2, transforming growth factor beta 2, transforming growth factor beta 3, transforming growth factor beta 5, latent transforming growth factor beta 1, transforming growth factor beta binding protein I, transforming growth factor beta binding protein II, transforming growth factor beta binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, and chimeric proteins and biologically or immunologically active fragments thereof.

[0138] In some embodiments, the conjugate comprises an antigen binding protein as described herein and a cytotoxic agent. A cytotoxic agent is any molecule (chemical or biochemical) that is toxic to cells. In some aspects, when a cytotoxic agent is conjugated to an antigen binding protein of the present disclosure, the results obtained are synergistic. That is, the efficacy of the combination therapy of the antigen binding protein and the cytotoxic agent is synergistic, i.e., the efficacy is higher than would be expected from the additive individual effects of each. Thus, the dosage of the cytotoxic agent can be reduced, thus simultaneously reducing the risk of toxicity issues and other side effects. In some embodiments, the cytotoxic agent is a chemotherapeutic agent. Chemotherapeutic agents are known in the art and include, but are not limited to, platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids, and difluoronucleosides, as described in U.S. Pat. No. 6,630,124.

[0139] In some embodiments, the chemotherapeutic agent is a platinum coordination compound. The term "platinum coordination compound" refers to any tumor cell growth inhibiting platinum coordination compound that provides platinum in the ionic form.

[0140] In some embodiments, the platinum coordination complex is a cis-diamminediaquoplatinum(II)-ion; chloro(diethylenetriamine)-platinum(II) chloride; dichloro(ethylenediamine)-platinum(II); diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin); spiroplatin; iproplatin; diammine(2-diethylmalonato)-platinum(II); ethylenediaminemalonatoplatinum(II); aqua(1,2-dia (1,2-diaminocyclohexane)cis(pyruvato)platinum(II); (1,2-diaminocyclohexane)oxalatoplatinum(II); ormaplatin; and tetraplatin.

[0141] In some embodiments, cisplatin is the platinum coordination compound used in the compositions and methods of the present disclosure. Cisplatin is commercially available from Bristol Myers-Squibb Corporation under the name PLATINOL™ and is available as a powder for reconstitution with water, sterile saline or other suitable vehicle. Other platinum coordination compounds suitable for use in the present invention are known, commercially available, and / or may be prepared by conventional techniques. Cisplatin, or cis-dichlorodiammineplatinum II, has been used successfully for many years as a chemotherapeutic agent in the treatment of various human solid malignancies. More recently, other diamino-platinum complexes have also shown efficacy as chemotherapeutic agents in the treatment of various human solid malignancies. Such diamino-platinum complexes include, but are not limited to, spiroplatinum and carboplatinum. Cisplatin and other diamino-platinum complexes have been widely used as chemotherapeutic agents in humans, but they have had to be delivered at high dosage levels that can lead to toxicity issues such as kidney damage.

[0142] In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerases are enzymes that can change DNA topology in eukaryotic cells. They are essential for cell function and cell proliferation. Usually, there are two classes of topoisomerases in eukaryotic cells: type I and type II. Topoisomerase I is a monomeric enzyme with a molecular weight of approximately 100,000. The enzyme binds to DNA, introduces a transient single-strand break, unwinds (or allows to unwind) the double helix, and then reseals the break before dissociating from the DNA strand. Various topoisomerase inhibitors have recently shown clinical efficacy in treating humans with ovarian cancer, esophageal cancer or non-small cell lung cancer.

[0143] In some embodiments, the topoisomerase inhibitor is camptothecin or a camptothecin analog. Camptothecin is a water-insoluble cytotoxic alkaloid produced by the Camptotheca accuminata tree native to China and the Nothapodytes foetida tree native to India. Camptothecin exhibits tumor cell growth inhibitory activity against a number of tumor cells. Camptothecin analog class compounds are typically specific inhibitors of DNA topoisomerase I. The term "topoisomerase inhibitor" refers to any tumor cell growth inhibitor compound structurally related to camptothecin. Camptothecin analog class compounds include, but are not limited to, topotecan, irinotecan, and 9-amino-camptothecin.

[0144] In additional embodiments, the cytotoxic agent is any of the compounds described in U.S. Patent No. 5,004,758, issued April 2, 1991, and European Patent Application Publication No. 88311366.4, published June 21, 1989, as Publication No. EP 0321122; U.S. Patent No. 4,604,463, issued August 5, 1986, and European Patent Application Publication No. EP 0137145, published April 17, 1985; No. 4,473,692, issued Sep. 25, 1982, and European Patent Application Publication No. EP 0074256, published Mar. 16, 1983; U.S. Patent No. 4,545,880, issued Oct. 8, 1985, and European Patent Application Publication No. EP 0074256, published Mar. 16, 1983; European Patent Application Publication No. EP 0088642, published Sep. 14, 1983; Wani et al., J. Med. Chem., 29, 2358-2363 (1986); Nitta et al., Proc. 14th International Congr. Chemotherapy, Kyoto, 1985, Tokyo Press, Anticancer Section 1, p. 28-30 (particularly the compound designated CPT-11). CPT-11 is a camptothecin analog with a 4-(piperidino)-piperidine side chain attached via a carbamate bond at C-10 of 10-hydroxy-7-ethylcamptothecin.CPT-11, also called irinotecan, is currently undergoing human clinical trials; Wani et al, J. Med. Chem., 23, 554 (1980); Wani et.al., J. Med. Chem., 30, 1774 (1987); U.S. Patent No. 4,342,776, issued August 3, 1982; U.S. Patent Application Serial No. 581,916, filed September 13, 1990, and European Patent Application Publication No. EP 418099, published March 20, 1991; U.S. Patent No. 4,513,138, issued April 23, 1985, and European Patent Application Publication No. EP 0074770, published March 23, 1983; U.S. Patent No. 4,399,276, issued August 16, 1983, and European Patent Application Publication No. 0056692, published July 28, 1982; the entire disclosures of each of which are incorporated herein by reference. All of the above compounds of the camptothecin analog class are commercially available and / or may be prepared by conventional techniques, including those described in the above references. The topoisomerase inhibitor may be selected from the group consisting of topotecan, irinotecan, and 9-aminocamptothecin.

[0145] In some embodiments, the camptothecin analog is an active metabolite of irinotecan (CPT-11). In some such embodiments, the camptothecin analog is 7-ethyl-10-hydroxycamptothecin (SN-38). As a metabolite, SN-38 is formed by hydrolysis of irinotecan by carboxylesterase. In some embodiments, SN-38 has one of the following structures: [ka] SN-38 is described in U.S. Patent Application Serial No. 7,999,083; U.S. Patent Application Serial No. 8,080,250; U.S. Patent Application Serial No. 8,759,496; U.S. Patent Application Serial No. 8,999,344; U.S. Patent Application Serial No. 10,195,288; and U.S. Patent Application Serial No. 9,808,537.

[0146] In some embodiments, the camptothecin analog is exatecan methanesulfonate. Exatecan methanesulfonate is a water-soluble camptothecin (CPT) that exhibits more potent topoisomerase I inhibitory activity and antitumor activity than other CPT analogs. Exatecan is also effective against p-glycoprotein (P-gp)-mediated multidrug resistance cells.

[0147] In some embodiments, the camptothecin analog is deruxtecan (Dxd), a potent derivative of exatecan, which has 10-fold greater topoisomerase I inhibitory potency than SN-38. In some embodiments, Dxd has the following structure: [ka] Dxd is described in U.S. Patent Application Serial No. 6,407,115; U.S. Patent Application Serial No. 10,195,288; U.S. Patent Application Serial No. 9,808,537; and U.S. Patent Application Serial No. 6,407,115.

[0148] Numerous compounds of the camptothecin analog class, including pharma- ceutically acceptable salts, hydrates and solvates thereof, and the preparation of oral and parenteral pharmaceutical compositions comprising such compounds of the camptothecin analog class and an inert, pharma- ceutically acceptable carrier or diluent, have been extensively described in U.S. Patent No. 5,004,758, issued April 2, 1991, and European Patent Application No. 88311366.4, published June 21, 1989 as Publication No. EP0321122, the teachings of which are incorporated herein by reference.

[0149] In yet another embodiment of the invention, the chemotherapeutic agent is an antibiotic compound. Suitable antibiotics include, but are not limited to, doxorubicin, mitomycin, bleomycin, daunorubicin, and streptozocin.

[0150] In some embodiments, the chemotherapeutic agent is an antimitotic alkaloid. Antimitotic alkaloids can generally be extracted from Cantharanthus roseus and have been shown to be effective as anticancer chemotherapeutic agents. Numerous semi-synthetic derivatives have been chemically and pharmacologically studied (see O. Van Tellingen et al, Anticancer Research, 12, 1699-1716 (1992)). Antimitotic alkaloids of the present invention include, but are not limited to, vinblastine, vincristine, vindesine, taxol, and vinorelbine. The latter two antimitotic alkaloids are commercially available from Eli Lilly and Company and Pierre Fabre Laboratories, respectively (see U.S. Patent No. 5,620,985). In one embodiment, the antimitotic alkaloid is vinorelbine.

[0151] In another embodiment of the invention, the chemotherapeutic agent is a difluoronucleoside. 2'-deoxy-2',2'-difluoronucleosides are known in the art as having antiviral activity. Such compounds are disclosed and taught in U.S. Patent Nos. 4,526,988 and 4,808,614. European Patent Application Publication No. 184,365 discloses that these same difluoronucleosides have oncolytic activity. In certain aspects, the 2'-deoxy-2',2'-difluoronucleoside used in the compositions and methods of the invention is 2'-deoxy-2',2'-difluorocytidine hydrochloride, also known as gemcitabine hydrochloride. Gemcitabine is commercially available or can be synthesized in a multi-step process as disclosed and taught in U.S. Patent Nos. 4,526,988, 4,808,614 and 5,223,608, which are incorporated herein by reference.

[0152] In various embodiments, the chemotherapeutic agent is an antimitotic agent that inhibits cell division by blocking tubulin polymerization, by destabilizing microtubules, or by altering microtubule dynamics, such as a maytansinoid or a derivative thereof (e.g., DM1 or DM4), an auristatin or a derivative thereof. In various examples, the chemotherapeutic agent is an auristatin. For example, the auristatin is, in some embodiments, a dolastatin, monomethyl auristatin E (MMAE), monomethyl auristatin E (MMAE), or PF-06380101. Auristatins are described in the art. See, for example, Maderna, A.; et al., Mol Pharmaceutics 12(6):1798-1812 (2015). In various embodiments, the conjugate comprises an antibody of the present disclosure in combination with MMAE. Optionally, the conjugate comprises a linker. In some embodiments, the linker comprises a cleavable linking site. In various examples, the conjugate comprises an antibody of the present disclosure linked to an attachment group linked to a cathepsin-cleavable linker, which in turn is linked to a spacer linked to MMAE. In an embodiment, the attachment group is attached to the antibody via a Cys residue in the Fc region of the antibody. In an exemplary embodiment, the attachment group is represented by Formula I: [ka] Includes the structure of. In an exemplary embodiment, the cathepsin-cleavable linker has Formula II: [ka] Includes the structure of. In an exemplary embodiment, the spacer has Formula III: [ka] Includes the structure of.

[0153] In some embodiments, the MMAE has the structure: [ka]

[0154] The present disclosure also provides a conjugate comprising the antigen-binding protein of the present disclosure linked to a polypeptide such that the conjugate is a fusion protein.Therefore, the present disclosure provides a fusion protein comprising the antigen-binding protein of the present disclosure linked to a polypeptide.In various embodiments, the polypeptide is a diagnostic label, for example, a fluorescent protein such as green fluorescent protein, or other tag, for example, a Myc tag.In various aspects, the polypeptide is one of the cytokines, lymphokines, growth factors, or other hematopoietic factors listed above.

[0155] Linker

[0156] In some embodiments, the conjugate is directly linked to the heterologous moiety. In alternative embodiments, the conjugate comprises a linker that attaches the compound of the present disclosure to the heterologous moiety. In some aspects, the linker comprises a chain of 1 to about 60 atoms, or 1 to 30 atoms or more, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms in length. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. The chain atoms and linkers may be selected according to their expected solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, the linker provides a functional group that is subject to cleavage by an enzyme or other catalyst or hydrolytic conditions found in the target tissue or organ or cell. In some embodiments, the length of the linker is long enough to reduce the potential for steric hindrance. In some embodiments, the linker is an amino acid or a peptidyl linker. Such peptidyl linkers may be of any length. Various linkers are about 1-50 amino acids in length, 5-50, 3-5, 5-10, 5-15, or 10-30 amino acids in length.

[0157] A variety of suitable linkers are known in the art. The linker can be cleavable under physiological conditions, e.g., intracellular conditions, such that cleavage of the linker releases the drug in the intracellular environment (cleavable linker). Alternatively, the linker can be cleavable under extracellular conditions, e.g., outside tumor cells or near tumor mass, such that cleavage of the linker releases the drug that preferentially penetrates inside tumor cells. In other embodiments, the linker is not cleavable (non-cleavable linker), and the drug is released by antibody degradation.

[0158] The linker may be attached to a chemically reactive group on the antibody site, for example, a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N- or C-terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid groups of one or more glutamic or aspartic acid residues, to the sulfhydryl groups of one or more cysteinyl residues, or to the hydroxyl groups of one or more serine or threonine residues). The site to which the linker is attached may be a natural residue in the amino acid sequence of the antibody site, or it may be introduced into the antibody site, for example, by recombinant DNA techniques (e.g., by introducing a cysteine ​​or a protease cleavage site in the amino acid sequence) or by protein biochemistry (e.g., reduction, pH adjustment, or proteolysis). The site to which the linker is attached may also be a non-natural amino acid. The site to which the linker is attached may also be a glycan on the antibody.

[0159] Typically, a linker is substantially inert under the conditions under which the two groups to which it is attached are linked. The term "bifunctional crosslinker", "bifunctional linker" or "crosslinker" refers to a modifying agent having two reactive groups at each end of the linker such that one reactive group can be reacted first with a cytotoxic compound to provide a compound bearing a linker site and a second reactive group, which can then be reacted with an antibody. Alternatively, one end of the bifunctional crosslinker can be reacted first with an antibody to provide an antibody bearing a linker site and a second reactive group, which can then be reacted with a cytotoxic compound. The linking site can contain a chemical bond that allows for the release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid labile bonds, photolabile bonds, protease / peptidase labile bonds, and esterase labile bonds. See, for example, U.S. Patent Nos. 5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and 7,414,073. In some embodiments, the bond is a disulfide bond, a thioether, and / or a protease / peptidase labile bond. Other linkers that may be used in the present invention include non-cleavable linkers, such as those described in US20050169933, charged linkers, or hydrophilic linkers, such as those described in US2009 / 0274713, US2010 / 0129314, and WO2009 / 134976, each of which is expressly incorporated herein by reference.

[0160] In some embodiments, the linker is a hydrophilic linker that confers hydrophilicity to the conjugate. In some embodiments, the hydrophilic linker comprises polyethylene glycol (PEG). In some embodiments, the hydrophilic linker is CLA2. In some embodiments, the CLA2 linker has the following structure: [ka] CLA2 is described in U.S. Patent Nos. 8,080,250; 8,759,496; and 10,195,288.

[0161] In some embodiments, the hydrophilic linker is CL2E. In some embodiments, CL2E has the following structure: [ka] CL2E is described in U.S. Patent Nos. 8,080,250; 8,759,496; and 10,195,288.

[0162] In some embodiments, the linker is cleavable by a cleavage factor present in the intracellular environment (e.g., in a lysosome or endosome or caveolae). The linker can be, for example, a peptide linker that is cleaved by an intracellular or extracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptide linker comprises at least 2, at least 3, at least 4, or at least 5 amino acids in length.

[0163] In some embodiments, the peptide linker is MC-VC-PAB, which contains valine and citrulline residues. In some embodiments, the MC-VC-PAB linker has the following structure: [ka] MC-VC-PAB is described in U.S. Patent Nos. 7,659,241; 7,829,531; 6,884,869; 6,214,345; and 6,214,345.

[0164] In some embodiments, the peptide linker is maleimidocaproylglycine-glycine-phenylalanine-glycine (MC-GGFG). In some embodiments, the MC-GGFG linker has the following structure: [ka] MC-GGFG is described in U.S. Patent Nos. 9,808,537 and 10,195,288.

[0165] In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at a certain pH value. In some embodiments, the pH sensitive linker is hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, for example, U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al, 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as those in blood, but are unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker, such as a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Patent No. 5,622,929).

[0166] In other embodiments, the linker is cleavable under reducing conditions (e.g., disulfide linker). Bifunctional crosslinkers that allow the antibody to be linked to a cytotoxic compound via a disulfide bond include, but are not limited to, N-succinimidyl-4-(4-nitropyridyl-2-dithio)butanoate, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB). Sulfo-SPDB is described, for example, in U.S. Pat. No. 8,236,319 (hereby incorporated by reference). Alternatively, crosslinkers that introduce thiol groups, such as 2-iminothiolane, homocysteine ​​thiolactone, or S-acetylsuccinic anhydride, may be used. In other embodiments, the linker may contain a combination of one or more of the peptide linkers, pH-sensitive linkers, or disulfide linkers described above.

[0167] A "heterobifunctional crosslinker" is a bifunctional crosslinker that has two different reactive groups. Heterobifunctional crosslinkers that contain both an amine-reactive N-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazine group can also be used to link cytotoxic compounds to antibodies. Examples of such commercially available heterobifunctional crosslinkers include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazid terephthalate hydrochloride (SHTH) and succinimidyl hydrazinium nicotinate hydrochloride (SHNH). Conjugates bearing acid-labile bonds can also be prepared using the hydrazine-bearing benzodiazepine derivatives of the present invention. Examples of bifunctional crosslinkers that can be used include succinimidyl-p-formylbenzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).

[0168] The linkers described herein may be used in any combination with the heterologous moieties described herein. All of the linkers and heterologous moieties described herein are commercially available and / or may be prepared by conventional techniques, including those described in the references above.

[0169] Conjugation

[0170] The heterogeneous site to antigen-binding protein ratio (HAR) represents the number of linked heterogeneous sites per antigen-binding molecule. In some embodiments, the HAR is in the range of 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2. In some embodiments, the HAR is in the range of 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3. In other embodiments, the HAR is about 2, about 2.5, about 3, about 4, about 5, or about 6. In some embodiments, the HAR is in the range of about 2 to about 4. The HAR can be characterized by conventional means, such as mass spectrometry, UV / Vis spectroscopy, ELISA assay, and / or HPLC.

[0171] In some embodiments, the conjugates are heterogeneous conjugates (also referred to as "conventional") in which the antigen binding proteins are conjugated to different numbers of heterogeneous sites. In some embodiments, the heterogeneous conjugates follow a Gaussian or quasi-Gaussian distribution of conjugates, where the distribution is centered around an average heterogeneous site loading value with some antigen binding proteins conjugated above the average and some antigen binding proteins conjugated below the average.

[0172] In some embodiments, the conjugate is a homogeneous conjugate in which a substantial proportion of the antigen binding protein is conjugated to a defined number of heterologous sites. In some embodiments, the homogeneous conjugate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HARs. In some embodiments, the homogeneous conjugate comprises 2, 4, 6, or 8 HARs. In preferred embodiments, the homogeneous conjugate comprises 4 HARs. In preferred embodiments, the homogeneous conjugate comprises 2 HARs. In some embodiments, the homogeneous conjugate comprises 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more, or 100 percent of the conjugates have a defined HAR. In some embodiments, the homogeneous conjugate comprises about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent of the conjugates having a defined HAR. In some embodiments, the homogeneous conjugate comprises at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent of the conjugates having a defined HAR. In some embodiments, the homogeneous conjugate comprises a HAR distribution that is not Gaussian or quasi-Gaussian. In some embodiments, the homogeneity of the homogeneous conjugate is determined by a chromatogram, for example, HPLC or any suitable chromatography. In some embodiments, the chromatogram is a HIC chromatogram. The homogeneous conjugate can be produced by site-specific conjugation.

[0173] In some embodiments, the heterologous moiety is conjugated to the antigen binding protein (eg, an antibody) in a site-specific manner. Various site-specific conjugation methods, such as conjugation at thiomab or TDC or unpaired cysteine ​​residues (Junutula et al. (2008) Nat. Biotechnol. 26:925-932; Dimasi et al. (2017) Mol. Pharm. 14:1501-1516; Shen et al. (2012) Nat. Biotechnol. 30:184-9); thiol bridge linkers (Behrens et al. (2015) Mol. Pharm. 12:3986-98); conjugation at glutamine using transglutaminase (Dennler et al. (2013) Methods Mol. Bio. 1045:205-15; Dennler et al. (2014) Bioconjug Chem. 25:569-78); conjugation with engineered non-natural amino acid residues (Axup et al. (2012) Proc Natl Acad Sci USA 104-16101-6; Tian et al. (2014) Proc Natl Acad Sci USA 111:1766-71; VanBrunt et al. (2015) Bioconjug Chem 26:2249-60; Zimmerman et al. (2014) Bioconjug Chem 25:351-61); selenocysteine ​​conjugation (Li et al. (2017) Cell Chem Biol 24:433-442); glycan-mediated conjugation (Okeley et al. (2013) Bioconjug Chem 24:1650-5); conjugation with galactose or GalNAc analogues (Ramakrishnan and Qasba (2002) J Biol Chem 277:20833-9; van Geel et al. (2015) Bioconjug Chem 26:2233-42); or via glycan engineering (Zhou et al. (2014) Bioconjug Chem 25:510-20; Tang et al.(2017) Nat Protoc 12:1702-1721); via short peptide tags, e.g., engineering glutamine tags or sortase-A mediated transpeptidation (Strop et al. (2013) Chem Biol 20:161-7; Beerli et al. (2015) PLoS One 10:e0131177); and via aldehyde tags (Wu et al. (2009) Proc Natl Acad Sci USA 106:3000-5) are known in the art.

[0174] Unpredictability of conjugates (e.g., ADCs)

[0175] Based solely on antibody profile or drug payload profile, it is not possible to predict in advance which antibody-drug conjugate will be safe and effective enough for clinical application. For example, a particular drug payload may work perfectly well when conjugated to an antibody directed to one target, but may not work nearly as well when conjugated to an antibody directed to a different target, or even when conjugated to a different antibody directed to the same target. Why different antibody-drug conjugates show different antitumor activity in vivo is not well understood enough to allow accurate prediction in the design of new antibody-drug conjugates. It is speculated that the unpredictable interplay of many factors plays a role. These factors may include, for example, the binding affinity of the antibody-drug conjugate to the target antigen, the ability of the conjugate to penetrate solid tumors, and the half-life in circulation for adequate exposure to the tumor without causing toxicity.

[0176] The complexity and unpredictability are well demonstrated with antibody affinity alone. Antibodies or antibody-drug conjugates with high affinity are associated with better cellular uptake, which results in higher levels of cytotoxic payload released inside the cell. Higher affinity is also known to enhance antibody-dependent cellular cytotoxicity (ADCC). All these attributes favor the cell killing properties of antibody-drug conjugates. However, it is also known that high affinity of antibodies or antibody-drug conjugates can hinder efficient tumor penetration via the "antigen barrier effect", suggesting that to achieve potent antitumor activity in vivo, the affinity of antibody-drug conjugates must be just right, not too high or too low. Currently, there is no known way to predict what level of affinity will be most effective or effective for antibody-drug conjugates.

[0177] Also, in vivo antitumor activity cannot be predicted by the mechanism of the linker and payload alone. For example, O. Ab et al, Mol. Cancer Ther. 14(&):1605-1613 (2015) demonstrated that the same antibody conjugated to the same antitubulin toxin via different linkers exhibited dramatically different antitumor activity when tested in preclinical cancer models. This example is particularly surprising since the chemical structures of the two linkers are very similar. Moreover, the linker present in the superior conjugate contained a hydrophilic site. It is believed that hydrophilic metabolites are usually less membrane permeable and more slowly efflux from lysosomes (sites of conjugate degradation), resulting in delayed antitubulin activity of the released payload. This finding claims an "ideal" kinetics of payload delivery, but to date, there is no insight into what constitutes such kinetics. Adding to this complexity is the open question of whether the ideal kinetics of payload delivery, even if defined for a specific cell type, applies to all cell types. Thus, it is not possible to predict the most effective in vivo anti-tumor activity simply from the chemical composition of the linker or payload.

[0178] Compositions, pharmaceutical compositions and formulations

[0179] Provided herein are compositions comprising the antigen binding proteins, nucleic acids, vectors, host cells, or conjugates of the present disclosure. The compositions in some embodiments comprise the antigen binding proteins in isolated and / or purified form. In some embodiments, the compositions comprise a single type (e.g., structure) of the antigen binding proteins of the present disclosure, or a combination of two or more antigen binding proteins of the present disclosure, the combination comprising two or more antigen binding proteins of different types (e.g., structures).

[0180] In some aspects, the composition comprises an agent that improves the chemical-physical properties of the antigen binding protein, e.g., by stabilizing the antigen binding protein at a given temperature, e.g., room temperature, extending the shelf life, reducing degradation, e.g., oxidative protease mediated degradation, extending the half-life of the antigen binding protein, etc. In some aspects, the composition comprises any of the agents disclosed herein as a heterologous or conjugated moiety, optionally in admixture with or conjugated to an antigen binding protein of the disclosure.

[0181] In various aspects of the present disclosure, the composition further comprises a pharma- ceutically acceptable carrier, diluent, or excipient.In some embodiments, the antigen-binding protein, nucleic acid, vector, host cell, or conjugate (hereinafter referred to as "active agent") of the present disclosure is formulated into a pharmaceutical composition comprising the active agent together with a pharma-ceutically acceptable carrier, diluent, or excipient.In this regard, the present disclosure further provides a pharmaceutical composition comprising the active agent intended for administration to a subject, for example, a mammal.

[0182] In some embodiments, the active agent is present in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the active agent has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%, and a pharma- ceutically acceptable diluent, carrier or excipient. In some embodiments, the composition contains the active agent at a concentration of about 0.001 to about 30.0 mg / ml.

[0183] In various embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier.As used herein, the term "pharma-ceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents.The term also includes any of the agents approved by the regulatory agency of the United States Federal Government or listed in the United States Pharmacopoeia for use in animals, including humans.

[0184] Pharmaceutical compositions may contain, for example, an acidifier, an additive, an adsorbent, an aerosol propellant, an exhaust agent, an alkalizing agent, an anti-caking agent, an anticoagulant, an antimicrobial preservative, an antioxidant, a preservative, a base, a binder, a buffer, a chelating agent, a coating agent, a colorant, a drying agent, a detergent, a diluent, a bactericide, a disintegrant, a dispersant, a dissolution enhancer, a pigment, an emollient, an emulsifier, an emulsion stabilizer, a filler, a film former, a flavor enhancer, a flavoring, a flow improver, a gel Any pharmaceutically acceptable ingredient may be included, including: viscoelastic agent, granulating agent, moisturizing agent, lubricant, mucoadhesive agent, ointment base, ointment, oily vehicle, organic base, lozenge base, pigment, plasticizer, abrasive, preservative, sequestering agent, skin penetrating agent, solubilizer, solvent, stabilizer, suppository base, surface active agent, surfactant, suspending agent, sweetener, therapeutic agent, thickening agent, tonicity agent, toxicity agent, viscosity increasing agent, water absorption agent, water miscible cosolvent, water softener, or humectant.See, for example, Handbook of Pharmaceutical Excipients, Third Edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000) (incorporated by reference in its entirety); Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) (incorporated by reference in its entirety).

[0185] In various aspects, pharmaceutical compositions include formulation materials that are non-toxic to recipients at the dosages and concentrations used. In certain embodiments, pharmaceutical compositions include an active agent and one or more pharma- ceutically acceptable salts; polyols; surfactants; osmotic balancing agents; tonicity agents; antioxidants; antibiotics; antifungal agents; bulking agents; lyoprotectants; antifoaming agents; chelating agents; preservatives; coloring agents; analgesics; or additional pharmaceutical agents. In various aspects, pharmaceutical compositions optionally include one or more polyols and / or one or more surfactants in addition to one or more excipients, including, but not limited to, pharma- ceutically acceptable salts; osmotic balancing agents (tonicity agents); antioxidants; antibiotics; antifungal agents; bulking agents; lyoprotectants; antifoaming agents; chelating agents; preservatives; coloring agents; and analgesics.

[0186] In certain embodiments, pharmaceutical compositions may contain formulatory substances to adjust, maintain or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, aroma, sterility, stability, rate of dissolution or release, adsorption or permeability of the composition. In such embodiments, suitable formulating agents include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); bulking agents; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium etc.); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal, etc.); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.See REMINGTON'S PHARMACEUTICAL SCIENCES, 18" Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0187] The pharmaceutical composition may be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition may be, for example, between about 4 or about 5 to about 8.0, or about 4.5 to about 7.5, or about 5.0 to about 7.5. In various embodiments, the pH of the pharmaceutical composition is between 5.5 to 7.5.

[0188] The present disclosure provides methods of producing pharmaceutical compositions. In various aspects, the methods include combining an antigen binding protein, a conjugate, a fusion protein, a nucleic acid, a vector, a host cell, or a combination thereof with a pharma- ceutically acceptable carrier, diluent, or excipient.

[0189] Route of administration

[0190] In accordance with the present disclosure, the active agent or pharmaceutical composition comprising it can be administered to a subject via any suitable administration route.For example, the active agent can be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal or rectal administration.The following discussion of administration routes is provided merely to illustrate various embodiments and should not be construed as limiting the scope in any manner.

[0191] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. The term "parenteral" means not through the digestive tract, but by some other route such as subcutaneous, intramuscular, intrathecal, or intravenous. The active agents of the present disclosure may be administered with a physiologically acceptable diluent in a pharmaceutical carrier such as a sterile liquid or mixture of liquids including water, saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2-dimethyl-153-dioxolane-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, with or without the addition of pharmaceutically acceptable surfactants, e.g., soaps or detergents, suspending agents, e.g., pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0192] The oil that can be used in parenteral formulations includes petroleum, animal, vegetable, or synthetic oil.Specific examples of oil include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral.Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0193] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents, such as, for example, dimethyldialkylammonium halides, and alkylpyridinium halides; (b) anionic detergents, such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates; (c) nonionic detergents, such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers; (d) amphoteric detergents, such as, for example, alkyl-β-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0194] Parenteral formulations, in some embodiments, contain about 0.5% to about 25% by weight of the active agent of the present disclosure in solution. Preservatives and buffers may be used. To minimize or eliminate irritation at the injection site, such compositions may contain one or more non-ionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations typically ranges from about 5 to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and high molecular weight adducts of hydrophobic bases with ethylene oxide formed by condensation of propylene oxide with propylene glycol. Parenteral formulations in some embodiments are provided in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition for injection, requiring only the addition of a sterile liquid excipient, such as water, immediately prior to use. Extemporaneous injection solutions and suspensions in some embodiments are prepared from sterile powders, granules, and tablets of the type described above.

[0195] Injectable formulations are subject to the present disclosure. The requirements of effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[0196] Dosage

[0197] The active agents of the present disclosure are believed to be useful in other methods further described herein, including methods of inhibiting tumor growth, as well as methods of treating or preventing cancer. For purposes of this disclosure, the amount or dose of the active agent administered should be sufficient to effect, e.g., a therapeutic or prophylactic response in a subject or animal over a reasonable time frame. For example, a dose of the active agent of the present disclosure should be sufficient to treat a cancer as described herein for a period of about 1-4 minutes, 1-4 hours, or 1-4 weeks or more, e.g., 5-20 weeks or more, from the time of administration. In certain embodiments, the time period may be even longer. The dose is determined by the efficacy of the particular active agent and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[0198] Many assays for determining dosage are known in the art. For the purposes herein, an assay that includes comparing the degree to which cancer is treated by administration of a given dose of the active agent of the present disclosure to a mammal with a set of mammals, each set being given a different dose of the active agent, can be used to determine the starting dose to be administered to the mammal. The degree to which cancer is treated by administration of a given dose can be represented, for example, by the degree of tumor regression achieved by the active agent in a mouse xenograft model. Methods for assaying tumor regression are known in the art and are described herein in the examples.

[0199] The dosage of the active agent of the present disclosure will also be determined by the presence, nature and extent of any adverse side effects that may accompany the administration of a particular active agent of the present disclosure. Typically, the attending physician will determine the dosage of the active agent of the present disclosure to treat each individual patient, taking into account a variety of factors such as age, body weight, general health, diet, sex, the active agent of the present disclosure being administered, the route of administration, and the severity of the condition being treated. By way of example and not intended to limit the present disclosure, the dosage of the active agent of the present disclosure may be about 0.0001 to about 1 g / kg body weight / day of the subject being treated, about 0.0001 to about 0.001 g / kg body weight / day, or about 0.01 mg to about 1 g / kg body weight / day.

[0200] Controlled-release formulations

[0201] In some embodiments, the active agents described herein can be modified into a depot form (see, e.g., U.S. Patent No. 4,450,150) so that the manner in which the active agent of the present disclosure is released into the body to which it is administered can be controlled according to time and location within the body. The depot form of the active agent of the present disclosure can be, for example, an implantable composition comprising an active agent and a porous or non-porous material, e.g., a polymer, in which the active agent is encapsulated or diffused throughout the material and / or diffuses by degradation of the non-porous material. The depot is then implanted into the subject's body at a desired location, and the active agent is released from the implant at a predetermined rate.

[0202] In certain embodiments, the pharmaceutical composition comprising the active agent is modified to have any type of in vivo release profile.In some embodiments, the pharmaceutical composition is an immediate release, controlled release, sustained release, extended release, delayed release, or biphasic release formulation.Methods of formulating peptides for controlled release are known in the art.See, for example, Qian et al., J Pharm 374:46-52(2009) and International Patent Application Publication Nos. WO2008 / 130158, WO2004 / 033036; WO2000 / 032218; and WO1999 / 040942.

[0203] The compositions may further comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in an extended release form to provide a prolonged depot and / or delivery effect.

[0204] use

[0205] The antigen binding proteins of the present disclosure are useful for inhibiting tumor growth. Without being bound to a particular theory, the inhibitory effects of the antigen binding proteins provided herein make such entities useful in methods of treating cancer.

[0206] Thus, provided herein are methods of inhibiting tumor growth in a subject and methods of reducing tumor size in a subject. In various embodiments, the methods include administering to a subject a pharmaceutical composition of the present disclosure in an amount effective to inhibit tumor growth or reduce tumor size in the subject. In various aspects, the growth of an ovarian tumor, a melanoma tumor, a bladder tumor, or an endometrial tumor is inhibited. In various aspects, the size of an ovarian tumor, a melanoma tumor, a bladder tumor, or an endometrial tumor is reduced.

[0207] As used herein, the terms "inhibit" or "reduce" and words derived therefrom may not be 100% or complete inhibition or reduction. Rather, there are various degrees of inhibition or reduction that one of skill in the art would recognize as having a potential benefit or therapeutic effect. In this regard, the antigen binding proteins of the present disclosure may inhibit tumor growth or reduce tumor size to any amount or level. In various embodiments, the inhibition provided by the methods of the present disclosure is at least or about 10% inhibition (e.g., at least or about 20% inhibition, at least or about 30% inhibition, at least or about 40% inhibition, at least or about 50% inhibition, at least or about 60% inhibition, at least or about 70% inhibition, at least or about 80% inhibition, at least or about 90% inhibition, at least or about 95% inhibition, at least or about 98% inhibition). In various embodiments, the reduction provided by the methods of the present disclosure is at least or about a 10% reduction (e.g., at least or about a 20% reduction, at least or about a 30% reduction, at least or about a 40% reduction, at least or about a 50% reduction, at least or about a 60% reduction, at least or about a 70% reduction, at least or about an 80% reduction, at least or about a 90% reduction, at least or about a 95% reduction, at least or about a 98% reduction).

[0208] Also provided herein is a method of treating a subject having cancer, for example, a cancer that expresses CLDN18.2. In various embodiments, the method comprises administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to treat the cancer in the subject.

[0209] For purposes herein, the cancer of the methods disclosed herein can be any cancer, for example, any malignant growth or tumor caused by abnormal and unregulated cell division that can spread to other parts of the body via the lymphatic system or bloodstream. In some embodiments, the cancer is acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck, gallbladder, or pleura cancer, nose, nasal cavity, or middle ear cancer, oral cavity cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor. , Hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, and bladder cancer. In certain aspects, the cancer is selected from the group consisting of head and neck, ovarian, cervical, bladder, and esophageal cancer, pancreatic, gastrointestinal cancer, gastric, breast, endometrial, and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder, lung cancer, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma. In various embodiments, the cancer is pancreatic cancer, gastrointestinal cancer, bladder cancer, colon cancer, lung cancer, liver cancer, endometrial cancer. In various embodiments, the cancer is any cancer characterized by moderate to high expression of CLDN18.2. See, for example, Figures 1-2. In various embodiments, the cancer is acute myeloid leukemia, large B-cell lymphoma, gastric cancer, prostate cancer, melanoma, colon cancer, rectal cancer, bladder cancer, cervical cancer, liver cancer, breast cancer, renal clear cell carcinoma, head and neck cancer, sarcoma, renal chromophobe carcinoma, low-grade glioma, adrenocortical carcinoma, glioblastoma, renal papillary cell carcinoma, lung squamous cell carcinoma, thyroid cancer, lung adenocarcinoma, pancreatic cancer, endometrioid carcinoma, uterine carcinosarcoma, or ovarian cancer. In various aspects, the cancer is selected from pancreatic cancer, gastrointestinal cancer, bladder cancer, colon cancer, lung cancer, liver cancer, ovarian cancer, endometrioid cancer, uterine cancer, lung cancer, gastric cancer, breast cancer, head and neck squamous cell carcinoma (HNSCC), and cervical cancer.

[0210] As used herein, the term "treat" and related words do not necessarily mean 100% or complete treatment. Rather, there are various degrees of treatment that one skilled in the art will recognize as having potential benefit or therapeutic effect. In this regard, the disclosed methods of treating cancer may provide any amount or level of treatment. Furthermore, the treatment provided by the disclosed methods may include treatment of one or more conditions or symptoms or signs of the cancer being treated. The treatment provided by the disclosed methods may also include delaying the progression of the cancer. For example, the methods may treat the cancer by improving T cell activity or immune response to the cancer, decreasing tumor or cancer growth, decreasing metastasis of tumor cells, increasing cell death of tumor or cancer cells, etc. In various embodiments, the methods treat with the intent of delaying the onset or recurrence of the cancer for at least 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, 2 months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, 4 years, or more. In various embodiments, the subject is treated with the intent of prolonging survival.

[0211] The antigen binding proteins of the present disclosure may also be used to detect CLDN18.2 in a sample or to diagnose CLDN18.2-positive cancer. Thus, the present disclosure provides a method of detecting CLDN18.2 in a sample. In various embodiments, the method comprises contacting the sample with an antigen binding protein, conjugate, or fusion protein described herein, and assaying for immune complexes comprising the antigen binding protein, conjugate, or fusion protein bound to CLDN18.2. The present disclosure also provides a method of diagnosing CLDN18.2-positive cancer in a subject. In various embodiments, the method comprises contacting a biological sample comprising cells or tissue obtained from the subject with an antigen binding protein, conjugate, or fusion protein described herein, and assaying for immune complexes comprising the antigen binding protein, conjugate, or fusion protein bound to CLDN18.2.

[0212] subject

[0213] In some embodiments of the present disclosure, the subject is a mammal, including but not limited to mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals of the order Carnivora, including felines (cats) and dogs (dogs), mammals of the order Artiodactyla, including bovines (cows) and porcines (pigs), or mammals of the order Perssodactyla, including equines (horses). In some aspects, the mammal is of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some aspects, the mammal is a human.

[0214] kit

[0215] In some embodiments, the antigen binding proteins of the present disclosure are provided in a kit. In various aspects, the kit includes the antigen binding protein(s) as a unit dose. For purposes herein, a "unit dose" refers to a discrete amount dispersed in a suitable carrier. In various aspects, the unit dose is an amount sufficient to provide a desired effect to a subject, e.g., inhibition of tumor growth, reduction of tumor size, treatment of cancer. Thus, provided herein are kits including the antigen binding proteins of the present disclosure, optionally provided in unit doses. In various aspects, the kit includes several unit doses, e.g., a weekly or monthly supply of unit doses, optionally each of which is individually packaged or otherwise separated from the other unit doses. In some embodiments, the components of the kit / unit dose are packaged with instructions for administration to a patient. In some embodiments, the kit includes one or more devices for administration to a patient, e.g., a needle and a syringe, etc. In some embodiments, the antigen binding protein of the present disclosure, its pharma- ceutically acceptable salt, conjugate comprising the antigen binding protein, or multimer or dimer comprising the antigen binding protein is pre-packaged in a ready-to-use form, e.g., a syringe, an intravenous bag, etc. In some embodiments, the kit further comprises other therapeutic or diagnostic agents or pharma- ceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of those described herein. In certain embodiments, the kit comprises the antigen binding protein of the present disclosure together with an agent, e.g., a therapeutic agent, used in chemotherapy or radiation therapy.

[0216] Various embodiments

[0217] In various embodiments of the present disclosure, the antigen binding protein binds to human CLDN18.2 protein (SEQ ID NO:1), and (a) the antigen binding protein binds to extracellular loop 1 (EL1) of the extracellular domain (ECD) of CLDN18.2, but not to extracellular loop 2 (EL2) of the ECD of CLDN18.2; or (b) does not bind to claudin 18.1 (CLDN18.1) or any other claudin protein, and binds to CLDN18.2 endogenously expressed by HUPT4 cells with an affinity 1.5 times higher than that of the reference antibody; or (c) a combination thereof. In various embodiments, the antigen binding protein herein binds to cells (e.g., HUPT4 or other cells) that endogenously express CLDN18.2 with an affinity at least 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 times higher or more than that of the reference antibody.

[0218] In various examples, the antigen binding protein binds to an epitope within the amino acid sequence GLWRSCVRESSGFTECRFYFTL (SEQ ID NO: 4), QGLWRSCVRESSGFTECRGYFTLK (SEQ ID NO: 5), DQWSTQDLYNNPVTAVFNYQGLWRSC (SEQ ID NO: 6) or CRGYFTLLFLPAMLQAVR (SEQ ID NO: 7) of CLDN18.2.

[0219] In various embodiments, the antigen binding protein comprises: (a) a heavy chain (CDR) 1 amino acid sequence as set forth in Table A or a sequence selected from the group consisting of SEQ ID NOs: 64, 88, 69, 89, 72, 75, 91, 94, 95, 97, 99, 101, 103, 106, 109; or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (b) a heavy chain (CDR) 1 amino acid sequence as set forth in Table A; (c) a CDR2 amino acid sequence or a sequence selected from the group consisting of SEQ ID NOs: 65, 67, 70, 90, 73, 76, 92, 73, 96, 98, 100, 102, 104, 107, 110; or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (d) the light chain (LC) CDR1 amino acid sequences shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 78, 81, 82, 86, 114, 120, 123, 126; or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; (e) the light chain (LC) CDR2 amino acid sequences shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 78, 81, 82, 86, 114, 120, 123, 126; or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; CDR2 amino acid sequences or sequences selected from the group consisting of SEQ ID NOs: 79, 112, 79, 84, 116, 118, 119, 129, 121, 124, 127; or variant sequences thereof differing by one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity;(f) a LC CDR3 amino acid sequence as set forth in Table A or a sequence selected from the group consisting of SEQ ID NOs: 80, 83, 113, 85, 87, 115, 117, 122, 125, 128, or a variant sequence thereof differing by one or two amino acids or having at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity; (g) a combination of any two or more of (a)-(f);

[0220] In various embodiments, the antigen binding protein comprises a light chain CDR1 amino acid sequence, a light chain CDR2 amino acid sequence, and a light chain CDR3 amino acid sequence shown in Table A and one or two of the heavy chain CDR amino acid sequences shown in Table A. In some examples, the antigen binding protein comprises a heavy chain CDR1 amino acid sequence, a heavy chain CDR2 amino acid sequence, and a heavy chain CDR3 amino acid sequence shown in Table A and one or two of the light chain CDR amino acid sequences shown in Table A. In various aspects, the antigen binding protein is selected from the group consisting of: (a) SEQ ID NO: 78, 79, 80, 64, 65, 66; (b) SEQ ID NO: 81, 112, 80, 88, 65, 66; (c) SEQ ID NO: 81, 79, 80, 64, 67, 68; (d) SEQ ID NO: 82, 79, 83, 39, 70, 71; (e) SEQ ID NO: 81, 79, 113, 89, 90, 77; (f) SEQ ID NO: 81, 84, 85, 72, 73, 74; (g) SEQ ID NO: 86, 79, 87, 75, 76, 77; (h) SEQ ID NO: 114, 79, 115, 91, 92, 93; (i) SEQ ID NO: 81, 116, 117, (k) SEQ ID NOs: 81, 119, 117, 97, 98, 74; (l) SEQ ID NOs: 81, 118, 85, 99, 100, 74; (m) SEQ ID NOs: 81, 129, 117, 101, 102, 74; (n) SEQ ID NOs: 120, 121, 122, 103, 104, 105; (o) SEQ ID NOs: 123, 124, 125, 106, 107, 108; and (p) SEQ ID NOs: 126, 127, 128, 109, 110, 111.

[0221] In various aspects, the antigen binding protein comprises (a) a heavy chain variable region amino acid sequence as set forth in Table B, or a sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 2, 34, 36, 38 and 40, or a variant sequence thereof which differs by one or two amino acids or has at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity; or (b) a light chain variable region amino acid sequence as set forth in Table B, or a sequence selected from the group consisting of SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and 41, or a variant sequence thereof which differs by one or two amino acids or has at least or about 70% (e.g., at least or about 85%, at least or about 90%) sequence identity; or both (a) and (b). In various aspects, the antigen binding protein comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 10 and 11; (b) SEQ ID NOs: 12 and 13; (c) SEQ ID NOs: 14 and 15; (d) SEQ ID NOs: 16 and 17; (e) SEQ ID NOs: 18 and 19; (f) SEQ ID NOs: 20 and 21; (g) SEQ ID NOs: 22 and 23; (h) SEQ ID NOs: 24 and 25; (i) SEQ ID NOs: 26 and 27; (j) SEQ ID NOs: 28 and 29; (k) SEQ ID NOs: 30 and 31; (l) SEQ ID NOs: 32 and 33; (m) SEQ ID NOs: 34 and 35; (n) SEQ ID NOs: 36 and 37; (o) SEQ ID NOs: 38 and 39; (p) SEQ ID NOs: 40 and 41.

[0222] In various embodiments, the antigen binding protein comprises: (a) a heavy chain variable region amino acid sequence as set forth in Table C, Table D, Table 6, Table 8, Table 9, Table 10, or a sequence selected from the group consisting of SEQ ID NOs: 42, 46, 49, 52, 55, 56, 57, and 131, or a variant sequence thereof differing by one or two amino acids or having at least or about 70%, or about 80%, or about 90%, or about 95% sequence identity; or (b) a heavy chain variable region amino acid sequence as set forth in Table C, Table D, Table 6, Table 8, Table 9, Table 10, or a sequence selected from the group consisting of SEQ ID NOs: 42, 46, 49, 52, 55, 56, 57, and 131, or a variant sequence thereof differing by one or two amino acids or having at least or about 70%, or about 80%, or about 90%, or about 95% sequence identity; or a light chain variable region amino acid sequence shown in Table D, Table 6, Table 8, Table 9, Table 10, or a sequence selected from the group consisting of SEQ ID NOs: 43-45, 47-48, 50-51, 53-54, 130, 132, 147, 149, and 150, or a variant sequence thereof differing by one or two amino acids or having at least or about 70%, or about 80%, or about 90%, or about 95% sequence identity; or (c) both (a) and (b). In various aspects, the antigen binding protein comprises a pair of amino acid sequences listed in Table D.

[0223] In various aspects, the antigen binding proteins of the present disclosure comprise a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 42 and 43; (b) SEQ ID NOs: 42 and 44; (c) SEQ ID NOs: 42 and 45; (d) SEQ ID NOs: 46 and 47; (e) SEQ ID NOs: 46 and 48; (f) SEQ ID NOs: 131 and 149; (g) SEQ ID NOs: 131 and 150; (h) SEQ ID NOs: 52 and 53; (i) SEQ ID NOs: 52 and 54; (j) SEQ ID NOs: 55 and 53; (k) SEQ ID NOs: 55 and 54; (l) SEQ ID NOs: 56 and 53; (m) SEQ ID NOs: 56 and 54; (n) SEQ ID NOs: 57 and 50; (o) SEQ ID NOs: 57 and 51; (p) SEQ ID NOs: 49 and 50; (q) SEQ ID NOs: 49 and 51; (r) SEQ ID NOs: 42 and 130; (s) SEQ ID NOs: 131 and 147; and (t) SEQ ID NOs: 131 and 132.

[0224] In some embodiments, the antigen binding protein of the disclosure comprises an Fc polypeptide. In some embodiments, the antigen binding protein of the disclosure comprises an Fc polypeptide comprising a non-fucosylated glycan.

[0225] In various embodiments, the antigen binding protein of the present disclosure is an antibody, e.g., a monoclonal antibody. In various examples, the antigen binding protein is an IgG. In various embodiments, the antigen binding protein inhibits at least about 50% colony growth in a soft agar 3D growth assay or inhibits tumor growth in xenograft mice injected with human cancer cells. In various embodiments, the antigen binding protein inhibits tumor growth in xenograft mice injected with ovarian cancer cells, melanoma cancer cells, bladder cancer cells, or endometrial cancer cells. In various examples, the antigen binding protein inhibits at least 50% tumor growth in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells.

[0226] The present disclosure provides a bispecific antigen binding protein that binds to CLDN18.2 and a second antigen, wherein the antigen binding protein that binds to CLDN18.2 is any one of the antigen binding proteins described herein. In some embodiments, the bispecific antigen binding protein comprises (a) a heavy chain variable region amino acid sequence shown in Table B, Table C, Table D, Table 6, Table 8, Table 9, Table 10, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% sequence identity; (b) a light chain variable region amino acid sequence shown in Table B, Table D, Table 6, Table 8, Table 9, Table 10, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% sequence identity; or (c) both (a) and (b). In some embodiments, the variant sequence has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the bispecific antigen binding protein comprises an Fc polypeptide. In some embodiments, the bispecific antigen binding protein comprises an Fc polypeptide that comprises a non-fucosylated glycan.

[0227] In various aspects, the bispecific antigen binding protein binds to CLDN18.2 and a second antigen. In some embodiments, the bispecific antigen binding protein comprises an antigen-binding fragment of an antibody specific for the second antigen. In various embodiments, the second antigen is a cell surface protein expressed by T cells, optionally a component of the T cell receptor (TCR), e.g., CD3. In some embodiments, the second antigen is CD3. In some embodiments, the second antigen is CD3E.

[0228] In various embodiments, the second antigen is a costimulatory molecule that assists in T cell activation, such as CD40 or 4-1BB (CD137). In various embodiments, the second antigen is an Fc receptor, optionally an Fc gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor. In some embodiments, the Fc receptor is CD64 (Fc-gamma RI), CD32 (Fc-gamma RIIA), CD16A (Fc-gamma RIIIA), CD16b (Fc-gamma RIIIb), FcεRI, CD23 (Fc-epsilon RII), CD89 (Fc-epsilon RI), Fcα / μR, or FcRn. In some embodiments, the Fc receptor is CD16A.

[0229] In various embodiments, the second antigen is an immune checkpoint molecule, e.g., a protein involved in an immune checkpoint pathway, optionally A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA, or SIGLEC7. In some embodiments, the immune checkpoint molecule is PD-1, LAG3, TIM3, or CTLA4. In various embodiments, the bispecific antigen binding protein comprises an scFv, Fab, or F(ab)2' of any of the CLDN18.2 antibodies of the present disclosure (e.g., Table B, Table C, Table D, Table 6, Table 8, Figure 13, or Figure 17).

[0230] In various embodiments, the bispecific antigen binding protein comprises an antigen binding protein comprising a sequence set forth in Table 11 or SEQ ID NO: 143, 144, 145, or 146, or a variant sequence thereof that differs by 1-5 amino acids or has at least or about 70% sequence identity. In some embodiments, the variant sequence has at least or about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In various embodiments, the bispecific antigen binding protein comprises a nanobody, diabody, BiTE®, DART, TandAb, CrossMab, or HSAbody structure. The present disclosure provides a conjugate comprising an antigen binding protein or bispecific antigen binding protein described herein and a heterologous moiety. In some embodiments, the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 42 and SEQ ID NO: 45. In some embodiments, the conjugate comprises a cytotoxic or chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is an antimitotic agent that inhibits cell division by inhibiting tubulin polymerization. In some embodiments, the antimitotic agent is an auristatin. In some embodiments, the auristatin is MMAE.

[0231] In various embodiments, the conjugates of the present disclosure are conjugated to the antigen binding protein via a cleavable linker. In some embodiments, the cleavable linker is MC-VC-PAB.

[0232] In some embodiments, the conjugate comprises an antigen-binding protein that is a monoclonal antibody, and optionally the monoclonal antibody is an IgG antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0233] In various embodiments, the conjugates of the present disclosure have an average number of units of drug conjugated per antigen binding protein ranging from 1 to 8, preferably the average number of units of drug conjugated per antigen binding protein is in the range of 3 to 8. In some embodiments, the conjugate is a heterologous conjugate. In other embodiments, the conjugate is a homologous conjugate. In some embodiments, the conjugate comprises a heterologous moiety or drug, where the drug is conjugated at a specific site of the antigen binding protein. In some embodiments, the specific site is an unpaired cysteine ​​residue. In some embodiments, the conjugate comprises a polypeptide comprising the amino acids set forth in SEQ ID NO: 42 and SEQ ID NO: 45 conjugated to MC-VC-PAB-MMAE.

[0234] The present disclosure also provides a fusion protein comprising the antigen binding protein or bispecific antigen binding protein described herein. The present disclosure further provides a nucleic acid comprising a nucleotide sequence encoding the antigen binding protein, bispecific antigen binding protein, conjugate, or fusion protein of the present disclosure. The present disclosure provides a vector comprising a nucleic acid comprising a nucleotide sequence encoding the antigen binding protein, conjugate, or fusion protein of the present disclosure. The present disclosure further provides a host cell comprising the nucleic acid or vector of the present disclosure.

[0235] The present disclosure provides a method of producing an antigen binding protein or bispecific antigen binding protein that binds to claudin 18.2 (CLDN18.2) protein, comprising (i) culturing a host cell of the present disclosure in a cell culture medium, the host cell comprising a nucleic acid comprising a nucleotide sequence encoding an antigen binding protein or bispecific antigen binding protein described herein, and (ii) harvesting the antigen binding protein or bispecific antigen binding protein from the cell culture medium. Also provided is a method of producing a fusion protein comprising an antigen binding protein or bispecific antigen binding protein that binds to claudin 18.2 (CLDN18.2) protein, comprising (i) culturing a host cell of the present disclosure in a cell culture medium, the host cell comprising a nucleic acid comprising a nucleotide sequence encoding a fusion protein of the present disclosure, and (ii) harvesting the fusion protein from the cell culture medium.

[0236] The present disclosure further provides a method of making a pharmaceutical composition comprising combining an antigen binding protein, bispecific antigen binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof of the present disclosure, and a pharma- ceutically acceptable carrier, diluent or excipient.Also provided are pharmaceutical compositions comprising an antigen binding protein, bispecific antigen binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof of the present disclosure, and a pharma- ceutically acceptable carrier, diluent or excipient.

[0237] Provided herein is a method of treating a subject with a cancer expressing CLDN18.2, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to treat the cancer. Also provided is a method of inhibiting tumor growth in a subject, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to inhibit tumor growth. The present disclosure provides a method of reducing tumor size in a subject, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to reduce tumor size. Additionally provided is a method of preventing recurrence of cancer in a subject, comprising administering to the subject a pharmaceutical composition described herein in an amount effective to prevent recurrence of the cancer.

[0238] The present disclosure provides a method of detecting claudin 18.2 (CLD18.2) in a sample, comprising contacting the sample with an antigen binding protein, bispecific antigen binding protein, conjugate, or fusion protein of the present disclosure, and assaying for immune complexes comprising the antigen binding protein, conjugate, or fusion protein bound to CLD18.2. Also provided herein is a method of diagnosing claudin 18.2 (CLD18.2) positive cancer in a subject, comprising contacting a biological sample comprising cells or tissue obtained from the subject with an antigen binding protein, bispecific antigen binding protein, conjugate, or fusion protein of the present disclosure, and assaying for immune complexes comprising the antigen binding protein, conjugate, or fusion protein bound to CLD18.2.

[0239] The present disclosure also provides a method of treating cancer in a subject diagnosed as a low overexpressor of CLDN18.2. In various embodiments, the method comprises administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to prevent recurrence of the cancer. In some aspects, the administration induces apoptosis in tumor cells, and optionally, the administration induces apoptosis in cells expressing CLDN18.2. In various aspects, the subject has a tumor, and the tumor is semi-quantitatively classified into one of four groups: high expressor, medium expressor, low expressor, and non-expressor. In various examples, a high expressor is defined as CLDN8.2 RNA greater than 12 log Fragments Per Kilobase Million (FPKM), where CLDN8.2 RNA is measured by RNASeq, or CLDN18.2 protein levels are greater than 3+ as measured by immunohistochemistry (IHC). In various examples, a medium expressor is defined as a CLDN8.2 RNA greater than 10 log FPKM, where the CLDN18.2 RNA is measured by RNASeq, or the CLDN18.2 protein level is greater than 2+ when measured by IHC. In various examples, a low expressor is defined as a CLDN18.2 RNA greater than 6 log FPKM, where the CLDN8.2 RNA is measured by RNASeq, or the CLDN18.2 protein level is greater than 1+ when measured by IHC. In various examples, a non-expressor is defined as a CLDN8.2 RNA less than 6 log FPKM, where the CLDN18.2 RNA is measured by RNASeq, or the CLDN18.2 protein level is less than the IHC detection limit. In various embodiments, the subject with the tumor is also described as a high expressor, medium expressor, low expressor, or non-expressor of CLDN18.2.

[0240] The following examples are provided merely to illustrate the present disclosure and are not intended to limit its scope in any way. EXAMPLES

[0241] Example 1 This example demonstrates the analysis of RNA levels of CLDN18.2 in different cell and tissue sources.

[0242] To establish a baseline for CLDN18.2 expression in different source materials, we assayed expression levels of CLDN18.2 expression in patient samples and cell lines generated by the Translational Oncology Research laboratory (TORL).

[0243] Information contained in The Cancer Genome Atlas (TCGA) database maintained by the National Cancer Institute (NCI) was used to measure CLDN8.2 RNA levels in patient samples. Information in the Genotype-Tissue Expression (GTEX) database maintained by the Common Fund was used to measure CLDN18.2 levels in normal tissues. Analysis of tissues from the GTEX database indicates that CLDN18.2 is detectable in a variety of sites, including lung, stomach, prostate, colon, pancreas, and breast, among other tissues.

[0244] CLDN18.2 expression levels were measured in TORL cancer cell lines using Agilent 44K microarrays (4x44K array chips, Agilent Technologies, Santa Clara, CA) and RNA sequencing (RNA-Seq) assays. RNASeq was performed by BGI Americas (Cambridge, MA) using their "RNASeq for Quantitation" service. As shown in Figures 1 and 2, upper gastrointestinal (GI), pancreatic and colon cancer cells expressed the highest levels of CLDN18.2, but CLDN18.2 expression levels were detectable in head and neck, melanoma, breast, lung, prostate, sarcoma and lymphoma cancer cells.

[0245] Figure 3 shows that those cells that are high CLDN18.2 expressors do not express CLDN18.1. Figure 4 shows the distribution of native cell lines expressing CLDN18.2, demonstrating that CLDN18.2 RNA positive cells have a positive flow signal.

[0246] Example 2 This example demonstrates the generation of cells engineered to overexpress CLDN18.2.

[0247] Models engineered to overexpress CLDN18.2 were generated. These models were used to determine the efficacy of the CLDN18.2 antibody described herein. Briefly, a nucleotide sequence encoding CLDN18.2 was engineered into a lentiviral vector with a CMV promoter and an attenuated internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). The IRES was placed between the cDNA of the gene of interest (GOI) (CLDN18.2) and the puromycin cDNA. A woodchuck posttranscriptional regulatory element (WPRE) was placed downstream of the puromycin cDNA. The vectors also expressed either a GFP marker sequence or a MycDDK tag.

[0248] The expression vector was virally transduced into HEK293T cells (for screening purposes) and NIH3T3 cells (for immunization). Positively transduced cells were selected based on survival in medium containing puromycin (1 μg / ml). Positive cells were subcloned to obtain a stable and homogenous clonal population of CLDN18.2-overexpressing cells.

[0249] Subclone CLDN18.2 expression was confirmed by fluorescence microscopy using the GFP tag to confirm overexpression of CLDN18.2.

[0250] Example 3 This example demonstrates the generation of a reference antibody and a control antibody.

[0251] Benchmark (reference) CLDN18.2-specific and control antibodies were generated by cloning the heavy and light chain variable regions of the antibodies into the ExpiCHO™ Expression System (ThermoFisher Scientific, Waltham, Mass.) to generate recombinant mouse IgG2A chimeric antibodies. These antibodies were tested in parallel with the newly generated CLDN18.2-specific antibodies described in Example 5.

[0252] Briefly, plasmids containing control and benchmark antibody sequences were transfected using the ExpiCHO™ Expression System (Cat. No.: A29133, ThermoFisher Scientific, USA) according to the manufacturer's protocol. Cells were incubated at 37°C and 8% CO for 1 day post-transfection in the medium provided in the kit. 2 and then incubated at 32°C and 5% CO 2The antibodies were purified by clarifying the ExpiCHO™ medium by centrifugation at 1,000g for 10 min followed by 5,000g for 30 min. The supernatant was then filtered using a 0.45 μm filter followed by a 0.22 μm filter. The supernatant was then subjected to affinity purification according to the manufacturer's protocol using Protein A / G resin (Life Technologies, Carlsbad, CA; Cat #20424). Prior to ELISA purification, the antibody titer in the medium was roughly determined to ensure that the amount of medium loaded occupied less than 80% of the resin binding capacity. After incubation, the resin was washed with PBS and eluted with elution buffer (Life Technologies, Cat #21004). The eluted fraction was immediately adjusted to physiological pH by adding Tris buffer, pH 8.0. The purified antibodies were then subjected to buffer exchange and protein concentration using an Amicon Ultra-15 Centrifugal Filter Unit (Life Technologies, Cat#UFC900324) in PBS buffer. Antibody concentration was determined by BCA protein assay. SDS-PAGE and Coomassie staining were performed to test antibody purity. Purified proteins were aliquoted and stored at -80°C for long-term storage or kept at 4°C for immediate use.

[0253] The integrity of the antibody was verified under non-reducing versus reducing conditions by SDS-PAGE followed by Coomassie staining; under non-reducing conditions, one predominant band was observed at around 150 kDa, whereas under reducing conditions, two bands at 50 kDa and 25 kDa were observed.

[0254] Example 4 This example demonstrates the characterization of a cell line with high endogenous CLDN18.2 expression.

[0255] A panel of cancer cell lines was analyzed for their endogenous expression of CLDN18.2 by FACS. Briefly, cells overexpressing CLDN18.2 (e.g., HEK293T cells overexpressing CLDN18.2 as described in Example 2) and cell lines endogenously expressing CLDN18.2 at high or low levels as described in Example 1 were used to verify the binding of the antibody to the target by FACS. Cells expressing CLDN18.2 were incubated with reference or control antibodies (as described in Example 3) for 30 minutes on ice, washed, and then incubated with Alexa Fluor® 647-conjugated goat anti-mouse IgG (minimum x reactivity) antibody, Biolegend cat#405322, for 30 minutes on ice. Fluorescence was read by a BD Biosciences Accuri™ flow cytometer (San Jose, CA).

[0256] The overexpressing lines were used to validate the control and reference antibodies, and then the endogenous cell lines were characterized using the control and reference antibodies. Cells overexpressing CLDN18.2 were included as a positive control in these assays.

[0257] FACS assays showed that four upper GI cancer cell lines, as well as pancreatic, colon and bladder cancer cell lines, expressed CLDN18.2 on their surfaces at high levels. The endogenous expression levels of CLDN18.2 by the tested cancer cell lines are summarized in Table 2. See also Figure 4. [Table 7]

[0258] Example 5 This example describes the immunization of mice for the generation of CLDN18.2-specific antibodies.

[0259] CLDN18.2-specific antibodies were generated by immunizing Balb / c and CD1 mice with a mixture of six different peptide immunogens according to the techniques of the Fred Hutchinson Cancer Research Center. The peptides spanned either the first or second loop (i.e., EL1 or EL2) in the CLDN18.2 extracellular domain. The peptides included the full length of EL1, a peptide spanning the first (N-terminal) half of EL1, and a peptide spanning the other (C-terminal) half of EL1. Table 3 provides the sequences of the peptides. [Table 8]

[0260] Mice were also immunized with 3T3 cells overexpressing full-length CLDN18.2 using a plasmid containing the human CLDN18.2-myc-DDK expression vector.

[0261] Splenocytes were harvested from immunized mice and fused with myeloma lines by BTX Electrofusion (BTX, Holliston, MA) to generate hybridomas. 7000 primary hybridoma cultures were generated and cultured in 384-well plates. The ability of antibodies to bind peptides was assessed by bead array using beads expressing three different peptide targets. Approximately 2000 potential positive antibodies were re-arrayed into 96-well plates and further screened by flow cytometry against endogenous and artificial cell line models.

[0262] Positive hybridoma supernatants were then counterscreened by flow cytometry against endogenous and artificial models of proteins with sequence similarity to the target region (e.g., other CLDN proteins). From the secondary screen and counterscreen, approximately 20 CLDN18.2-specific antibodies were selected for further study. These antibodies were subcloned and the heavy and light chain sequences were determined. See Table B and the Sequence Listing.

[0263] CLDN18.2 antibody was formatted as a full-length IgG antibody using ExpiCHO™ expression. The heavy and light chain variable regions of the antibody were cloned into a laboratory-engineered antibody expression vector based on the pcDNA™ 3.4-TOPO® vector (Cat. No.: A14697, ThermoFisher Scientific, USA) and transfected into CHO cells (following the protocol provided in the kit (ExpiCHO™ Expression System, Cat. No.: A29133, ThermoFisher Scientific, USA)). The antibody was purified and the cell surface binding of the antibody to CLDN18.2 and the antibody IC50 were determined by FACS with the CLDN18.2 antibody directly conjugated to Alexa Fluor® 647 NHS Ester (succinimidyl ester), Cat#A20106 (ThermoFisher Scientific) according to the manufacturer's protocol. CLDN18.2 antibodies were tested from 0.32 nM to 1000 nM (serial dilution 1:5, 6 points) in a volume of 50 μl with 150,000 cell lines.

[0264] CLDN18.2 expressing cells were used in FACS assays to determine the ability of CLDN18.2 antibodies to bind to CLDN18.2 on the surface of cells and cross-react with other CLDN family members. HEK293T cells engineered to express human CLDN18.2 fused to GFP, mouse CLDN18.2 fused to GFP, CLDN18.1-GFP, or GFP alone (no CLDN18.2) were used as artificial models of CLDN18.2 expression. HUPT4, SNU601, and PATU8988S cells were used as endogenous models of CLDN18.2 expression.

[0265] For each type of test cell and for each mAb, cells were detached from the surface of the culture flask by Versene (instead of trypsin) to protect cell surface proteins. The detached cells were then incubated with Adlexa Fluor®-labeled CLDN18.2 mAb at the indicated concentrations for 30 minutes in the dark on ice. CLDN18.2 mAb was directly labeled with Alexa Fluor® 647 NHS Ester (succinimidyl ester). After washing, cells were read on a BD Accuri™ Flow Cytometer C6 to detect antibody-antigen protein binding in channel FL4H. Each antibody was tested at various concentrations to establish a dose-fluorescence curve. The EC50 / IC50 (concentration of antibody at half maximum) of the antibody was calculated based on the FL4H (gated on live singlet cells) values ​​using a very simple IC50 toolkit available online that can plot biological dose-response data and fit the curve type to obtain the EC50 / IC50. The maximum was the lowest concentration of antibody at which fluorescence was maximal. Antibodies were screened for their ability to cross-react with the other CLDN18 isoform, CLDN18.1. These values ​​were used to determine the relative affinity of each antibody among the set of antibodies tested. Cross-reactivity data was obtained using a similar methodology.

[0266] Relative affinity data determined by this method are shown in Table 4. [Table 9]

[0267] Example 6 This example demonstrates the characterization of a chimeric mouse IgG mAb.

[0268] In vivo binding studies were performed in xenograft mice injected with human cancer cell lines. Briefly, xenograft models of human cancer cell lines were established in 6-week-old CD-1 athymic nude mice (Charles River Laboratories). The following conditions were followed for subcutaneous injection of each cell line: HUPT4 1.0 × 10 7 Cells, SNU601 1.0×10 7 Cells (with 50% Matrigel (BD Biosciences)). A sufficient number of mice were injected to achieve 8 mice per treatment arm. Tumors were 150-300 mm 3 Mice were randomized into treatment groups when tumors reached an average size of 100 mm. For treatment, each therapeutic antibody (Ab384, Ab400, reference Ab1, reference Ab2, and non-targeting IgG2 control) was diluted in sterile saline to a working concentration of 1 mg / ml for intravenous tail vein (IV) injection. Tumor xenografts were measured three times per week with calipers and tumor volumes (mm 3 ) was determined by multiplying height × width × length. Mice were treated for 2 to 5 weeks. At the end of the study, animals were euthanized and tumor tissues were excised and sectioned for storage as flash-chilled or formalin-fixed paraffin-embedded (FFPE) tissues for biomarker analysis. All animal experiments were performed under protocols approved by the IACUC and the University of California at Los Angeles Animal Research Committee. Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA). Results are provided as the mean volume for each group. Error bars represent the standard error of the mean (SE).

[0269] The results of the xenograft assays are shown in Figures 5-6. As shown in Figures 5A and 5B, Ab384 and Ab400 each caused a substantial mean change in tumor volume at day 30 compared to the control IgG2 antibody in mice bearing gastrointestinal tumors. As shown in Figures 6A and 6B, clones Ab384, Ab376, Ab307, and Ab432 caused a substantial mean change in tumor volume at day 28 compared to the control IgG2 antibody in mice bearing pancreatic tumors.

[0270] Example 7 This example demonstrates the humanization of an antibody of the present disclosure.

[0271] A subset of antibodies listed in Table A / Table B was selected for humanization analysis. The heavy chain variable (VH) and light chain variable (VL) sequences of Ab307, Ab376, Ab358, Ab360 and Ab432 antibodies were compared to a library of known human germline sequences from human VH genes and human VL kappa genes (IMGT® the international ImMunoGeneTics information system® www.imgt.org; Founder and President: Marie-Paule Lefranc, Montpellier, France); the databases used were IMGT human VH genes (F+ORF, 273 germline sequences) and IMGT human VL kappa genes (F+ORF, 74 germline sequences). Acceptor human germlines were selected based on the closest sequence to the parent antibody.

[0272] CDRs were defined according to AbM definitions (see Dr Andrew CRMartin's website www.bioinf.org.uk / abs / for a table comparing CDR definitions).

[0273] Changes to the corresponding parent murine sequences of human germline framework (i.e., non-CDR residues in VH and VL) positions may be required to optimize binding of the humanized antibody. Sequences of versions of the humanized antibodies are provided as SEQ ID NOs: 42-57.

[0274] Table 6 shows a scheme for combining humanized VH and VL in case none of the humanized versions are equivalent to the chimeric mAb. [Table 10]

[0275] The humanized antibodies listed in Table 6 were constructed and expressed essentially as described in Example 5. FACS assays were performed to determine the relative antigen binding strength of the humanized antibodies (either 1.5 μg or 0.3 μg) binding to CLDN18.2 expressed by the indicated cancer cell lines. The results of the assays are provided in Table 7. Zolbetuximab, 163E12 and 175D10 were used as reference antibodies. The corresponding parent antibodies (antibodies prior to humanization) were used as controls and designated "chim". [Table 11]

[0276] Based on the in vitro antigen binding data, five humanized antibodies were selected for further testing and development. The antibodies were derived from Ab307, Ab376, Ab358, Ab360, and Ab432.

[0277] In vivo binding studies of humanized versions of Ab307, Ab376, Ab358, Ab360, and Ab432 were performed in xenograft mice injected with the pancreatic cancer cell line HUPT4 essentially as described in Example 6. Briefly, a xenograft model of HUPT4 was established in 6-week-old CD-1 athymic nude mice (Charles River Laboratories). 3 Mice were randomized into treatment groups after reaching an average size of 1 mg / ml. Humanized antibodies were diluted in sterile saline to a working concentration of 1 mg / ml for intravenous tail vein (IV) injection. Tumor xenografts were measured three times per week with calipers and tumor volumes (mm 3) was determined by multiplying height × width × length. Mice were treated for 2–7 weeks. At the end of the study, animals were euthanized and tumor tissue was excised and sectioned for storage as flash-chilled or formalin-fixed, paraffin-embedded (FFPE) tissue for biomarker analysis.

[0278] The results of the xenograft assay are shown in Figures 7-12. Figures 7A-7B show the results of the xenograft assay for humanized Ab307, Ab376, Ab358, Ab360, Ab432, where the antibodies were administered once a week for 5 weeks. Controls included vehicle control (PBS), human IgG1 (10 mg / kg Q4D), and the mouse version of 307 (10 mg / kg Q4D). As shown in Figure 7, animals treated with humanized Ab307 (HuAb307) and HuAb358 demonstrated the greatest reduction in tumor volume over the 42-day treatment period, whereas HuAb360 and HuAb432 treatment induced a moderate reduction in tumor volume.

[0279] Figures 8A-8B show the results of a xenograft assay for the treatment of HUPT4 pancreatic cancer with various doses of HuAb307 antibody, e.g., 10 mg / kg given every 4 days (q4d), every week (qw), every 3 weeks (q3w), or 2.5 mg / kg given every 4 days. Treatment every week or every 4 days showed better results compared to antibody given every 3 weeks by day 42 after treatment.

[0280] Example 8 The antibodies were also tested for their ability to carry a cytotoxic payload. Humanized antibodies carrying MMAE were tested at various doses and dose schedules to assess the ability of the antibodies to mediate cytotoxicity in models for pancreatic or bladder cancer.

[0281] Briefly, CD-1 nude mice (8 mice per group) were injected subcutaneously in the right flank with HUPT4 or PATU8988S pancreatic cells, or SNU601 bladder cells. Tumors were 150–300 mm 3Mice were randomized into treatment groups when tumors reached an average size of 100 mm. For treatment, mice were administered either humanized anti-CLDN18.2 MMAE antibody or human IgG control antibody by tail vein injection once a week. Tumor xenografts were measured three times a week with calipers and tumor volumes (mm 3 ) was determined by multiplying height × width × length. Mice were treated for 20 to 42 days. At the end of the study, animals were euthanized and tumor tissues were excised and sectioned for storage as flash-chilled or formalin-fixed paraffin-embedded (FFPE) tissues for biomarker analysis. All animal experiments were performed under protocols approved by the IACUC and the University of California at Los Angeles Animal Research Committee. Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA). Results are provided as the mean volume for each group. Error bars represent the standard error of the mean (SE).

[0282] Figures 9A-9B show the results of treatment in a pancreatic tumor model in which antibodies HuAb307-MMAE and HuAb358-MMAE were administered at 5 mg / kg, 2.5 mg / kg, or 1 mg / kg once a week for 3 weeks. The anti-CLDN18.2-ADC was more effective at reducing tumor volume than the payload alone. A single dose of HuAb307 at 5 mg / kg was also effective at killing tumor cells by day 40.

[0283] The efficacy of antibodies HuAb307, HuAb368, HuAb360 and HuAb432 administered at 10 mg / kg was compared to HuAb307 and HuAb358-ADCs conjugated to MMAE, administered at 5 mg / kg once a week in a pancreatic cancer cell model. Antibodies alone reduced tumor volume size, but anti-CLDN18.2-ADCs showed improved tumor killing out to 48 days (Figure 10A-B). Similar results were observed in a gastric cancer model (Figure 11).

[0284] In additional experiments, Ab307 and Ab358-ADC conjugates were administered intravenously at different doses, and the results show that intravenous treatment was dose-dependent (Figures 12A-B).

[0285] These results indicate that antibodies are useful delivery systems for cytotoxic agents to solid tumors.

[0286] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0287] Use of the terms "a" and "an" and "the" and similar referents in the context of describing this disclosure (particularly in the context of the claims which follow) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise stated.

[0288] The recitation of ranges of values ​​herein, unless otherwise stated herein, is intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, and each separate value and endpoint is incorporated herein as if it were individually recited herein.

[0289] All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any examples provided herein or the use of various language (e.g., "such as") are intended merely to better illustrate the disclosure and do not limit the scope of the disclosure unless otherwise stated. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0290] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate those skilled in the art to adopt such variations as necessary, and the inventors intend for the present disclosure to be practiced other than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, the present disclosure encompasses any combination of the above-described elements in all possible variations thereof, unless otherwise indicated herein or clearly contradicted by context.

[0291] Example 9 This example demonstrates the in vivo activity of a humanized anti-CLDN18.2-307 antibody drug conjugate (ADC) in pancreatic cancer patient-derived xenografts (PDX).

[0292] Methods: The antitumor activity of a CLDN18.2-directed ADC (CLDN18.2-307-ADC (conjugated to MC-VC-PAB-MMAE)) and a CLDN18.2-directed mAb CLDN18.2-307 was evaluated in two patient-derived xenografts of pancreatic cancer. Each PDX had previously been shown to be positive (XWR6) or negative (XWR187) for CLDN18.2 protein by IHC (Figure 14C and Figure 15C). Patient-derived xenografts were established through in vivo passaging of tumor tissue in mice by subcutaneously implanting tumor pieces in the right hind flank of the animals in 6-week-old CD-1 athymic nude mice (Charles River Laboratories). A sufficient number of xenograft tumor-bearing mice was achieved, with tumor volumes ranging from 200 to 300 mm. 3 After tumors reached a range of 10 mg / kg QW IV, mice were randomized into treatment groups receiving either non-targeting IgG2A control antibody (10 mg / kg QW IV), humanized mAb CLDN18.2-307 (10 mg / kg QW IV) or CLDN18.2-307-ADC (5 mg / kg QW IV) for three repeated doses. For all experiments, tumor xenografts were measured three times per week with calipers and tumor volumes (mm 3 ) was determined by multiplying height × width × length. All animal experiments were performed under protocols approved by the IACUC and the UCLA Animal Research Committee. Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA).

[0293] Brief summary: In the CLDN18.2-positive XWR6 model, treatment with mAb CLDN18.2-307 slowed the average rate of PDX progression compared to non-targeted controls, whereas 307-ADC induced complete regression of xenograft tumor burden in each of the four mice in that group (Figures 14A and 14B). In contrast to the CLDN18.2-positive model, CLDN18.2-307-ADC or mAb CLDN18.2-307 showed no effect on xenograft tumor progression in the CLDN18.2-negative PDX, XWR187 (Figures 15A and 15B).

[0294] Example 10 This example demonstrates evaluation of the activity of humanized CLDN18.2 mAb and humanized CLDN18.2 ADC in target-negative cancer cell line xenografts.

[0295] Methods: The target-selective activity of two CLDN18.2 ADCs (conjugated to MC-VC-PAB-MMAE) and two CLDN18.2 mAbs was evaluated in a cell line xenograft model of human melanoma (M202) previously shown to be negative for (i.e., lacking expression of) the CLDN18.2 protein. Six-week-old CD-1 athymic nude mice (Charles River Laboratories) were implanted with 1.0 × 10 mAbs in 50% Matrigel (BD Biosciences) on the right hind flank of the animals. 7 Cancer cell line xenografts were established by injecting cells into the tumor. 3 ~300mm 3 When tumors reached an average size of 100 mm, mice were randomized into treatment groups with non-targeting humanized IgG1 control antibody (10 mg / kg QW IV), mAb CLDN18.2-307 (10 mg / kg QW IV), mAb CLDN18.2-358 (10 mg / kg QW IV), CLDN18.2-307-ADC (5 mg / kg QW IV) or CLDN18.2-338-ADC (5 mg / kg QW IV) for three repeated doses. For all experiments, tumor xenografts were measured three times per week with calipers and tumor volumes (mm3 ) was determined by multiplying height × width × length. All animal experiments were performed under protocols approved by the IACUC and the UCLA Animal Research Committee. Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA).

[0296] Brief summary: No significant antitumor activity was observed with either of these mAbs or ADCs in this CLDN18.2-negative model (Figures 16A and 16B). These data indicate that these molecules have minimal off-target activity.

[0297] Example 11 This example demonstrates humanized antibody binding activity against native positive cells (cells with endogenous expression of CLDN18.2) or artificially overexpressing cells (cells engineered to overexpress CLDN18.2) as analyzed by flow cytometry.

[0298] Methods: Humanized antibodies at three different concentrations were incubated with 150,000 cells in 50 μl of 2% FBS / PBS for 30 minutes at 4° C., followed by washing with 2% FBS / PBS; then incubated with Alexa Fluor® 647 anti-human IgG Fc antibody (409320) from Biolegend for 30 minutes at 4° C. Samples were then measured by Intellicyt® iQue Advanced Flow Cytometry Platform.

[0299] Brief summary: Eight humanized antibodies engineered based on 307 (h1F06) and eight humanized antibodies engineered based on 358 against CLDN18.2 show varying binding affinities by flow cytometry compared to their parental antibodies in native CLDN18.2-expressing HUPT4 cells or in artificial CLDN18.2-overexpressing HEK293T cells, whereas they show no activity against negative control M202 cells and parental HEK293T cells (Figure 18).

[0300] Example 12 Table 8 below shows further humanized antibodies and sequences. [Table 12]

[0301] Example 13 This example demonstrates humanized antibody binding activity to species orthologs of CLDN18.1 and CLDN18.2 on cells engineered to overexpress the orthologs. Binding activity was analyzed by flow cytometry.

[0302] Methods: Four humanized antibodies at three different concentrations were incubated with 150,000 cells in 50 μl of 2% FBS / PBS at 4° C. for 30 minutes, followed by washing with 2% FBS / PBS; then incubated with Alexa Fluor® 647 anti-human IgG Fc antibody (409320) from Biolegend for 30 minutes at 4° C. Samples were then measured by Intellicyt® iQue Advanced Flow Cytometry Platform.

[0303] Brief summary: Four humanized antibodies engineered based on 307 and 358 show significantly different binding affinities to human CLDN18.2, human CLDN18.1, mouse CLDN18.2, canine CLDN18, and rat CLDN18 by flow cytometry (Figure 19). The monkey CLDN18.2 OE strain was not included in this assay because both loop 1 and loop 2 are identical between humans and Macaca fascicularis (cynomolgus macaque).

[0304] Example 14 This example demonstrates the in vitro characterization of antibody internalization using CLDN18.2 humanized antibody in HUPT4 cells.

[0305] Methods: Nuclei were stained with DNA dye Hoechst33342 at 1ug / ml in medium overnight at 37°C. Lysosomes were stained with CellLight™ Lysosomes-GFP, BacMam 2.0 (C10507) and transfected at 37°C overnight at 2ul / 10k cells. CLDN18.2 antibody is primary labeled with AF647. HUPT4 cells are stained with 8ug antibody / 300ul growth medium for 30 minutes at 4°C.

[0306] Brief summary: All four antibodies tested (02-0307-h4, 02-0307-h6, 02-0358-h3 and 02-0358-h5) were fully internalized between 24 and 48 hours after staining (Figure 20). After internalization, the antibodies (red) integrated with the lysosomes (green) and formed a yellow spot around the nucleus (blue) (Figure 20).

[0307] Example 15 This example demonstrates the binding affinity (KD) of humanized CLDN18.2 antibody and the expression levels of CLDN18.2 on the surface of various cancer cell lines.

[0308] Methods: Cell-based antibody affinity KD was measured by KinExA 4000 from Sapidye (Figure 21A). For each KD measurement, at least two antibody concentration curves were usually prepared using the same cell batch (same passage) to avoid changes in expression levels between passages. Briefly, either HEK293T CLDN18.2-mGFP C12 cells (engineered overexpressing cell line) or HUPT4 cells (native positive cell line) were detached using Versene. The cells were then equilibrated in different concentrations of antibody solutions overnight at 4°C in 2% FBS / DMEM or 2% FBS / RPMI, respectively. The cell concentration was 1.8×10 7 Starting with cells / ml, two-fold serial dilutions were performed up to 10 points. Also, antibody solution alone (100% signal) and non-specific binding (NSB) buffer alone were included in each curve. The next day, cells were spun down at 1500 rpm for 10 min and the supernatant was saved for measurement. PMMA beads (Sapidye, 440176) were pre-coated with Goat-Anti-Human IgG (Jackson ImmunoResearch Labs, 109-005-003) at 30 μg / ml, followed by incubation with 10 mg / ml BSA / PBS at room temperature for 1 h. Fluorescent Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG (Jackson ImmunoResearch Labs, 109-605-088) was diluted to 0.5 μg / ml in 1% BSA / PBS. KD and antigen expression levels were calculated using two antibody curves analyzed by n-curve analysis.

[0309] Cell surface antigen expression levels were performed via quantitative flow cytometry measurements (Figure 21B) by using a combination of the Quantum MESF kit (four fluorescent microsphere populations labeled with various amounts of Alexa Fluor 647, Bangs Laboratories Inc, catalog #647) and Quantum Simply Cellular (four standards, Bangs Laboratories Inc, catalog #816) or Simply Cellular Kit (one standard, catalog #812). Briefly, cells were detached using Versene solution; cells and QSC beads were stained with CLDN18.2 antibody, primarily labeled with Alexa Fluor 647 dye, for 30 minutes at 4°C; after washing with 2% FBS / PBS, cells, MESF, and QSC beads were analyzed on an Accuri C6 with identical settings gating around the singlet population. Standard curves were generated using the MESF kit and fluorescence was quantified using the QuickCal analysis template provided with each kit; cell samples were then read against the curves for expression determination. We also quantified the fluorophore:protein (F / P) ratio using the Quantum Simply Cellular or Simply Cellular Kit. Using the QuickCal analysis template. Results were then converted from MESF to ABC units using the F / P ratio. Finally, the calculated monovalent ABC was multiplied by 2 to obtain the ABC value for bivalent binding.

[0310] Brief summary: The four humanized CLDN18.2 antibodies showed different binding affinities to CLDN18.2-positive cell lines; higher binding affinity to native CLDN18.2-positive cells compared to CLDN18.2-overexpressing artificial cells (Figure 21A). The expression levels of CLDN18.2 on the cell surface varied in endogenous cell lines among different tissues (Figure 21B).

[0311] Example 16 This example demonstrates the efficacy of CLDN18.2 humanized mAb in CLDN18.2+ pancreatic cancer cell line (HUPT4) xenografts.

[0312] Methods: The antitumor activity of four CLD18.2-directed antibodies was compared in a HUPT4 cell line xenograft model of human pancreatic cancer. Six-week-old CD-1 athymic nude mice (Charles River Laboratories) were treated with 1.0 × 10 7 Cancer cell line xenografts were established by injecting cells into the tumor. 3 Mice were randomized into treatment groups when tumor volume reached an average size of 100 mm. Mice were treated once weekly (QW) with either non-targeting humanized IgG1 control antibody, CLDN18.2 antibody clones #307-4, #02-307-6h, #358-3 or #02-358-5h by intravenous (IV) injection at 10 mg / kg. For all experiments, tumor xenografts were measured three times per week with calipers and tumor volumes (mm 3 ) was determined by multiplying height × width × length. All animal experiments were performed under protocols approved by the IACUC and the UCLA Animal Research Committee. Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA).

[0313] Brief summary: Treatment with each antibody induced complete inhibition of xenograft tumor progression 24 days after dosing. Of the four CLDN18.2 mAbs tested, clone #307-4 showed slightly improved efficacy over the other three clones (Figures 22A and 22B).

[0314] Example 17 This example demonstrates CLDN18.2-CD3-bispecific T cell engager (BiTE) binding activity to native positive or artificially overexpressing cells by flow cytometry.

[0315] Method: #307-CD3E-BiTE (2 batches) and 358-CD3E-BiTE were incubated with 150,000 cells in 50 μl of 2% FBS / PBS for 30 min at 4° C., followed by washing with 2% FBS / PBS; then incubated with anti-his Alexa Fluor® 647 (Biolegend #652513) for 30 min at 4° C. Samples were then measured on a BD Accuri™ C6 Flow Cytometer.

[0316] Brief summary: CLDN18.2 BiTEs [#307-CD3E-BiTE (2 batches) and 358-CD3E-BiTE] showed good binding affinity with native CLDN18.2-expressing HUPT4 cells and artificial CLDN18.2-overexpressing HEK293T cells by flow assay, whereas they showed no activity against negative control ARK2 cells (Figure 24A-E). However, anti-his-AF647 (Biolegend #652513) has very high background / non-specific binding with parental HEK293T cells.

[0317] Example 18 This example demonstrates CLDN18.2-CD16-TandAb (tandem diabody) binding activity to native positive or artificially overexpressing cells by flow cytometry.

[0318] Methods: TandAbs containing antigen binding sites binding to CD16A or CLDN18.2, respectively, were constructed using the antigen binding proteins of the 307 or 358 antibodies. The structure of the TandAb described herein was CD16A VH-(G2S)3-CLDN18.2AbVL-(G2S)3-CLDN18.2AbVH-(G2S)3-CD16A VL-His tag (Figures 23A-C). TandAb307-CD16A and TandAb358-CD16A as well as AFM13 were incubated with 150,000 cells in 50 μl of 2% FBS / PBS for 30 minutes at 4°C, followed by washing with 2% FBS / PBS; then incubated with anti-his Alexa Fluor® 647 (Biolegend #652513) for 30 minutes at 4°C. The samples were then measured using a BD Accuri™ C6 Flow Cytometer.

[0319] Brief summary: TandAb307-CD16A and TandAb358-CD16A showed good binding affinity by flow assay with native CLDN18.2 expressing HUPT4 cells, whereas they showed lower activity on negative control ARK2 cells. However, anti-his-AF647 (Biolegend #652513) has very high background / non-specific binding. AFM13 antibody (bispecific for CD16A and CD30) was used as a positive control for binding to CD16A in Jurkat-Lucia-NFAT-CD16A cells. The results in this flow assay demonstrated that 307-CD16A and 358-CD16A TandAbs successfully bound to both CLDN18.2 and CD16A (Figure 25A-E).

[0320] Example 19 This example demonstrates CLDN18.2-CD16-TandAb binding activity to native positive or artificially overexpressing cells by flow cytometry.

[0321] Methods: Tandb307-CD16A and Tandb358-CD16A and AFM13 were incubated with 150,000 cells in 50 μl of 2% FBS / PBS for 30 minutes at 4° C., followed by washing with 2% FBS / PBS; then incubated with anti-his Alexa Fluor® 647 (Biolegend#652513) for 30 minutes at 4° C. Samples were then measured on a BD Accuri™ C6 Flow Cytometer.

[0322] Brief summary: Tandab307-CD16A and Tandab358-CD16A showed good binding affinity with native CLDN18.2-expressing HUPT4 cells, whereas they showed lower activity against CLDN18.2-negative ARK2 cells (Figure 26A-E). However, anti-his-AF647 (Biolegend #652513) has high background / non-specific binding. AFM13 antibody (bispecific for CD16A and CD30) was used as a positive control for binding to CD16A in Jurkat-Lucia-NFAT-CD16A cells. Results in this flow assay demonstrated that Tandab307-CD16A and Tandab358-CD16A successfully bound to both CLDN18.2 and CD16A (Figure 26A-E).

[0323] Example 20 This example shows CLDN18.2-CD3 BiTE binding activity to Jurkat cells by flow cytometry.

[0324] Methods: Jurkat cells intrinsically positive for CD3 were incubated with 1ug of each BiTE of interest (Blincyto-His Tag, p307 BiTE-His Tag, p358 BiTE-His Tag), incubated at 4°C for at least 30 minutes, washed, and incubated with 0.5ug of anti-His secondary antibody. Samples were washed and flow cytometry was performed on the same day. For negative controls, Jurkat cells were incubated only with anti-His secondary antibody.

[0325] Brief summary: Flow results demonstrate the anti-CD3 binding capacity of the benchmark antibody (Blincyto) as well as our CLDN18.2 BiTE p307 and p358 (as shown in Figure 27A). 40X Keyence images show consistent surface staining (Figure 27B).

[0326] Example 21 This example shows a T cell activation assay using Jurkat cells with an NFAT-RE reporter and a CLDN18.2-CD3 BiTE using native or artificially overexpressing cells.

[0327] Methods: The assay was performed using Promega's T cell activation bioassay kit (NFAT-RE J1621). Target cells were seeded in wells of a white 96-well plate at a density of 40,000 cells / well and incubated overnight at 37°C, after which thaw-and-use Jurkat T cells (engineered to emit light when activated) included in the assay kit were added to the seeded cells along with BiTE treatment (1:1 cell ratio). BiTE treatment was given in nine 1:4 serial dilutions at a starting concentration of 10 nM. Treated plates were incubated at 37°C for a period of 6 hours, after which Bio-Glo reagent (included in the kit) was added and immediately read in units of counts per second (CPS) on a Victor 3V luminometer.

[0328] Brief summary: Luminescence data demonstrate that p307 BiTE induces CLDN18.2-specific activation of CD3+ Jurkat cells (Figure 28). That is, cells positive for the tumor-associated antigen (TAA) must be present for the BiTE to induce a relative level of activation.

[0329] Example 22 This example demonstrates a T cell activation assay using Jurkat cells with an NFAT-RE reporter and a CLDN18.2-CD3 BiTE using artificially overexpressing cells and an endogenous cell line.

[0330] Methods: (Same as FIG. 28, except with both CLDN18.2 BiTEs) The assay was performed using Promega's T cell activation bioassay kit (NFAT-RE J1621). Target cells were seeded in wells of a white 96-well plate at a density of 40,000 cells / well and incubated overnight at 37° C., after which thawed Jurkat T cells (engineered to emit light when activated) included in the assay kit were added to the seeded cells along with the BiTE treatment (1:1 cell ratio). BiTE treatments were given in nine 1:4 serial dilutions at a starting concentration of 10 nM. Treated plates were incubated at 37° C. for a period of 6 hours, after which Bio-Glo reagent (included in the kit) was added and immediately read in units of counts per second (CPS) on a Victor 3V luminometer.

[0331] Brief summary: Luminescence data demonstrate that p307 and p358 BiTEs induce CLDN18.2-specific activation of CD3+ Jurkat cells (Figure 29). That is, cells positive for tumor-associated antigens (TAA) are required for BiTEs to induce relative levels of activation.

[0332] Example 23 This example shows representative images of a CLDN18.2-CD3 bispecific cytotoxicity assay after 5 days of treatment.

[0333] Methods: Pan CD3+ human PBMCs (purchased from HumanCells Bio) were co-cultured with HUPT4, Cldn18.2+ cell lines at different effector:target ratios in 96-well plates and treated with either p307 CLDN18.2 BiTE or negative control Blincyto. Treatment concentrations started at 125ng / mL and were serially diluted at a ratio of 1:10. Five days after treatment, plates were imaged with a Cellavista imaging system to determine whether and to what extent the co-culture and treatment induced a cytotoxic effect on HUPT4 cells.

[0334] Brief summary: Cellavista images demonstrate that p307 CLDN18.2 BiTE induces a cytotoxic effect on HUPT4 cells. In contrast, the negative control Blincyto, specific for CD19 but not CLDN18.2, had no effect on HUPT4 cells at the same concentration (Figure 30A-F).

[0335] Example 24 This example demonstrates the LDH activity of CLDN18.2-CD3 BiTEs after 5 days of treatment.

[0336] Methods: Media from the experiment detailed in Example 22 was collected and spun down at 14,500 rpm for 10 min to prepare for LDH assay (Sigma: MAK066). 6 μl of media from each well was used for LDH activity assay. RPMI with 10% FBS was used as a negative control. Assays were performed at 37° C. and OD450 nM was measured every 15 min at 30 s intervals using a microplate reader (Molecular Device).

[0337] Brief summary: The LDH colorimetric assay was used as a simple method to determine the relative levels of cytotoxicity. Results demonstrate target-specific cytotoxicity and a dose-dependent response; HUPT4 cells co-cultured with CD3+ human PBMCs and treated with p307 BiTE released significantly higher levels of LDH compared to cells treated with the negative control Blinatumomab (Blincyto). A response dependent on the effector to target cell ratio was also observed, indicating that CD3+ cells are required for the cytotoxic effect (Figure 31).

[0338] Example 25 This example demonstrates the in vivo activity of a CLDN18.2-directed BiTE(CD3) in CLDN18.2-positive HUPT4 pancreatic cancer cell line xenografts in mice injected with human PBMCs.

[0339] Methods: We evaluated the antitumor activity of CLDN18.2-directed BiTEs in cell line xenograft studies using immunodeficient mice supplemented with human PBMC injections. Immunodeficient (NSG) mice were injected with 1.0 × 10 7 HUPT4 pancreatic cancer cells were injected, and 12 days later, 1.0 × 10 7 The day after injection of PBMCs, mice were cultured at 4°C for 12 h at 4°C until the mean tumor volume (150–200 mm) was reached. 3 ) and treated with daily intravenous (IV) injections of either 1 mg / kg Blincyto (non-targeting control), 1 mg / kg CLDN18.2-BiTE-307 or 1 mg / kg CLDN18.2-BiTE-358 for seven consecutive days. Additional control groups were treated with a non-targeting human IgG1 antibody (10 mg / kg IV QW), CLDN18.2-mAb-307 (10 mg / kg IV QW) or CLDN18.2-mAb-358 (10 mg / kg IV QW).

[0340] Brief summary: Significant tumor regression of CLDN18.2-positive HUPT4 xenografts was observed only in mice treated with CLDN18.2-BiTE-307 or CLDN18.2-BiTE-358 (Figure 32). No antitumor activity was observed in response to treatment with the CD19-directed BiTE, Blincyto. HUPT4 cells do not express CD19 protein. mAbs directed against CLDN18.2 also had no activity in this model (Figure 32). These data demonstrate the potent activity of CLDN18.2 BiTEs and the advantages of using BiTE technology to target CLDN18.2.

[0341] Example 26 This example demonstrates the in vivo activity of a CLDN18.2-directed BiTE(CD3) in CLDN18.2-positive HUPT4 pancreatic cancer cell line xenografts in humanized BLT mice.

[0342] Methods: The antitumor activity of CLDN18.2-directed BiTEs was evaluated in cell line xenograft studies using immunodeficient (NSG) mice implanted with fetal donor CD38+ stem cells and fragments of donor liver and thymus to generate a fully humanized immune system. Reconstitution of human immune cells was confirmed in these BLT (bone marrow liver thymus) mice before administration of 1.0 × 10 7 HUPT4 pancreatic cancer cells were injected. The tumors were 200 mm 3 After reaching an average volume of 10 mg / kg, mice were treated with either Hu-IgG1 control IV QW or 1 mg / kg CLDN18.2-BiTE-307 IV for 5 consecutive days.

[0343] Brief summary: Significant tumor regression was observed in mice treated with CLDN18.2-BiTE-307 (Figures 33A and 33B). These data demonstrate that the CLDN18.2-targeted BiTEs described herein induce significant antitumor activity in humanized mice bearing CLDN18.2-positive cancer cell line xenografts.

[0344] Tables 9-11 below show various antigen binding proteins or bispecific antigen binding proteins that bind to CLDN18.2. [Table 13] TIFF2025032078000024.tif220165TIFF2025032078000025.tif222165TIFF2025032078000026.tif231165TIFF2025032078 000027.tif223165TIFF2025032078000028.tif228165TIFF2025032078000029.tif242160TIFF2025032078000030.tif58160 [Table 14] TIFF2025032078000032.tif213159TIFF2025032078000033.tif191160TIFF2025032078000034.tif136164

[0345] The antibodies in Table 10 represent the sequences for antibodies 307-4h and 358-h3 as well as additional sequence variants. These antibodies were generated to provide additional information on the potential manufacturability sequence burden. [Table 15] TIFF2025032078000036.tif180163

Claims

1. An antigen binding protein that binds to human claudin 18.2 (CLDN18.2) protein (SEQ ID NO: 1), a. the antigen binding protein binds to extracellular loop 1 (EL1) of the extracellular domain (ECD) of CLDN18.2 and does not bind to extracellular loop 2 (EL2) of the ECD of CLDN18.2; or b. does not bind to claudin 18.1 (CLDN18.1) and binds to CLDN18.2 endogenously expressed by HUPT4 cells with greater than 1.5-fold higher affinity than a reference antibody; or c. A combination thereof. The antigen-binding protein.

2. The antigen binding protein of claim 1, which binds to an epitope within the amino acid sequence of residues 28 to 74 of CLDN18.2 (SEQ ID NO:1).

3. The antigen-binding protein of claim 2, which binds to the amino acid sequence of GLWRSCVRESSGFTECRFYFTL (SEQ ID NO: 4), QGLWRSCVRESSGFTECRGYFTLK (SEQ ID NO: 5), DQWSTQDLYNNPVTAVFNYQGLWRSC (SEQ ID NO: 6) or CRGYFTLLFLPAMLQAVR (SEQ ID NO: 7) of CLDN18.

2.

4. 2. The antigen binding protein of any one of the preceding claims, wherein said reference antibody comprises a light chain variable sequence of SEQ ID NO: 58 and a heavy chain variable sequence of SEQ ID NO: 59, a light chain variable sequence of SEQ ID NO: 60 and a heavy chain variable sequence of SEQ ID NO: 61, or a light chain variable sequence of SEQ ID NO: 62 and a heavy chain variable sequence of SEQ ID NO:

63.

5. a. the heavy chain (CDR) 1 amino acid sequence shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 64, 88, 69, 89, 72, 75, 91, 94, 95, 97, 99, 101, 103, 106, 109; or a variant sequence thereof differing by one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; b. The HC CDR2 amino acid sequences shown in Table A or sequences selected from the group consisting of SEQ ID NOs: 65, 67, 70, 90, 73, 76, 92, 73, 96, 98, 100, 102, 104, 107, 110; or variant sequences thereof differing by one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; c. A HC CDR3 amino acid sequence as set forth in Table A, or a sequence selected from the group consisting of SEQ ID NOs: 66, 68, 71, 77, 74, 77, 93, 74, 105, 108, 111; or a variant sequence thereof differing by one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; d. A light chain (LC) CDR1 amino acid sequence shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 78, 81, 82, 86, 114, 120, 123, 126; or a variant sequence thereof differing by one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; e. The LC CDR2 amino acid sequences shown in Table A or sequences selected from the group consisting of SEQ ID NOs: 79, 112, 79, 84, 116, 118, 119, 129, 121, 124, 127; or variant sequences thereof differing by one or two amino acids or having at least or about 70% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%) sequence identity; f. a LC CDR3 amino acid sequence shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 80, 83, 113, 85, 87, 115, 117, 122, 125, 128; and g. Any combination of two or more of (a) to (f) 2. An antigen-binding protein according to any one of the preceding claims, comprising:

6. 6. The antigen binding protein of claim 5, wherein the variant sequence has at least about 80% or at least or about 85% sequence identity.

7. 6. The antigen binding protein of claim 5, wherein the variant sequence has at least about 90% sequence identity or at least or about 95% sequence identity.

8. 6. The antigen binding protein of claim 5, comprising one or two of the light chain CDR1 amino acid sequence, the light chain CDR2 amino acid sequence, and the light chain CDR3 amino acid sequence shown in Table A and the heavy chain CDR amino acid sequence shown in Table A.

9. 6. The antigen binding protein of claim 5, comprising a heavy chain CDR1 amino acid sequence, a heavy chain CDR2 amino acid sequence, and a heavy chain CDR3 amino acid sequence shown in Table A and one or two of the light chain CDR amino acid sequences shown in Table A.

10. SEQ ID NOs: 78, 79, 80, 64, 65, 66; b. SEQ ID NOs: 81, 112, 80, 88, 65, 66; c. SEQ ID NOs: 81, 79, 80, 64, 67, 68; d. SEQ ID NOs: 82, 79, 83, 39, 70, 71; e. SEQ ID NOs: 81, 79, 113, 89, 90, 77; f. SEQ ID NOs: 81, 84, 85, 72, 73, 74; g. SEQ ID NOs: 86, 79, 87, 75, 76, 77; h. SEQ ID NOs: 114, 79, 115, 91, 92, 93; i. SEQ ID NOs: 81, 116, 117, 94, 73, 74; j. SEQ ID NOs: 81, 118, 117, 95, 96, 74; k. SEQ ID NOs: 81, 119, 117, 97, 98, 74; SEQ ID NOs: 81, 118, 85, 99, 100, 74; m. SEQ ID NOs: 81, 129, 117, 101, 102, 74; n. SEQ ID NOs: 120, 121, 122, 103, 104, 105; o. SEQ ID NOs: 123, 124, 125, 106, 107, 108; and p. SEQ ID NOs: 126, 127, 128, 109, 110, 111 10. The antigen-binding protein of any one of claims 5 to 9, comprising six CDR amino acid sequences selected from the group consisting of:

11. a. a heavy chain variable region amino acid sequence shown in Table B, or a sequence selected from the group consisting of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 2, 34, 36, 38, and 40, or a variant sequence thereof that differs by 1 or 2 amino acids or has at least or about 70% sequence identity; or b. a light chain variable region amino acid sequence shown in Table B, or a sequence selected from the group consisting of 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% sequence identity; or c. Both (a) and (b) The antigen-binding protein of any one of claims 5 to 10, comprising:

12. 12. The antigen binding protein of claim 11 , wherein the variant sequence has at least about 80% or at least about 85% sequence identity.

13. 12. The antigen binding protein of claim 11 , wherein the variant sequence has at least about 90% or at least about 95% sequence identity.

14. a. SEQ ID NOs: 10 and 11; b. SEQ ID NOs: 12 and 13; c. SEQ ID NOs: 14 and 15; d. SEQ ID NOs: 16 and 17; e. SEQ ID NOs: 18 and 19; f. SEQ ID NOs: 20 and 21; g. SEQ ID NOs: 22 and 23; h. SEQ ID NOs:24 and 25; i. SEQ ID NOs:26 and 27; j. SEQ ID NOs:28 and 29; k. SEQ ID NOs: 30 and 31; l. SEQ ID NOs: 32 and 33; m. SEQ ID NOs: 34 and 35; n. SEQ ID NOs: 36 and 37; o. SEQ ID NOs: 38 and 39; and p. SEQ ID NOs: 40 and 41 12. The antigen-binding protein of claim 11, comprising a pair of amino acid sequences selected from the group consisting of:

15. 10. An antigen-binding protein according to any one of the preceding claims which is an antibody.

16. 16. The antigen-binding protein of claim 15 which is a monoclonal antibody.

17. 17. The antigen-binding protein of claim 15 or 16, which is an IgG antibody.

18. 2. An antigen binding protein according to any one of the preceding claims, which inhibits colony growth by at least about 50% in a soft agar 3D growth assay.

19. 10. An antigen-binding protein according to any one of the preceding claims which inhibits tumour growth in xenograft mice injected with human cancer cells.

20. 20. The antigen binding protein of claim 19, which inhibits tumor growth in xenograft mice injected with pancreatic, gastrointestinal, bladder, or colon cancer cells.

21. 21. The antigen binding protein of claim 20, which inhibits at least 50% tumor growth in xenograft mice injected with pancreatic, gastrointestinal, bladder, or colon cancer cells.

22. (a) a HC CDR1 amino acid sequence as shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 64, 88, 69, 89, 72, 75, 91, 94, 95, 97, 99, 101, 103, 106, 109, or a variant sequence thereof differing by one or two amino acids or having at least or about 70% sequence identity; (b) a HC CDR2 amino acid sequence as shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 65, 67, 70, 90, 73, 76, 92, 73, 96, 98, 100, 102, 104, 107, 110; or a variant sequence thereof differing by one or two amino acids or having at least or about 70% sequence identity; (c) a HC CDR3 amino acid sequence as shown in Table A or a sequence selected from the group consisting of SEQ ID NOs: 65, 67, 70, 90, 73, 76, 92, 73, 96, 98, 100, 102, 104, 107, 110; or a variant sequence thereof differing by one or two amino acids or having at least or about 70% sequence identity; (d) the LC CDR1 amino acid sequences shown in Table A or sequences selected from the group consisting of SEQ ID NOs: 78, 81, 82, 86, 114, 120, 123, 126; or variant sequences thereof differing by one or two amino acids or having at least or about 70% sequence identity; (e) the LC CDR2 amino acid sequences shown in Table A or sequences selected from the group consisting of SEQ ID NOs: 79, 112, 79, 84, 116, 118, 119, 129, 121, 124, 127; or variant sequences thereof differing by one or two amino acids or having at least or about 70% sequence identity; (f) the LC CDR3 amino acid sequences shown in Table A or sequences selected from the group consisting of SEQ ID NOs: 66, 68, 71, 77, 74, 77, 93, 74, 105, 108, 111; or variant sequences thereof differing by one or two amino acids or having at least or about 70% sequence identity; (g) an antigen binding protein comprising a CDR3 amino acid sequence or a sequence selected from the group consisting of SEQ ID NOs: 80, 83, 113, 85, 87, 115, 117, 122, 125, 128; or a variant sequence thereof differing by one or two amino acids or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f).

23. SEQ ID NOs: 78, 79, 80, 64, 65, 66; b. SEQ ID NOs: 81, 112, 80, 88, 65, 66; c. SEQ ID NOs: 81, 79, 80, 64, 67, 68; d. SEQ ID NOs: 82, 79, 83, 39, 70, 71; e. SEQ ID NOs: 81, 79, 113, 89, 90, 77; f. SEQ ID NOs: 81, 84, 85, 72, 73, 74; g. SEQ ID NOs: 86, 79, 87, 75, 76, 77; h. SEQ ID NOs: 114, 79, 115, 91, 92, 93; i. SEQ ID NOs: 81, 116, 117, 94, 73, 74; j. SEQ ID NOs: 81, 118, 117, 95, 96, 74; k. SEQ ID NOs: 81, 119, 117, 97, 98, 74; SEQ ID NOs: 81, 118, 85, 99, 100, 74; m. SEQ ID NOs: 81, 129, 117, 101, 102, 74; n. SEQ ID NOs: 120, 121, 122, 103, 104, 105; o. SEQ ID NOs: 123, 124, 125, 106, 107, 108; and p. SEQ ID NOs: 126, 127, 128, 109, 110, 111 An antigen binding protein comprising six CDR amino acid sequences selected from the group consisting of:

24. a. a heavy chain variable region amino acid sequence shown in Table B or a sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 2, 34, 36, 38, and 40, or a variant sequence thereof differing by one or two amino acids or having at least or about 70% sequence identity; or b. a light chain variable region amino acid sequence shown in Table B or a sequence selected from the group consisting of SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% sequence identity; or c. Both (a) and (b) 1. An antigen-binding protein comprising:

25. 25. The antigen binding protein of claim 24, wherein the variant sequence has at least about 85% sequence identity, or about 90% or about 95% sequence identity.

26. a. SEQ ID NOs: 10 and 11; b. SEQ ID NOs: 12 and 13; c. SEQ ID NOs: 14 and 15; d. SEQ ID NOs: 16 and 17; e. SEQ ID NOs: 18 and 19; f. SEQ ID NOs: 20 and 21; g. SEQ ID NOs: 22 and 23; h. SEQ ID NOs:24 and 25; i. SEQ ID NOs:26 and 27; j. SEQ ID NOs:28 and 29; k. SEQ ID NOs: 30 and 31; l. SEQ ID NOs: 32 and 33; m. SEQ ID NOs: 34 and 35; n. SEQ ID NOs: 36 and 37; o. SEQ ID NOs: 38 and 39; and p. SEQ ID NOs: 40 and 41 An antigen-binding protein comprising a pair of amino acid sequences selected from the group consisting of:

27. a. a heavy chain variable region amino acid sequence shown in Table C, Table D, Table 6, Table 8, Table 9, Table 10, or a sequence selected from the group consisting of SEQ ID NOs: 42, 46, 49, 52, 55, 56, 57, and 131, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% sequence identity; or b. a light chain variable region amino acid sequence shown in Table C, Table D, Table 6, Table 8, Table 9, Table 10, or a sequence selected from the group consisting of SEQ ID NOs: 43, 44, 45, 47, 48, 50, 51, 53, 54, 130, 132, 147, 149, and 150, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% sequence identity; or c. Both (a) and (b) 1. An antigen-binding protein comprising:

28. 28. The antigen binding protein of claim 27, wherein the variant sequence has at least about 85% sequence identity.

29. 28. The antigen binding protein of claim 27, wherein the variant sequence has at least about 90% or about 95% sequence identity.

30. a. SEQ ID NOs: 42 and 43; b. SEQ ID NOs: 42 and 44; c. SEQ ID NOs: 42 and 45; d. SEQ ID NOs: 46 and 47; e. SEQ ID NOs: 46 and 48; f. SEQ ID NOs: 131 and 149; g. SEQ ID NOs: 131 and 150; h. SEQ ID NOs:52 and 53; i. SEQ ID NOs:52 and 54; j. SEQ ID NOs: 55 and 53; k. SEQ ID NOs: 55 and 54; l. SEQ ID NOs: 56 and 53; m. SEQ ID NOs: 56 and 54; n. SEQ ID NOs: 57 and 50; o. SEQ ID NOs: 57 and 51; p. SEQ ID NOs: 49 and 50; q. SEQ ID NOs: 49 and 51; r. SEQ ID NOs: 42 and 130; s. SEQ ID NOs: 131 and 147; and t. SEQ ID NOs: 131 and 132 An antigen-binding protein comprising a pair of amino acid sequences selected from the group consisting of:

31. 2. The antigen binding protein of any one of the preceding claims, comprising an Fc polypeptide comprising non-fucosylated glycans.

32. A bispecific antigen-binding protein that binds to CLDN18.2 and a second antigen, wherein the antigen-binding protein that binds to CLDN18.2 is an antigen-binding protein according to any one of the preceding claims or as described herein.

33. The antigen-binding protein that binds to CLDN18.2 is a. a heavy chain variable region amino acid sequence shown in Table B, Table C, Table D, Table 6, Table 8, Table 9, Table 10, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% sequence identity; b. a light chain variable region amino acid sequence shown in Table B, Table C, Table D, Table 6, Table 8, Table 9, Table 10, or a variant sequence thereof that differs by one or two amino acids or has at least or about 70% sequence identity; c. Both (a) and (b) 33. The bispecific antigen-binding protein of claim 32, comprising:

34. 34. The bispecific antigen-binding protein of claim 33, wherein the variant sequences have at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

35. 35. The bispecific antigen-binding protein of any one of claims 32 to 34, comprising an Fc polypeptide.

36. 36. The bispecific antigen-binding protein of any one of claims 32 to 35, comprising an Fc polypeptide comprising a non-fucosylated glycan.

37. 37. The bispecific antigen-binding protein of any one of claims 32 to 36, comprising an antigen-binding fragment of an antibody specific for the second antigen.

38. 38. The bispecific antigen-binding protein of any one of claims 32 to 37, wherein said second antigen is a cell surface protein expressed by T cells, optionally a component of the T cell receptor (TCR), such as CD3.

39. 39. The bispecific antigen-binding protein of any one of claims 32 to 38, wherein the second antigen is CD3.

40. 40. The bispecific antigen-binding protein of any one of claims 32 to 39, wherein the second antigen is CD3E.

41. 41. The bispecific antigen-binding protein of any one of claims 32 to 40, wherein the second antigen is a costimulatory molecule that supports T cell activation, such as CD40 or 4-1BB (CD137).

42. 38. The bispecific antigen-binding protein of any one of claims 32 to 37, wherein the second antigen is an Fc receptor, optionally an Fc gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor.

43. 43. The bispecific antigen-binding protein of claim 42, wherein the Fc receptor is CD64 (Fc-gamma RI), CD32 (Fc-gamma RIIA), CD16A (Fc-gamma RIIIA), CD16b (Fc-gamma RIIIb), FcεRI, CD23 (Fc-epsilon RII), CD89 (Fc-epsilon RI), Fcα / μR, or FcRn.

44. 44. The bispecific antigen-binding protein of claim 43, wherein the Fc receptor is CD16A.

45. 38. The bispecific antigen binding protein of any one of claims 32 to 37, wherein the second antigen is an immune checkpoint molecule, such as a protein involved in an immune checkpoint pathway, optionally A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA, or SIGLEC7.

46. 46. ​​The bispecific antigen binding protein of claim 45, wherein said immune checkpoint molecule is PD-1, LAG3, TIM3, or CTLA4.

47. Any scFv, Fab, or F(ab) of the CLDN18.2 antibodies of the present disclosure (e.g., Table B, Table C, Table D, Table 6, Table 8, Table 9, or Table 10). ２ 47. The bispecific antigen-binding protein of any one of claims 32 to 46, comprising:

48. 48. The bispecific antigen binding protein of any one of claims 32 to 47, comprising an antigen binding protein comprising a sequence as shown in Table 11 or SEQ ID NO: 143, 144, 145, or 146, or a variant sequence thereof differing by 1 to 5 amino acids or having at least or about 70% sequence identity.

49. 49. The bispecific antigen-binding protein of claim 48, wherein the variant sequences have at least or about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

50. 50. The bispecific antigen-binding protein of any one of claims 32 to 49, comprising the structure of a Nanobody, a Diabody, a BiTE®, a DART, a TandAb, a CrossMab, or an HSAbody.

51. 13. An antigen-binding protein or a bispecific antigen-binding protein according to any one of the preceding claims or a conjugate comprising such as described herein.

52. 52. The conjugate of claim 51 , wherein the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO: 42 and SEQ ID NO:

45.

53. 53. The conjugate of claim 51 or 52, comprising a cytotoxic or chemotherapeutic agent.

54. 54. The conjugate of claim 53, wherein the chemotherapeutic agent is an antimitotic agent that inhibits cell division by inhibiting tubulin polymerization.

55. 55. The conjugate of claim 54, wherein the antimitotic agent is an auristatin.

56. 56. The conjugate of claim 55, wherein the auristatin is MMAE.

57. 57. The conjugate of any one of claims 51 to 56, wherein the agent is conjugated to the antigen binding protein via a cleavable linker.

58. 58. The conjugate of claim 57, wherein the cleavable linker is MC-VC-PAB.

59. The conjugate of any one of claims 51 to 58, wherein the antigen-binding protein is an antibody.

60. 60. The conjugate of claim 59, wherein the antibody is a monoclonal antibody, optionally wherein the monoclonal antibody is an IgG antibody.

61. The conjugate of claim 59 or 60, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

62. 62. The conjugate of any one of claims 51 to 61, wherein the average number of units of said agent conjugated per antigen binding protein is in the range of 1 to 8, preferably said average number of units of said agent conjugated per antigen binding protein is in the range of 3 to 8.

63. The conjugate according to any one of claims 51 to 62, wherein the conjugate is a heterologous conjugate.

64. The conjugate according to any one of claims 51 to 62, wherein the conjugate is a homogeneous conjugate.

65. 65. The conjugate of any one of claims 51 to 62 and 64, wherein the agent is conjugated at a specific site on the antigen binding protein.

66. 66. The conjugate of claim 65, wherein the specific site is an unpaired cysteine ​​residue.

67. 67. The conjugate of any one of claims 51 to 66, wherein the conjugate comprises a polypeptide comprising the amino acids set forth in SEQ ID NO: 42 and SEQ ID NO: 45 conjugated to MC-VC-PAB-MMAE.

68. 13. An antigen-binding protein or a bispecific antigen-binding protein according to any one of the preceding claims or a fusion protein comprising such as described herein.

69. 68. A nucleic acid comprising a nucleotide sequence encoding an antigen binding protein according to any one of claims 1 to 31, a bispecific antigen binding protein according to any one of claims 32 to 50, a conjugate according to any one of claims 51 to 67, or a fusion protein according to claim 68.

70. 70. A vector comprising the nucleic acid of claim 69.

71. 71. A host cell comprising the nucleic acid of claim 69 or the vector of claim 70.

72. 72. A method for producing an antigen binding protein or a bispecific antigen binding protein that binds to Claudin 18.2 (CLDN18.2) protein, the method comprising: (i) culturing a host cell of claim 71 in a cell culture medium, the host cell comprising a nucleic acid comprising a nucleotide sequence encoding the antigen binding protein or bispecific antigen binding protein of any one of the preceding claims; and (ii) harvesting the antigen binding protein or bispecific antigen binding protein from the cell culture medium.

73. 73. A method for producing a fusion protein comprising an antigen-binding protein or a bispecific antigen-binding protein that binds to claudin 18.2 (CLDN18.2) protein, comprising: (i) culturing a host cell of claim 71 in a cell culture medium, the host cell comprising a nucleic acid comprising a nucleotide sequence encoding the fusion protein of claim 69; and (ii) harvesting the fusion protein from the cell culture medium.

74. 70. A method of producing a pharmaceutical composition comprising combining an antigen binding protein according to any one of claims 1 to 31, a bispecific antigen binding protein according to any one of claims 32 to 50, a conjugate according to any one of claims 51 to 67, a fusion protein according to claim 68, a nucleic acid according to claim 69, a vector according to claim 70, a host cell according to claim 71, or combinations thereof, and a pharma- ceutically acceptable carrier, diluent or excipient.

75. 70. A pharmaceutical composition comprising an antigen binding protein according to any one of claims 1 to 31, a bispecific antigen binding protein according to any one of claims 32 to 50, a conjugate according to any one of claims 51 to 67, a fusion protein according to claim 68, a nucleic acid according to claim 69, a vector according to claim 70, a host cell according to claim 71, or a combination thereof, and a pharma- ceutically acceptable carrier, diluent or excipient.

76. A method for treating a subject having a cancer that expresses CLDN18.2, comprising administering to the subject a pharmaceutical composition of claim 75 in an amount effective to treat the cancer.

77. 76. A method of inhibiting tumor growth in a subject, comprising administering to the subject a pharmaceutical composition of claim 75 in an amount effective to inhibit tumor growth.

78. 76. A method for reducing tumor size in a subject, comprising administering to the subject a pharmaceutical composition of claim 75 in an amount effective to reduce tumor size.

79. 76. A method for preventing recurrence of cancer in a subject, comprising administering to the subject the pharmaceutical composition of claim 75 in an amount effective to prevent recurrence of cancer.

80. A method for treating cancer in a subject diagnosed as a low overexpressor of CLDN18.2, comprising administering to the subject a pharmaceutical composition of claim 75 in an amount effective to prevent recurrence of the cancer.

81. 82. The method of any one of claims 76-81, wherein said administering induces apoptosis in tumor cells.

82. The method of any one of claims 76 to 82, wherein the administering induces apoptosis in cells expressing CLDN18.

2.

83. 10. A method for detecting Claudin 18.2 (CLDN18.2) in a sample, comprising contacting the sample with an antigen binding protein according to any one of claims 1 to 31, a bispecific antigen binding protein according to any one of claims 32 to 50, a conjugate according to any one of claims 51 to 67, or a fusion protein according to claim 68, and assaying for an immune complex comprising the antigen binding protein, conjugate or fusion protein bound to CLDN18.

2.

84. 13. A method of diagnosing claudin 18.2 (CLDN18.2) positive cancer in a subject, comprising contacting a biological sample comprising cells or tissue obtained from the subject with an antigen binding protein of any one of claims 1 to 31, a bispecific antigen binding protein of any one of claims 32 to 50, a conjugate of any one of claims 51 to 67, or a fusion protein of claim 68, and assaying for an immune complex comprising the antigen binding protein, conjugate or fusion protein bound to CLDN18.2.

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