Anti-claudin 18.2 antibody, and its manufacturing method and use.

Abstract

An isolated monoclonal antibody (mAb) or antigen-binding fragment thereof having binding specificity to CLDN18, wherein the isolated mAb or antigen-binding fragment selectively binds to CLDN18.2 with measurable affinity and avidity, but does not bind to CLDN18.1.

Description

[Technical Field]

[0001] Cross-reference of related applications This application asserts the benefit as of the filing date of U.S. Provisional Application No. 63 / 468,198, filed on 22 May 2023 pursuant to 35 U.S. Code § 119(e), the entirety of which disclosure is incorporated herein by reference.

[0002] This disclosure relates to the technology of antibody cancer therapies in general, and more specifically to anti-claudin 18.2 antibodies. [Background technology]

[0003] Gastric and pancreatic cancers are among the most common malignancies worldwide. Gastric cancer is a leading cause of death in many parts of Asia, Central and South America, and Eastern Europe. In 2020, more than one million new cases were reported, ranking it as the fifth most common type of cancer. 1 In the United States, 26,380 cases were diagnosed in 2022, with an estimated 11,090 deaths. Between 2011 and 2017, the overall five-year survival rate was 32%. 2 Pancreatic cancer is the 12th most common cancer in the world, with over 495,000 cases reported in 2020. 3 In the United States, pancreatic cancer is more common than stomach cancer, accounting for 3% of all cancers and 7% of cancer deaths. 4 Depending on the stage of disease progression after diagnosis, the prognosis is poor, with a 5-year survival rate of only 11%. 2 Although targeted therapies are advancing, these malignancies clearly represent areas of unmet medical needs.

[0004] Overexpression of claudin 18.2 (also known as CLDN18.2) is associated with several human malignancies, including gastric and pancreatic cancer. CLDN18.2 is a selective marker of gastric mucosal epithelial cells and, during normal expression, is embedded within tight junctions in healthy tissue. 5However, during the carcinogenesis process, the breakdown of the epithelial cell barrier exposes the CLDN18.2 epitope, making it accessible to targeted therapeutic agents. 5-7 Due to this characteristic (distinction), CLDN18.2 is a unique and gastric cancer-specific target for immunotherapy. In addition to gastric cancer, overexpression of CLDN18.2 has been detected in several primary cancers, including breast cancer, colon cancer, esophageal cancer, biliary tract cancer, and pancreatic cancer. 6 CLDN18.2 is not detected in normal pancreatic tissue, but in cases of primary pancreatic ductal adenocarcinoma, 59.2% of cases had cells showing positive expression of CLDN18.2. 5 Several antibodies targeting CLDN18.2 for cancer immunotherapy are under development, but none have been approved for clinical use. 5 Therefore, there remains a need for the development of highly specific anti-CLDN18.2 antibodies for treating malignant tumors such as gastric cancer and pancreatic cancer.

Summary of the Invention

[0005] The present disclosure generally relates to the technical field of antibody therapeutic agents, and more specifically to antibodies against specific epitopes of claudin 18, including CLDN18.1 and / or CLDN18.2.

[0006] In one aspect, the present disclosure provides an isolated monoclonal antibody (mAb) or an antigen-binding fragment thereof having binding specificity for CLDN18.1, CLDN18.2, or a combination thereof. In one embodiment, the mAb or its antigen-binding fragment has binding affinity for CLDN18.1. In one embodiment, the mAb or its antigen-binding fragment has binding affinity for CLDN18.2. In one embodiment, the mAb or its antigen-binding fragment has binding affinity for CLDN18.1 and CLDN18.2.

[0007] In one embodiment, the isolated mAb or its antigen-binding fragment contains an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 2, 4, 6, 8, 10, or 12. In one embodiment, the isolated mAb or its antigen-binding fragment contains an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 14 or 16. In one embodiment, the isolated mAb or its antigen-binding fragment contains an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 18, 20, 22, 24, 26, or 28. In one embodiment, the isolated mAb or its antigen-binding fragment contains an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 30, 32, 34, or 36. In one embodiment, the isolated mAb or its antigen-binding fragment contains an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, or 52. In one embodiment, the isolated mAb or its antigen-binding fragment contains an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 54 or 56.

[0008] CLDN18.2 expression is associated with several human malignancies, including gastric and pancreatic cancer. This disclosure further discloses the development of anti-CLDN18.2 antibodies for the treatment of cancer.

[0009] In another embodiment, the Disclosure provides an isolated mAb or antigen-binding fragment thereof having binding specificity to CLDN18, comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56.

[0010] In one embodiment, the isolated mAb or its antigen-binding fragment selectively binds to CLDN18.2 but not to CLDN18.1. In one embodiment, the isolated mAb or its antigen-binding fragment has a binding affinity to CLDN18.2 with a KD value of 5, 3, 2, 1.5, 1.45, 1.4, 1, or 0.8 nM or less. In one embodiment, the isolated mAb or its antigen-binding fragment has a binding avidity to CLDN18.2 with a KD value of 0.5, 0.4, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.2, or 0.18 nM or less. In one embodiment, the binding ratio of the isolated mAb or its antigen-binding fragment to CLDN18.2, compared to the binding ratio to CLDN18.1, is at least 4, 5, 8, 10, 15, 20, 30, or 40. In one embodiment, the binding ratio of the isolated mAb or its antigen-binding fragment to CLDN18.2, compared to the binding ratio to CLDN18.1, is approximately 4 to 10, approximately 3 to 12, approximately 10 to 25, or approximately 12 to 22.

[0011] In one embodiment, the isolated mAb or antigen-binding fragment further comprises a human framework region.

[0012] In one embodiment, the isolated mAb may be a humanized antibody, a chimeric antibody, or a recombinant antibody. In one embodiment, the isolated mAb is IgG. In one embodiment, the isolated mAb may be a bispecific antibody, a tripspecific antibody, or a multispecific antibody.

[0013] In one embodiment, the antigen-binding fragment may include Fv, Fab, F(ab')2, scFv, or scFv2 fragments.

[0014] In one embodiment, the Disclosure provides an IgG monohelic acid chain for an isolated mAb or its antigen-binding fragment, comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, or 54.

[0015] In one embodiment, the Disclosure provides kappa light chains for isolated mAbs or antigen-binding fragments disclosed herein, comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, or 56.

[0016] In one embodiment, the Disclosure provides a variable light chain domain for an isolated mAb or antigen-binding fragment disclosed herein, comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, or 56.

[0017] In one embodiment, the Disclosure provides a variable heavy chain domain for an isolated mAb or antigen-binding fragment disclosed herein, comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, or 54.

[0018] In one embodiment, the present disclosure provides an isolated mAb having binding specificity for CLDN18 or an antigen-binding fragment thereof. In one embodiment, the isolated mAb or antigen-binding fragment thereof comprises a variable heavy chain domain and a variable light chain domain having complementarity-determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of SEQ ID NOs: 57, 58, 59, 60, 61, and 62; 63, 64, 65, 66, 67, and 68; 69, 70, 71, 72, 73, and 74; 75, 76, 77, 78, 79, and 80; or 81, 82, 83, 84, 85, and 86.

[0019] In a further aspect, the present disclosure provides an isolated nucleic acid encoding an isolated mAb or antigen-binding fragment, IgG1 heavy chain, kappa light chain, variable light chain domain, or variable heavy chain domain disclosed herein. In one embodiment, the isolated nucleic acid has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55.

[0020] In a further aspect, the present disclosure provides an expression vector comprising an isolated nucleic acid disclosed herein, wherein the vector is capable of being expressed intracellularly.

[0021] In a further aspect, the present disclosure provides a host cell comprising a nucleic acid disclosed herein, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

[0022] In a further aspect, the present disclosure provides a method for manufacturing an antibody, comprising culturing a host cell disclosed herein under conditions for producing an antibody.

[0023] In a further embodiment, the disclosure provides an immunoconjugate comprising the isolated mAb or its antigen-binding fragment, conjugated to a drug unit via a linker. In one embodiment, the linker comprises a covalent bond selected from ester bonds, ether bonds, amine bonds, amide bonds, disulfide bonds, imide bonds, sulfone bonds, phosphate bonds, phosphate ester bonds, peptide bonds, hydrazone bonds, or combinations thereof.

[0024] In one embodiment, the drug unit comprises a cytotoxic agent, an immunomodulatory reagent, or a combination thereof. In one embodiment, the cytotoxic agent is selected from proliferation inhibitors, tubulin binders, DNA intercalators, DNA alkylating agents, enzyme inhibitors, immunomodulators, antimetabolites, chemotherapeutic agents derived from radioisotopes, or a combination thereof. In one embodiment, the cytotoxic agent is selected from calicheamicin, ozogamicin, monomethyl auristatin E, emtansine derivatives, or a combination thereof. In one embodiment, the immunomodulatory reagent activates or inhibits immune cells, T cells, NK cells, B cells, macrophages, or dendritic cells.

[0025] In a further embodiment, the Disclosure provides a pharmaceutical composition comprising the isolated mAb or its antigen-binding fragment or the immunoconjugate, and a pharmaceutically acceptable carrier.

[0026] In one embodiment, the pharmaceutical composition further comprises a radioisotope, a radionuclide, a toxin, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

[0027] In one embodiment, the therapeutic agent includes an anti-estrogen agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA, or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof.

[0028] In further embodiments, the Disclosure provides a method for treating a subject having cancer. In one embodiment, the method comprises administering to the subject an effective amount of the isolated mAb or its antigen-binding fragment. In one embodiment, the method comprises administering to the subject an effective amount of the immunoconjugate disclosed herein. In one embodiment, the method comprises administering to the subject an effective amount of the pharmaceutical composition disclosed herein.

[0029] In one embodiment, the cancer comprises cells expressing claudin 18.2. In one embodiment, the cancer includes colorectal cancer, pancreatic cancer, esophageal cancer, nasopharyngeal cancer, anal cancer, rectal cancer, gastric cancer, or bladder cancer.

[0030] In one embodiment, the method for treating the cancer further comprises co-administering an effective amount of a therapeutic agent. In one embodiment, the therapeutic agent includes an antibody, a chemotherapeutic agent, an enzyme, an anti-estrogen agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA, or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof.

[0031] In one embodiment, the subject is a human being.

[0032] In a further embodiment, the Disclosure provides a solution comprising an effective concentration of the isolated mAb or its antigen-binding fragment, wherein the solution is plasma in the subject. In one embodiment, the Disclosure provides a solution comprising an effective concentration of the immunoconjugate disclosed herein. In one embodiment, the solution is plasma in the subject. [Brief explanation of the drawing]

[0033] The above and other features of this disclosure will become more apparent upon consideration of the following description and the attached claims in conjunction with the attached drawings. While understanding that these drawings depict only a few embodiments of this disclosure and should not be considered limiting, this disclosure is described with additional specificity and detail using the attached drawings. [Figure 1] This shows the alignment of the extracellular claudin domains (ECD1 and ECD2) in the full-length amino acid sequences encoding claudin 18.1 and claudin 18.2 in humans and cynomolgus monkeys. [Figure 2] This shows the detection of anti-claudin 18.2 antibody in B cell culture screening using direct sandwich ELISA. [Figure 3] This shows the detection of anti-claudin 18.2 antibodies in B cell culture screening by flow cytometry using 293HEK transfected with claudin 18.2 or claudin 18.1 expression vectors. [Figure 4] This report describes the characterization of chimeric anti-claudin 18.2 antibodies by flow cytometry using ExpiCHO cells transfected with claudin 18.2 or claudin 18.1 expression vectors. [Figure 5] This paper presents the characterization of the binding affinity of chimeric anti-claudin 18.2 antibodies, 5208H10(5A) and 5103F3(5B), using biolayer interferometry followed by fitting and calculations using manufacturer-provided software. [Figure 6] This paper demonstrates the characterization of the binding avidity of a chimeric anti-claudin 18.2 antibody using biolayer interferometry followed by fitting and calculations using manufacturer-provided software. [Figure 7] This paper describes the characterization of the CLDN18.2 specificity of a humanized anti-claudin 18.2 antibody using flow cytometry and ExpiCHO cells transiently expressing the BCMA-claudin 18 fusion protein. [Figure 8]This paper describes the characterization of the CLDN18.2 specificity of a humanized anti-claudin 18.2 antibody using direct ELISA with recombinant protein VLP. [Figure 9] This shows the characterization of epitope binning of an anti-claudin 18.2 antibody using biolayer interferometry (OCTET). [Figure 10] This shows the lysis of CLDN18.2-expressing CHO cells (CHO-Claudin18.2) in response to dose-increasing of humanized anti-claudin 18.2 antibody in a complement-dependent cell-mediated cytotoxicity (CDC) assay. [Figure 11] This shows the lysis of claudin 18.2-expressing CHO cells (CHO-Claudin18.2) in response to dose-increasing of humanized anti-claudin 18.2 antibody in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, in the presence of donor NK cells (5:1 ratio to target cells) as effector cells. [Figure 12] This paper presents the characterization of cross-specific interactions between human, rat, and mouse-derived anti-claudin 18.2 antibodies using flow cytometry. [Figure 13] This paper describes the characterization of lysosomal transport of anti-claudin 18.2 antibodies at concentrations of 5 nM and 10 nM, using time-series live-cell fluorescence microscopy. [Figure 14] Sequence alignment between 5103F3 BSM and the comparison antibody (14A); identity % of all CDRs and framework only (14B, upper and lower panels, respectively), and identity % of the entire variable region (14C) are shown. [Modes for carrying out the invention]

[0034] This disclosure describes the production, characterization, and application of anti-CLDN18.2 antibodies for the treatment of malignant tumors, such as gastrointestinal tumors. References to the accompanying drawings are made in the following detailed description, and these drawings form part of this specification. In the drawings, similar symbols identify typically similar components unless the context indicates otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to limit the scope. Other embodiments may be utilized, and other modifications may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily apparent that the aspects of this disclosure generally described herein and shown in the drawings can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly intended herein.

[0035] Claudins (also known as CLDNs) are a family of proteins that play a direct role in the formation of tight junctions. Tight junctions form intercellular space barriers that control the flow of molecules between cells. CLDNs are crucial for the formation of these barriers and help maintain the polarity of the epithelial cell layer. 8 CLDN is a tetraspanin protein composed of four transmembrane domains, intracellular N-terminus and C-terminus, and two extracellular domain loops. 8 CLDN is expressed in normal tissues, but changes in its function and expression are associated with cancer development. 5-6,8 Of particular interest is claudin 18.2 (CLDN18.2), which is associated with several human malignancies, including gastric and pancreatic cancer. 5-6 The human CLDN18 gene contains six exons and five introns. Alternative splicing of exons 1a and 1b forms two isoforms, CLDN18.1 and CLDN18.2. 9 These splice variants are limited to different tissues; CLDN18.1 is primarily expressed in normal lung tissue, while CLDN18.2 is expressed in gastric tissue. 5-6The sequence of CLDN18.1 is almost identical to that of CLDN18.2, with only a difference of eight amino acids in the extracellular loop D1 (ECD in Figure 1). This high degree of sequence similarity makes the discovery of CLDN18.2-specific antibodies extremely difficult.

[0036] Several antibodies targeting CLDN18.2 are under development for cancer immunotherapy, including monospecific monoclonal antibodies (mAbs), bispecific antibodies (BsAbs), chimeric antigen receptor T cells (CAR-T), and antibody-drug conjugates (ADCs). Zolbetuximab (Astellas Pharma) is a mouse chimeric mAb that specifically binds to CLDN18.2 and has demonstrated safety and efficacy in patients with HER2-positive advanced gastric cancer. 5 TST001 (Mabspace Bio-Sciences) is a humanized mAb that showed activity equivalent to zolbetuximab. 5 While most antibodies under development are mAbs, BsAbs and ADCs, including AMG-910 (Amgen, bispecific T-cell engagement) and CMG901 (Keymed Biosciences, ADC), have also proven to be effective treatments for both gastric and pancreatic cancer. 5 However, there are no CLDN18.2-targeted antibodies approved for clinical use.

[0037] This disclosure provides, in particular, isolated antibodies, methods for producing such antibodies, monoclonal and / or recombinant monospecific antibodies, pharmaceutical compositions containing antibodies, methods for producing monoclonal and / or recombinant monospecific antibodies, antibodies and compositions, and methods for treating cancer using the antibodies and compositions disclosed herein. Specifically, this disclosure provides isolated monospecific monoclonal antibodies (mAbs) or antigen-binding fragments thereof having binding specificity to human claudin 18.2, wherein the isolated mAb or antigen-binding fragment comprises an amino acid sequence having identity with a sequence selected from SEQ ID NOs: 2, 4, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56.

[0038] As used herein, the terms “a,” “an,” and “the” are defined to mean “one or more,” and include the plural form unless the context is appropriate.

[0039] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and are defined to mean biomolecules composed of amino acids linked by peptide bonds.

[0040] The term "antigen" refers to an entity or fragment thereof that can induce an immune response in living organisms, particularly animals, and more specifically mammals, including humans. This term includes immunogens and the regions of the organism involved in antigenicity or antigenic determinants.

[0041] The terms “antigen or epitope binding portion or fragment,” “variable region,” “variable region sequence,” or “binding domain” refer to fragments of an antibody that can bind to an antigen (such as Claudin 18.2 in this application). These fragments may perform antigen-binding functions and additional functions of an intact antibody. Examples of binding fragments include, but are not limited to, single-chain Fv fragments (scFv) consisting of a variable light chain (VL) domain and a variable heavy chain (VH) domain of one arm of an antibody linked in a single polypeptide chain by a synthetic linker, or monovalent fragments called Fab fragments consisting of VL, light chain constant (CL), VH, and heavy chain constant 1 (CH1) domains. Antibody fragments may also be smaller subfragments and may consist of small domains such as a CDR3 region derived from a single CDR domain, particularly either the VL and / or VH domain (see, for example, Beiboer et al., J. Mol. Biol. 296:833-49 (2000)). Antibody fragments are produced using conventional methods known to those skilled in the art. Antibody fragments can be screened for usefulness using the same techniques employed for intact antibodies.

[0042] The “antigen or epitope binding portion or fragment,” “variable region,” “variable region sequence,” or “binding domain” may be derived from the antibody of this disclosure by a number of known techniques. For example, a purified monoclonal antibody can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. Papain digestion of the antibody produces two identical antigen-binding fragments called “Fab” fragments, each having a single antigen-binding site, and the remaining “Fc” fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites and still capable of crosslinking antigens. A suitable fraction containing the Fab fragments can then be recovered and concentrated by membrane filtration or the like. For further explanation of general techniques for isolating antibody active fragments, see, for example, Khaw, BA et al., J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al., Methods Enzymology, 121:663-69, Academic Press, 1986.

[0043] The term "antibody" is used in its broadest sense and specifically includes single monoclonal antibodies and / or recombinant antibodies (including agonist and antagonist antibodies), antibody compositions having multiepitope specificity, and antibody fragments (e.g., Fab, F(ab')2, and Fv) insofar as they exhibit the desired biological activity. In some embodiments, the antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a single-chain antibody, a multispecific or multi-effective antibody, a human or humanized antibody, and / or an active fragment thereof. Examples of active fragments of molecules that bind to known antigens include Fab, F(ab')2, scFv, and Fv fragments, which include products of Fab immunoglobulin expression libraries, as well as epitope-binding fragments of any of the antibodies and fragments described above.

[0044] The term "Fv" refers to the smallest antibody fragment containing the complete antigen recognition and binding site. This region consists of a dimer formed by the strong, non-covalent association of one heavy-chain variable domain and one light-chain variable domain. It is in this arrangement that the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind to an antigen, albeit with lower affinity than the entire binding site.

[0045] In some embodiments, an antibody may include an immunoglobulin molecule and a molecule that includes an immunoactive portion of the immunoglobulin molecule, i.e., a binding site, which can bind immunospecifically to an antigen. A typical antibody refers to a heterotetrameric protein typically composed of two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable domain (abbreviated as VH) and three heavy chain constant domains (abbreviated as CH1, CH2, and CH3). Each light chain consists of a light chain variable domain (abbreviated as VL) and a light chain constant domain (abbreviated as CL). The light chains of antibodies (immunoglobulins) derived from any vertebrate species can be assigned to one of two distinct types called kappa and lambda, based on the amino acid sequence of their constant domains. The VH and VL regions can be further subdivided into a hypervariable complementarity-determining region (CDR) and a more conserved region called the framework region (FR). Each variable domain (either VH or VL) typically consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. Within the variable regions of the light and heavy chains are binding regions that interact with the antigen.

[0046] Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional arrangements of the different classes of immunoglobulins are well known.

[0047] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for any spontaneous mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed to a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier “monoclonal” indicates the nature of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by a specific method. For example, monoclonal antibodies used in accordance with this disclosure may be produced by the hybridoma method first described by Kohler & Milstein, Nature, 256:495 (1975), or by the recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567). "Recombinant" means that antibodies are produced in exogenous host cells using recombinant nucleic acid technology.

[0048] Monoclonal antibodies can be produced using a variety of methods, including, but not limited to, mouse hybridomas, phage display, recombinant DNA, direct molecular cloning of antibodies from primary B cells, and antibody discovery methods (see Siegel. Transfus. Clin. Biol. 2002; Tiller. New Biotechnol. 2011; Seeber et al. PLOS One. 2014). Monoclonal antibodies include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to a corresponding sequence in an antibody from a different species or belonging to a different antibody class or subclass, and fragments of such antibodies, insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855

[1984] ).

[0049] The term "humanized antibody" refers to a type of manipulated antibody in which its CDR is derived from a non-human donor immunoglobulin, and the remaining immunoglobulin-derived portion of the molecule is derived from one (or more) human immunoglobulins. In addition, framework supporting residues may be modified to preserve binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art [see, for example, Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson et al., Bio / Technology, 9:421 (1991)].

[0050] The terms “antigen or epitope binding portion or fragment,” “variable domain,” “variable region,” “variable region sequence,” or “binding domain” refer to fragments of an antibody that can bind to an antigen (such as EGFR and HER3 in this application). An antigen-binding fragment (Fab) is a region on an antibody that binds to an antigen (Fab region). These fragments can perform antigen-binding functions and additional functions for intact antibodies. Examples of binding fragments include, but are not limited to, single-chain Fv fragments (scFv) consisting of a light chain variable (VL) domain and a heavy chain variable (VH) domain on one arm of an antibody linked in a single polypeptide chain by a synthetic linker, or monovalent Fab fragments consisting of VL, light chain constant (CL), VH, and heavy chain constant 1 (CH1) domains.

[0051] The “antigen or epitope binding portion or fragment,” “variable region,” “variable region sequence,” or “binding domain” may be derived from the antibody of this disclosure by some known techniques. For example, a purified monoclonal antibody can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. Papain digestion of the antibody produces two identical antigen-binding fragments called “Fab” fragments, each having a single antigen-binding site, and the remaining “Fc” fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites and still capable of crosslinking antigens. A suitable fraction containing the Fab fragments can then be recovered and concentrated by membrane filtration or the like. For further explanation of general techniques for isolating antibody active fragments, see, for example, Khaw, BA et al., J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al., Methods Enzymology, 121:663-69, Academic Press, 1986.

[0052] The term "Octet" refers to biolayer interferometry (BLI), a label-free method for measuring biomolecular interactions. Dip and Read technology eliminates the need for microfluidics and means that antibodies do not need to be purified to obtain valuable antigen-binding and specificity information.

[0053] This disclosure may be more readily understood by referring to the following detailed descriptions of the specific embodiments and examples contained herein. Although this disclosure has been described with reference to certain details of its specific embodiments, it is not intended that such details should be considered limitations on the scope of this disclosure.

[0054] Examples Example 1: Generation of rabbit antibodies against claudin 18 immunity CLDN18.2-specific antibodies were obtained by immunization of New Zealand white rabbits. The rabbits were divided into two cohorts. Cohort 1 was 1 × 10⁶ during complete Freund's adjuvant. 7The rabbits received primary immunization with 293 human fetal kidney (HEK) cells transfected with claudin 18.2. The 293 HEK cells were transfected with a eukaryotic expression vector encoding full-length human CLDN18.2 (Figure 1, SEQ ID NO: 87). Following the immunization protocol in Table 1, the rabbits then received four additional immunizations at 7-day intervals, each containing a doubling of the cell count using alternating incomplete Freund's adjuvant and Alum CpG2007. After the fourth immunization, the rabbits received seven additional immunizations at 21-day intervals. All additional immunizations contained a doubling of the cell count using alternating incomplete Freund's adjuvant and Alum CpG2007 (except on days 49 and 70). On these days, the rabbits received 25 μg of recombinant human claudin 18.2 (Kactus Biosystems Cat. No. CLD-HE1822) aggregated in virus-like particles (VLPs). Cohort 2 also employed the same immunization strategy, except that the rabbits received a tenfold increase in cell count and 50 μg of recombinant human claudin 18.2 VLP. For both cohorts, whole peripheral blood was collected three weeks after each booster immunization, and PBMCs were isolated and stored in liquid nitrogen for sorting.

[0055] PBMC isolation Diluted blood was overlaid on Lympholyte cell isolation medium (Cedarlane, Cat. No. CL5050) and centrifuged at 2500 rpm for 30 minutes. After centrifugation, the PBMC layer was carefully aspirated and washed. Remaining RBCs in the pellet were lysed using RBC lysis buffer (Qiagen, Cat. No. 158904). After lysis, the cells were washed, and 5 × 10⁴ cells per ml were extracted. 7 The cells were resuspended in 10% DMSO in FBS at a subcellular concentration.

[0056] Isolation of rabbit B cells by FACS: PBMCs were thawed in immunotherapy medium (ICM) and then incubated with an antibody staining mix containing recombinant human CLDN18.2 VLP and reagents specific to rabbit B cell and T cell markers. After incubation, the PBMCs were washed and incubated with fluorescent dye-specific microbeads. B cells were enriched from the PBMCs by magnetically activated cell sorting (MACS) using a QuadroMACS Separator magnet (Miltenyi Biotec, Cat. No. 130-090-976). The cells were then stained with efluor780 viability dye (ebioscience, Cat. No. 65-0865-14), washed, resuspended in buffer, and stored at 4°C until sorting by fluorescence-activated cell sorting (FACS).

[0057] To sort B cells, appropriate compensation was performed using both sorter software and manual adjustment. Gates were adjusted to obtain live IgG-positive rabbit B cells. B cells from the final gate were sorted as single cells into 96-well plates containing pre-prepared B cell culture media including ICM, rabbit splenocyte condition medium, and feeder cells. Up to 30 plates were sorted for each sort. Plates were incubated at 37°C and 5% CO2 for 12 days prior to screening.

[0058] Example 2: Identification of anti-claudin 18.2 antibody Antibody screening using ELISA B cell cultures were screened for IgG secretion and claudin 18.2 binding by direct sandwich ELISA. Plates were coated with 1 μg / ml anti-rabbit IgG Fc fragment (Jackson ImmunoResearch, Cat. No. 111-005-046) or claudin 18.2 VLP (Kactus Biosystems Cat. No. CLD-HE1822). Plates were washed three times with 1X PBST and then blocked with 2% BSA in DPBS. B cell supernatant was collected from the culture plates and used for screening. B cells remaining in the culture flask were stored at -80°C in RNA Later (Invitrogen, Cat. No. AM7021) for RT-PCR amplification of the antibody variable region. After blocking, plates were washed as described above, and B cell supernatant was added to each plate at a 1:10 dilution. After 1 hour incubation, the plates were washed as described above and then incubated with HRP-conjugated anti-Rb IgG secondary antibody (Jackson ImmunoResearch, Cat. No. 111-035-046) for 30 minutes. The plates were washed and then incubated with TMB substrate (ThermoFisher Scientific, Cat. No. 34029). The reaction was stopped by adding a commercially available stop solution (ThermoFisher Scientific, Cat. No. SS04), and the plates were read at 450 nm using a spectrophotometer. Detection of rabbit IgG antibodies specific to Claudin 18.2 was confirmed based on OD values ​​exceeding the negative control of the secondary antibody alone.

[0059] Rabbit IgG antibodies were grouped and plotted in Figure 2. In Sort #1, 17.4% of the selected B cells were IgG-positive. A total of 11 clones specifically bound to claudin 18.2 VLP. In Sort #2, 28.5% of the selected B cells were IgG-positive. A total of 24 clones specifically bound to claudin 18.2 VLP. In Sort #3, 60% of the selected B cells were IgG-positive. A total of 54 clones specifically bound to claudin 18.2 VLP.

[0060] Antibody screening by flow cytometry To confirm specificity, clones were tested for binding to claudin 18.2 and claudin 18.1 expressed on the surface of transfected 293HEK cells. Detection of antibody binding to surface-expressed claudin 18.2 was achieved by flow cytometry. 293HEK cells were transiently transfected with eukaryotic expression vectors encoding human full-length claudin 18.2 and claudin 18.1 (Figure 1, SEQ ID NOs: 87 and 88, respectively). 293HEK cells transfected with unrelated DNA were used as negative controls. Two days after transfection, cells were harvested and stained with cell tracking dye (ThermoFisher Scientific, Cat. No. C34565), yielding approximately 1.5 × 10⁶ cells per well. 5 Cells were seeded individually. B cell supernatant was added to the cells at a 1:5 dilution and incubated at 4°C for 1 hour. The cells were washed and then incubated with Alexa Fluor 647 conjugate anti-rabbit IgG Fc secondary antibody (Jackson ImmunoResearch, Cat. No. 111-605-008) at 4°C for 30 minutes. After one wash, the cells were resuspended in FACS buffer. Binding was determined by flow cytometry using a BD Fortessa flow cytometer. Clones expressing antibodies that bind to claudin 18.2 but not to claudin 18.1 or the negative control were selected for molecular "rescue" of the antibody variable region.

[0061] To identify anti-claudin 18.2 antibodies, B cell clones that bound to surface-expressed claudin 18.1 and claudin 18.2 but not to negative controls were selected for further screening. A representative clone bound to claudin 18.2 but not to claudin 18.1. Binding activity of B cell supernatants obtained from sorts #2 and #3 is shown (Figure 3). One clone that bound to both claudin 18.1 and claudin 18.2 was specifically expressed for use as a screening reagent (5208H10). Selected disclosed antibodies are identified by black arrows (sort #2: 5103F3, sort #3: 5208H10).

[0062] Example 3: Production of rabbit / human chimeric monoclonal antibodies Amplification of the antibody variable region B cell clones identified by ELISA and flow cytometry were thawed from storage at -80°C. Heavy and light chain variable sequences were amplified by multiplex RT-PCR using primers specific to the leader sequences and constant regions of rabbit IgG and rabbit kappa sequences. The amplified products (amplicons) were further amplified by secondary PCR using nested primers containing restriction sites. The heavy and light chain amplicons were cloned into expression vectors containing human IgG1 or human IgK, respectively. After sequence confirmation, the expression plasmids were transiently co-transfected into 293HEK cells to produce rabbit / human chimeric antibodies.

[0063] Characterization of a chimeric anti-claudin 18.2 antibody - Flow cytometry Recombinant antibody supernatants were screened for binding to surface-expressed claudin 18.2 by flow cytometry. ExpiCHO cells were transiently transfected with eukaryotic expression vectors encoding human full-length claudin 18.2 and claudin 18.1 (SEQ ID NOs: 87 and 88, respectively). ExpiCHO cells transfected with unrelated DNA were used as negative controls. Two days after transfection, cells were stained for flow cytometry as described for B cell culture, and the cells were incubated with chimeric antibodies serially diluted from 10 μg / ml to 0.0625 μg / ml.

[0064] As shown in Figure 4, representative antibodies bind to claudin 18.2 with high MFI but not to claudin 18.1 or the negative control. All chimeric antibodies bound to claudin 18.2. Binding to claudin 18.1 was not detected for any of the antibodies except 5208H10. 5208H10, a pan-claudin 18 conjugate, serves as a control for claudin 18.1 expression. Detection of both claudin 18.1 and claudin 18.2 on transiently transfected ExpiCHO supports the specificity of the reported anti-claudin 18.2 clones for claudin 18.2.

[0065] Characterization of Chimeric Anti-Claudin 18.2 Antibodies - OCTET Affinity Recombinant antibody supernatants were validated for claudin 18.2 binding using biolayer interferometry (OCTET) with a ForteBio Octet Red 384 instrument. Antibody (10 μg / ml) was captured on an anti-human Fc biosensor and then associated with recombinant human claudin 18.2-His full-length protein (Acro Biosystems, Cat. No. CL2-H82E3) diluted 1:3 from 300 nM. The sensor was transferred to a well containing kinetic buffer, and the dissociation of the antibody from claudin 18.2 was measured. Curve fitting and affinity calculations were performed using manufacturer-provided software. As shown in Figure 5, the chimeric antibody bound to recombinant human claudin 18.2 with high affinity, exhibiting KD values ​​in the range of 1–3 nM, as determined by biolayer interferometry.

[0066] Characterization of a chimeric anti-claudin 18.2 antibody - OCTET avidity: Biolayer interferometry (i.e., OCTET) was also used to evaluate the avidity of chimeric antibodies against recombinant human claudin 18.2. Biotinylated recombinant human claudin 18.2 (10 μg / ml) was captured on a streptavidin biosensor and then associated with a chimeric antibody diluted 1:3 from 300 nM. The sensor was transferred to a well containing kinetic buffer, and the dissociation of the antibody / claudin 18.2 complex was analyzed. Curve fitting and avidity calculations were performed using manufacturer-provided software. As shown in Figure 6, the avidity of chimeric antibodies against recombinant human claudin 18.2 was in the range of 1–2 nM. Avidity was not evaluated for 5208H10 as it was merely a control reagent.

[0067] Example 4: Production of humanized monoclonal antibodies Humanization of the heavy and light chain variable regions The heavy and light chain variable regions of antibodies were humanized using the "Predict Humanizing Mutations" algorithm in Discovery Studio. Methods selected for humanization included best single mutation (BSM), frequent residue substitution (FRS), or germline transplantation (GS). The DNA sequences encoding the VH and VL regions of the humanized antibodies were cloned into mammalian expression vectors containing the human IgG1 / kappa constant region using gene fragments (Genewiz). The pan-claudin 18 conjugate (5208H10) was not humanized for use as a research tool.

[0068] Protein stability using UPLC-SEC Immediately after the first stage of protein A purification, antibodies were analyzed by analytical SEC using a Waters Acquity UPLC H-Class column equipped with an ACQUITY UPLC® Protein BEH SEC 200A, 4.6 mm × 150 mm, 1.7 μm column. 10 μg of protein was injected, and PBS (125 mM sodium phosphate, 137 mM sodium chloride, pH 6.8) was used as the mobile phase for a 10-minute run at 0.3 ml / min. The protein was buffered with 25 mM sodium acetate, 125 mM NaCl, 10% sucrose, pH 5.5. To test stability over time, antibody samples were injected into the same column approximately 3 and 6 weeks after the initial purification. UPLC-SEC traces were integrated into three categories: %POI (target protein), %HMW (high molecular weight), and %LMW (low molecular weight). Changes in various parameters (POI, HMW, LMW) were calculated based on the difference in integrated area between the initial and final stability points. As summarized in Table 2, the data demonstrate that most humanized anti-claudin 18.2 antibodies are stable for at least 4–6 weeks under these conditions and show only negligible changes in the target protein species over time.

[0069] Example 5: Characterization of humanized antibodies Flow cytometry and OCTET Humanized antibodies were screened by flow cytometry and OCTET as described above. For comparison, anti-CLDN18.2 antibodies included: humanized antibody 5103F3 BSM; pan-claudin 18-conjugated antibody 5208H10 (SI-64CH5); zolbetuximab (SI-64C8); and other positive control antibodies SI-64C9, SI-64C10, and SI-64C11. Specifically, anti-CLDN18.2 antibodies were expressed by transiently transfected ExpiCHO, and their expression levels were scored (Table 3). To evaluate the binding affinity and avidity of anti-CLDN antibodies, KD values ​​were measured using biolayer interferometry, as shown in Table 4. The KD values ​​of the anti-CLDN18.2 antibody showed accumulation within a range of 2x for affinity, between 0.7 nM and 1.45 nM, but the KD values ​​for avidity ranged between 0.22 nM and 5.31 nM, indicating a more than 20-fold improvement in functional affinity of 5103F3 BSM compared to zolbetuximab.

[0070] Verification of claudin 18.2 specificity by flow cytometry To further confirm that the specificity of the representative antibody is against claudin 18.2 and not against claudin 18.1, the BCMA-claudin 18 fusion protein was expressed in transiently transfected ExpiCHO. Flow cytometry was performed as described above. BCMA expression was detected using an anti-BCMA antibody. As shown in Figure 7, detection of BCMA confirmed the expression of both BCMA-claudin 18.1 and BCMA-claudin 18.2. Expression was also confirmed using the pan-claudin 18 conjugate 5208H10. Both controls confirmed that the lack of binding by the humanized antibody was not due to a lack of expression. Clone 5103F3 bound to BCMA-claudin 18.2 but not to BCMA-claudin 18.1, further supporting the specificity for claudin 18.2.

[0071] Verification of claudin 18.2 specificity by ELISA The binding specificity of representative antibodies to claudin 18.2 was further validated by direct sandwich ELISA as described above, with the following modifications: Plates were coated with 1 μg / ml recombinant protein VLPs: claudin 18.2 VLP (Acro Biosystems, Cat. No. CL2-H52P7), claudin 18.1 VLP (Acro Biosystems, Cat. No. CL1-H52P3), or an empty VLP isotype control (Acro Biosystems, Cat. No. VLP-N5213). Antibodies were tested at 10 μg / ml, and binding was detected using an HRP-conjugated anti-Hu IgG secondary antibody (Jackson ImmunoResearch, Cat. No. 309-035-003). The results showed that 5103F3 BSM bound to CLDN18.2-VLP but not to CLDN18.1-VLP or the VLP-only control (Figure 8). The pan-claudin 18 conjugate 5208H8 bound to both CLDN18.1 and CLDN18.2 but not to the VLP-only control. Taken together, the flow cytometry and ELISA data demonstrate the specificity of 5103F3 BSM for claudin 18.2.

[0072] Antibody binning using biolayer interferometry (OCTET) Antibody epitopes were binned by a biolayer interferometry (i.e., OCTET) intandem assay using a ForteBio OCTET Red 384 instrument. Biotinylated recombinant human claudin 18.2-His full-length protein (Acro Biosystems, Cat. No. CL2-H82E3, 10 μg / ml) was captured on a streptavidin-conjugated biosensor and then associated with 10 μg / ml of antibody (i.e., first association). The sensor was then moved to a well containing a different antibody (i.e., second association). Antibodies were binned based on hierarchical clustering of the change in response from the first to the second association using the instrument's software. A threshold was set for the appropriate autobinding signal to determine blocking or non-blocking antibody pairs. Antibodies that blocked binding were clustered in the same bin and were considered to have similar epitopes. Antibodies that did not block binding were clustered in separate bins and were considered to have different epitopes.

[0073] The hierarchical clustering of the disclosed antibody, candidate antibody, and positive control comparison antibody (SI-64C8~SI-64C11) is shown in the binning matrix table (Figure 9). Several candidate antibodies blocked the binding of the comparison antibody, indicating that these clones have epitopes similar to those already under development. However, the disclosed clone 5103F3 BSM showed unidirectional binding activity to all antibodies, including most of the comparison antibodies. When bound to claudin 18.2 as antibody #1, 5103F3 BSM did not block the binding of any candidate or comparison antibody, except for SI-64C8. When bound as antibody #2, 5103F3 BSM was blocked by all antibodies. After clustering, 5103F3 BSM was classified into a bin (circle) distinct from all candidate and comparison antibodies. This suggests that 5103F3 BSM has a unique epitope blocking profile compared to other blocking antibodies.

[0074] Example 6: Anti-claudin 18.2 antibody-dependent cell-mediated cytotoxicity Complement-dependent cell injury (CDC) The Fc-mediated CDC activity of antibody drugs can be compared in vitro to determine whether antibody features exist that enable a greater potential therapeutic mechanism. To compare the CDC function of 5103F3 BSM and zolbetuximab, Chinese hamster ovary cells (CHO) were transduced to express claudin 18.2 tagged with a fluorescent reporter. Target cells CHO-claudin 18.2 were treated with 5103F3 BSM and zolbetuximab in the presence and absence of normal human serum (NHS), and CDC was evaluated at 2 hours. In the assay, cells were treated with antibodies ranging from 6.4 pM to 500 nM across a 5-fold dilution curve. CDC was assessed by the uptake of membrane-impermeable fluorescent dyes measured using FACS.

[0075] The CDC activity of zolbetuximab was detectable in this assay system. 5103F3 BSM showed comparable CDC activity. This is consistent with comparable binding activity throughout the assay period and access for assembling the CDC multimer complement cascade.

[0076] Antibody-dependent cellular cytotoxicity (ADCC) The Fc-mediated ADCC activity of antibody drugs can be compared in vitro to determine whether antibody features exist that enable a greater potential therapeutic mechanism. In this test system, a low ADCC response may be due to the available antigen expression level over the assay period. Consequently, different binding properties or antibody internalization may affect ADCC activity. 10 .

[0077] To test this, primary human NK (natural killer) cells were concentrated from whole blood by negative screening and then density gradient centrifugation to create "effector" cells. CHO cells transduced to express claudin 18.2 tagged with a fluorescent reporter were used as target cells. CHO-claudin 18.2 target cells were treated with 5103F3 BSM, zolbetuximab, or rituximab in 10-fold serial dilutions ranging from 100 nM to 0.00001 nM. NK cells were added in a 5:1 E:T (effector:target) ratio to evaluate ADCC within a 24-hour assay timeframe. ADCC was assessed by the disappearance of the CHO-claudin 18.2 cell reporter signal using time-series fluorescence microscopy.

[0078] In this assay system, the ADCC activity of zolbetuximab was detectable, while CD20-specific rituximab did not exhibit ADCC activity. 5103F3 BSM showed significantly different pharmacokinetics, characterized by prolonged ADCC activity. The enhanced ADCC activity of 5103F3 BSM compared to zolbetuximab is consistent with greater antigen availability throughout the trial, enabling greater NK cell engagement and cytolytic activity.

[0079] Example 7: Internalization and lysosome transport of anti-CLDN18.2 antibody For many antibody-drug conjugates (ADCs), a crucial step in their mechanism of action involves internalization. 11 Following antibody binding, internalization can facilitate the uptake of cytotoxic payloads and their transport to cancer cell lysosomes, which function as intracellular recycling centers. Depending on the type of cytotoxic payload, cell lysis follows different mechanisms of action. Claudin 18.2 is a tight junction protein normally expressed in gastric epithelial cells, and has also been detected in gastric cancer, pancreatic cancer, biliary tract cancer, and colorectal cancer. 12Therefore, while the mechanisms of internalization and transport to lysosomes by anti-claudin 18.2 may be the same, its effectiveness may depend on antibody binding.

[0080] Anti-claudin 18.2 antibody, 5103F3 BSM, and zolbetuximab were labeled with FabFluor-pH red, an acid-sensitive fluorescent reagent that enables the quantification of internalized protein transport into acidic lysosomes. The fluorescence signal of FabFluor-pH red depends on the low pH environment of lysosomes. In this way, the accumulation of internalized antibodies in the lysosomal compartment can be quantified over time using time-series fluorescence microscopy. NUGC-4 cell lines were thawed and cultured in RPMI medium supplemented with Pen / Strep and 10% FBS. Cells were seeded at 10,000 cells / well in 50 μL of culture medium in a 96-well round-bottom ULA plate (Corning). Vehicle buffer, 5103F3 BSM, and zolbetuximab were labeled with FabFluor-pH according to the manufacturer's instructions. 50 microliters of FabFluor-pH-labeled antibody or control treatment were added to seeded cells at a 2x concentration (10 nM or 20 nM) per 100 μl final assay volume / well. The cells were cultured for 16 hours in an Incucyte S3 live cell imager (Sartorius) in a 37°C, 5% CO2 humidified incubator.

[0081] Using time-series live-cell fluorescence microscopy, internalization and transport signals were readily detectable for both 5103F3 BSM and zolbetuximab at both 5 nM and 10 nM concentrations. Signals were automatically counted and plotted over time. The results indicate that 5103F3 BSM was internalized more efficiently than zolbetuximab and transported more signal (indicating a cytotoxic payload in the case of ADCs) to lysosomes (Figure 13).

[0082] Example 8: Characterization of 5103F3 BSM Cross-species conjugation to mouse and rat claudin 18.2 Humanized anti-claudin 18.2 antibodies were analyzed for cross-species binding to mouse and rat claudin 18 by flow cytometry. Plasmids expressing mouse or rat claudin 18.1 and claudin 18.2 were transiently transfected into ExpiCHO. Binding of surface-expressed claudin 18 was determined using the method described above. Table 5 shows that antibody 5103F3 bound to mouse and rat claudin 18.2 but not to claudin 18.1. Due to its high sequence similarity to human claudin 18 (98.9% for both extracellular domains), cynomolgus monkey claudin 18 was not included in the assay. Binding of 5103F3 BSM to mouse claudin 18.2 was confirmed by OCTET with a binding affinity of 1.73 nM (Figure 13).

[0083] Zolbetuximab is a human / mouse chimeric antibody therapy used in combination with chemotherapy to treat gastric cancer patients. In contrast, 5103F3 BSM is a humanized antibody. Its higher humanness may allow 5103F3 BSM to exhibit improved therapeutic efficacy by reducing post-administration immunogenicity or other undesirable side effects. In addition, 5103F3 BSM recognized different epitope bins of claudin 18 isoform 2 compared to zolbetuximab (SI-64C8) and other anti-CLDN18.2 antibodies including AB011 (SI-64C10), LM-102 (SI-64C9), and MIL93 (SI-64C11) (Figure 9). This binding property of 5103F3 BSM may lead to unique binding specificity, clustering, internalization, and other biological activities. In fact, 5103F3 BSM and zolbetuximab exhibited similar CDC activity but different ADCC activity (Figures 10 and 11), as well as different pharmacokinetics in internalization and lysosomal transport (Figure 12).

[0084] Sequence alignment of lead antibody and comparison antibody To investigate the structural basis of the unique features of 5103F3 BSM, the coding sequences of 5103F3 BSM, 5208H10, and the comparison antibody were aligned in the Kabat numbering scheme using Geneious Prime bioinformatics software and are shown in Figure 14. The heavy and light chain complementarity-determining regions (CDRs) were aligned across all antibodies (framed in the upper and lower panels of Figure 14A). The identity percentages for all CDRs only, framework regions only, and the entire variable region were calculated for comparison in Figures 14B and 14C. Of the aligned CDR sequences, 5103F3 BSM shared 17–28% identity with each comparison antibody, while each comparison antibody shared 50–82% identity with another comparison antibody. Of all framework regions, 5103F3 BSM shared up to 70% identity with each comparison antibody. Next, a two-sided, one-sample t-test was used to compare the identity percentage of 5103F3 BSM to any given comparison antibody with the mean identity percentage among the remaining comparison antibodies. The differences were significant (P<0.05) in both the CDR and the complete variable region, but not in all framework regions (P>0.05) (Table 6). Significant differences in the amino acid sequence encoding the primary structure of the CDR underlie the distinctive properties of 5103F3 BSM compared to comparison antibodies in epitope binding (Figure 9) and consequently in biological activity.

[0085] In summary, 5103F3 BSM is a humanized anti-CLDN18.2 antibody with high affinity, avidity, and specificity to claudin 18.2, while 5208H10 is a pan-claudin 18 conjugate capable of binding to both claudin 18.1 and claudin 18.2. This pair of antibodies is useful for the development of antibody therapies and for the diagnosis of claudin expression in various forms of cancer. Furthermore, due to the significant technical advantages of 5103F3 BSM over other antibodies in the same category, including its high selectivity for claudin 18.2, 5103F3 BSM offers significant therapeutic advantages for treating cancers with claudin 18.2 overexpression.

[0086] References 1.Stomach cancer statistics | WCRF International (https: / / www.wcrf.org / cancer-trends / stomach-cancer-statistics / ) 2.American Cancer Society: Cancer Facts and Figures 2022. American Cancer Society, 2022. (Cancer Facts & Figures 2022) 3.Pancreatic cancer statistics - WCRF International (https: / / www.wcrf.org / cancer-trends / pancreatic-cancer-statistics / ) 4.Key Statistics for Pancreatic Cancer (https: / / www.cancer.org / cancer / pancreatic-cancer / about / key-statistics.html) 5.Cao W, Xing H, Li Y, Tian W, Song Y, Jiang Z, Yu J. Claudin18.2 is a novel molecular biomarker for tumor-targeted immunotherapy. Biomark Res. 2022 May 31;10(1):38. doi: 10.1186 / s40364-022-00385-1. PMID: 35642043; PMCID: PMC9153115. 6.Sahin U, Koslowski M, Dhaene K, Usener D, Brandenburg G, Seitz G, Huber C, Tureci O. Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development. Clin Cancer Res. 2008 Dec 1;14(23):7624-34. doi: 10.1158 / 1078-0432.CCR-08-1547. PMID: 19047087. 7.Niimi T, Nagashima K, Ward JM, Minoo P, Zimonjic DB, Popescu NC, Kimura S. claudin-18, a novel downstream target gene for the T / EBP / NKX2.1 homeodomain transcription factor, encodes lung- and stomach-specific isoforms through alternative splicing. Mol Cell Biol. 2001 Nov;21(21):7380-90. doi: 10.1128 / MCB.21.21.7380-7390.2001. PMID: 11585919; PMCID: PMC99911. 8.Li J. Targeting claudins in cancer: diagnosis, prognosis and therapy. Am J Cancer Res. 2021 Jul 15;11(7):3406-3424. PMID: 34354852; PMCID: PMC8332862. 9.Tureci O, Koslowski M, Helftenbein G, Castle J, Rohde C, Dhaene K, Seitz G, Sahin U. Claudin-18 gene structure, regulation, and expression is evolutionary conserved in mammals. Gene. 2011 Aug 1;481(2):83-92. doi: 10.1016 / j.gene.2011.04.007. Epub 2011 May 4. PMID: 21571049. 10.Garvin D, Stecha P, Gilden J, Wang J, Grailer J, Hartnett J, Fan F, Cong M, Cheng ZJ. Determining ADCC Activity of Antibody-Based Therapeutic Molecules using Two Bioluminescent Reporter-Based Bioassays. Curr Protoc. 2021 Nov;1(11):e296. 11.Yu et al. Frontiers Molecular Biosciences 2022. 12. Yong Hong et al. Trans Cancer Res 2020.

[0087] Table 1: Immunotherapy protocols for generating anti-CLDN18.2 antibodies - Cohort 1 and Cohort 2 [Table 1]

[0088] Table 2: Stability of humanized anti-claudin 18.2 antibody at time 0 and time 1 (top), and at time 2 and change (bottom). [Table 2] JPEG2026518213000004.jpg83170

[0089] Table 3: Anti-CLDN18.2 antibody and expression levels in CHO cells [Table 3]

[0090] Table 4: OCTET affinity and avidity of anti-CLDN18.2 antibodies [Table 4]

[0091] Table 5: Binding specificity of humanized clones and controls to mouse and rat CLDN18.2 [Table 5]

[0092] Table 6: Comparison of variable region CDR and framework coding sequences between 5103F3 BSM and other anti-CLDN18.2 antibodies [Table 6] Sequence List

[0093] [Table 7]

[0094] [Table 8] >Sequence ID 1: 5103F3 Rabbit VH Nucleic Acid Sequence CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACAGCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATTCAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATATCTTAATACTGGTGGTAGCACATACTACGCGAGCTGGGTGAATGGTCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTACCAGGGCCGGGGGTGTTAGTAGCAATATGGGCTTTAACATCTGGGGCCAAGGGACCCTGGTCACCGTCTCGAGC >Sequence ID 2: 5103F3 Rabbit VH Amino Acid Sequence QSVEESGGRLVTPGTPLTLTCTVSGIDLS GFAMG WVRQAPGKGPEYIG FIDSGGGAFYATWARG RFTISRTSSTTVDLKMTSLTAADTATYFCAR HGGNTYYYAMDP WGPGTLVTVSS >Sequence ID 3: 5103F3 Rabbit VL Nucleic Acid Sequence GCCCTTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGAGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTAACTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGTGTATAGTGTCACTAATATTGATAATGCTTTCGGCGGAGGCACCGAGGTGGAGTTCAAA >Sequence ID 4: 5103F3 Rabbit VL Amino Acid Sequence ALVMTQTPASVEAAVRGTVTIKC QASQSISNYLA WYQQKPGQPPKLLIY SAST LAS GVSSRFKGSGSGTQFTLTISGVECADAATYYC QQVYSVTNIDNA FGGGTEVEFK >Sequence ID 5: 5103F3 BSM VH Nucleic Acid Sequence CAGGTGCAGCTGCAGGAGTCCGGCGGCAGGCTGATCAAGCCCGGCGAGCCCCTGAGGCTGTCCTGCAAGACCTCCGGCATCGACCTGTCCGGCTTCGCCATGGGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTACATCGGCTTCATCGACTCCGGCGGCGGCGCCTTCTACGCCACCTGGGCCAGGGGCAGGTTCACCATCTCCAGGACCTCCACCAACACCGTGTACCTGCAGATGAACTCCCTGACCGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCACGGCGGCAACACCTACTACTACGCCATGGACCCCTGGGGCCCCGGCACCCTGGTGACCGTGTCCTCC >Sequence ID 6: 5103F3 BSM VH Amino Acid Sequence QVQLQESGGRLIKPGEPLRLSCKTSGIDLS GFAMG WVRQAPGKGLEYIG FIDSGGGAFYATWARG RFTISRTSTNTVYLQMNSLTAEDTAVYYCAR HGGNTYYYAMDP WGPGTLVTVSS >Sequence ID 7: 5103F3 BSM VL Nucleic Acid Sequence GCCCTGAGGATGACCCAGTCCCCCTCCTCCCTGGCCGCCACCACCGGCCAGAGGGTGACCATCACCTGCCAGGCCTCCCAGTCCATCTCCAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTCCGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCAAGGGCTCCGGCTCCGGCACCCAGTTCACCCTGACCATCTCCAGCGTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACTCCGTGACCAACATCGACAACGCCTTCGGCGGCGGCACCAGGGTGGAGATCAAG >Sequence ID 8: 5103F3 BSM VL Amino Acid Sequence ALRMTQSPSSLAATTGQRVTITC QASQSISNYLA WYQQKPGQPPKLLIY SAST LAS GVPSRFKGSGSGTQFTLTISCVQCEDFATYYC QQVYSVTNIDNA FGGGTRVEIK > Sequence ID 9: 5103F3 FRS VH Nucleic Acid Sequence CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCATCGACCTGTCCGGCTTCGCCATGGGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCTTCATCGACTCCGGCGGCGGCGCCTTCTACGCCACCTGGGCCAGGGGCAGGTTCACCATCTCCAGGACCTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCACGGCGGCAACACCTACTACTACGCCATGGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 10: 5103F3 FRS VH Amino Acid Sequence QVQLVESGGGLVKPGGSLRLSCAASGIDLS GFAMG WVRQAPGKGLEWVG FIDSGGGAFYATWARG RFTISRTSKNTVYLQMNSLRAEDTAVYYCAR HGGNTYYYAMDP WGQGTLVTVSS > Sequence ID 11: 5103F3 FRS VL Nucleic Acid Sequence GCCCTGGTGATGACCCAGTCCCCCTCCTCCCTGTCCGCCTCCGTGGGCGACAGGGTGACCATCTCCTGCCAGGCCTCCCAGTCCATCTCCAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACTCCGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCGTGGAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACTCCGTGACCAACATCGACAACGCCTTCGGCGGCGGCACCAAGGTGGAGATCAAG > Sequence ID 12: 5103F3 FRS VL Amino Acid Sequence ALVMTQSPSSLSASVGDRVTISC QASQSISNYLA WYQQKPGQAPKLLIY SAST LAS GVPSRFSGSGSGTDFTLTISSVEPEDFATYYC QQVYSVTNIDNA FGGGTKVEIK >Sequence ID 13: 5208H10 Rabbit VH Nucleic Acid Sequence CAGTCGGTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCGACGGATACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTACCTATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATGGATCGGAATCATTGATGCTGTTGGTCGCACATATTACGCGAGTTGGGCGAAAAGTCGATCCACCATCACCAGGAACACCAACCTGAACACGGTGACTCTGAAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGATGGTGGGATCTCTGGGGCCAAGGCACCCTGGTCACCGTCTCGAGC >Sequence ID 14: 5208H10 Rabbit VH Amino Acid Sequence QSVKESEGGLFKPTDTLTLTCTVSGFSLS TYAMS WVRQAPGNGLEWIG IIDAVGRTYYASWAKS RSTITRNTNLNTVTLKMTSLTAADTATYFCAR WWDL WGQGTLVTVSS >Sequence ID 15: 5208H10 Rabbit VL Nucleic Acid Sequence GCGCAAGTGCTGACCCAGACTGCATCGTCCGTGTCTGCCGCTGTGGGAGGCACTGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTTATGATAGCAACGACTTAACCTGGTATCAGCAGAAACCAGGGCAGTCTCCCAAGCTCCTGATCTATCTGGCATCCACTCTGGCATCTGGGGTCCCATCGCGATTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGACGATTCTGCCACTTACTACTGTGCAGCCCATTATAATAGTGATATTTATACTTTCGGCGGAGGGACCGAGGTGGAGGTCAAA >Sequence ID 16: 5208H10 Rabbit VL Amino Acid Sequence AQVLTQTASSVSAAVGGTVTINC QSSQSVYDSNDLT WYQQKPGQSPKLLIY LAST LAS GVPSRFKGSGSGTQFTLTISDLECDDSATYYC AAHYNSDIYT FGGGTEVEVK >Sequence ID 17: 5046A2 Rabbit VH Nucleic Acid Sequence TTCTCTCCACAGGTGTCCACTCCCAGGTCCAAGTTTAAACGGATCTCTAGCGAATTCAAGCTTACGCTCACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGATAACGCCTGGAGGATCCCTGACACTCACCTGCACCGTCTCTGGAATCGACCTCAGTAGCAATGGAATGAGCTGGGTCCGCCAGTCTCCAGGGAAGGGGCTGGAATACATCGGATTCATTGATACTGATGGTAGCGCATACTCCGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAGCCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGCCTATGTTAGTGGTAGTACTGGTTACAATTTTAACATCTGGGGCCCGGGGACCCTGGTCACCGTCTCGAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCT > Sequence ID 18: 5046A2 Rabbit VH Amino Acid Sequence FSPQVSTPRSKFKRISSEFKLTLTMETGLRWLLLVAVLKGVQCQSVEESGGRLITPGGSLTLTCTVSGIDLS SNGMS WVRQSPGKGLEYIG FIDTDGSAYSASWAKG RFTISKASSTTVDLKMTSLTTEDTATYFCAR AYVSGSTGYNFNI WGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC > Sequence ID 19: 5046A2 Rabbit VL Nucleic Acid Sequence TTCTCTCCACAGGTGTCCACTCCCAGGTCCAAGTTTAAACGGATCTCTAGCGAATTCAAGCTTCGAATCGACATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCATTCGAATTGACCCAGACTCCATCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTAGCTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGTCCCTGATCTACAAGGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGGTTATACTATTCGTAATATTGATAATGCTTTCGGCGGAGGGACCGAGGTGGAGTTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCT > Sequence ID 20: 5046A2 Rabbit VL Amino Acid Sequence FSPQVSTPRSKFKRISSEFKLRIDMDTRAPTQLLGLLLLWLPGARCAFELTQTPSSVEAAVGGTVTIKC Q ASQSISSYLA WYQQKPGQPPKSLIY KASTLAS GVPSRFKGSGSGTEFTLTISDLECADAATYYC QQGYTIRNIDNA FGGGTEVEFKRTVAAPSVFIFPPSDEQLKSGTAS > Sequence ID 21: 5046A2 BSM VH Nucleic Acid Sequence CAGGTGCAGCTGGTGGAGTCCGGCGGCAGGCTGATCAAGCCCGGCGGCTCCCTGAGGCTGACCTGCAAGACCTCCGGCATCGACCTGTCCTCCAACGGCATGTCCTGGGTGAGGCAGCCCCCCGGCAAGGGCCTGGAGTACATCGGCTTCATCGACACCGACGGCTCCGCCTACTCCGCCTCCTGGGCCAAGGGCAGGTTCACCATCTCCAAGGCCTCCATCAACACCGTGTTCCTGCAGATGACCTCCCTGAAGTCCGAGGACACCGCCATCTACTACTGCGCCAGGGCCTACGTGTCCGGCTCCACCGGCTACAACTTCAACATCTGGGGCCCCGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 22: 5046A2 BSM VH Amino Acid Sequence QVQLVESGGRLIKPGGSLRLTCKTSGIDLSSNGMSWVRQPPGKGLEYIGFIDTDGSAYSASWAKGRFTISKASINTVFLQMTSLKSEDTAIYYCARAYVSGSTGYNFNIWGPGTLVTVSS > Sequence ID 23: 5046A2 BSM VL Nucleic Acid Sequence GCCTTCAGGCTGACCCAGACCCCCTCCTCCTTCGCCGCCACCCTGGGCCAGAGGGTGACCATCACCTGCCAGGCCTCCCAGTCCATCTCCTCCTACCTGGCCTGGTACCAGCAGAAGCCCGGCAAGCCCCCCAAGTCCCTGATCTACAAGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCAAGGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGGGCTACACCATCAGGAACATCGACAACGCCTTCGGCGGCGGCACCAGGGTGGAGATCAAG > Sequence ID 24: 5046A2 BSM VL Amino Acid Sequence AFRLTQTPSSFAATLGQRVTITC Q ASQSISSYLA WYQQKPGKPPKSLIY KASTLAS GVPSRFKGSGSGTDFTLTISSLQPDDFATYYC QQGYTIRNIDNA FGGGTRVEIK > Sequence ID 25: 5046A2 FRS VH Nucleic Acid Sequence CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGATCAAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCATCGACCTGTCCTCCAACGGCATGTCCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCTTCATCGACACCGACGGCTCCGCCTACTCCGCCTCCTGGGCCAAGGGCAGGTTCACCATCTCCAAGGACTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGCCTACGTGTCCGGCTCCACCGGCTACAACTTCAACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 26: 5046A2 FRS VH Amino Acid Sequence QVQLVESGGGLIKPGGSLRLSCAASGIDLSSNGMSWVRQAPGKGLEWVGFIDTDGSAYSASWAKGRFTISKDSKNTVYLQMNSLRAEDTAVYYCARAYVSGSTGYNFNIWGQGTLVTVSS > Sequence ID 27: 5046A2 FRS VL Nucleic Acid Sequence GCCTTCGTGCTGACCCAGTCCCCCTCCTCCCTGTCCGCCTCCGTGGGCGACAGGGTGACCATCTCCTGCCAGGCCTCCCAGTCCATCTCCTCCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGTCCCTGATCTACAAGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGGAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCTACACCATCAGGAACATCGACAACGCCTTCGGCGGCGGCACCAAGGTGGAGATCAAG > Sequence ID 28: 5046A2 FRS VL Amino Acid Sequence AFVLTQSPSSLSASVGDRVTISC Q ASQSISSYLA WYQQKPGQAPKSLIY KASTLAS GVPSRFSGSGSGTDFTLTISSLEPEDFATYYC QQGYTIRNIDNA FGGGTKVEIK > Sequence ID 29: 5111D12 Rabbit VH Nucleic Acid Sequence TTCTCTCCACAGGTGTCCACTCCCAGGTCCAAGTTTAAACGGATCTCTAGCGAATTCAAGCTTACGCTCACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGNNCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACAGCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATTCAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATATCTTAATACTGGTGGTAGCACATACTACGCGAGCTGGGTGAATGGTCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTACCAGGGCCGGGGGTGTTAGTAGCAATATGGGCTTTAACATCTGGGGCCAAGGGACCCTGGTCACCGTCTCGAGCGCTAGCACCAAGGNNCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGC > Sequence ID 30: 5111D12 Rabbit VH Amino Acid Sequence FSPQVSTPRSKFKRISSEFKLTLTMETGLRWLLLXAVLKGVQCQSVEESGGRLVTPGTALTLTCTVSGFSLS SYSMG WVRQAPGKGLEWIG YLNTGGSTYYASWVNG RFTISKTSSTTVDLKITSPTTEDTATYFCTR AGGVSSNMGFNI WGQGTLVTVSSASTKXPSVFPLAPSSKSTSGGTAALG > Sequence ID 31: 5111D12 Rabbit VL Nucleic Acid Sequence TTCTCTCCACAGGTGTCCACTCCCAGGTCCAAGTTTAAACGGATCTCTAGCGAATTCAAGCTTCGAATCGACATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGACCCTGTGCTGACCCAGACTCCAGCCTCCGTGTCTGAGCCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTAGCTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGTACTGATTATGGCTACAATTATCTTGGGGCTTTCGGCGGAGGGACCGAGGTGGAGTTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTG > Sequence ID 32: 5111D12 Rabbit VL Amino Acid Sequence FSPQVSTPRSKFKRISSEFKLRIDMDTRAPTQLLGLLLLWLPGARCDPVLTQTPASVSEPVGGTVTIKC Q ASQSISSYLA WYQQKPGQPPKLLIY RASTLAS GVPSRFKGSGSGTQFTLTISDLECADAATYYC QCTDYGYNYLGA FGGGTEVEFKRTVAAPSVFIFPPSDEQLKSGTAS > Sequence ID 33: 5111D12 BSM VH Nucleic Acid Sequence CAGGTGCAGCTGCTGGAGTCCGGCGGCAGGCTGATCAAGCCCGGCGAGTCCCTGAAGCTGACCTGCAAGACCTCCGGCTTCTCCCTGTCCTCCTACTCCATGGGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCTACCTGAACACCGGCGGCTCCACCTACTACGCCTCCTGGGTGAACGGCAGGTTCACCATCTCCAAGACCTCCATCTCCACCGTGTTCCTGCAGCTGAGGTCCCCCAGGTCCGAGGACACCGCCCTGTACTACTGCACCAGGGCCGGCGGCGTGTCCTCCAACATGGGCTTCAACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 34: 5111D12 BSM VH Amino Acid Sequence QVQLLESGGRLIKPGESLKLTCKTSGFSLS SYSMG WVRQAPGKGLEWIG YLNTGGSTYYASWVNG RFTISKTSISTVFLQLRSPRSEDTALYYCTR AGGVSSNMGFNI WGQGTLVTVSS > Sequence ID 35: 5111D12 BSM VL Nucleic Acid Sequence GACCCCGTGCTGACCCAGTCCCCCTCCTTCCTGTCCCTGACCGTGGGCCAGAGGGTGACCATCAACTGCCAGGCCTCCCAGTCCATCTCCTCCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACAGGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGAGTTCACCCTGACCATCTCCTCCCTGCAGTCCGACGACTTCGCCACCTACTACTGCCAGTGCACCGACTACGGCTACAACTACCTGGGCGCCTTCGGCGGCGGCACCAGGGTGGAGATCAAG > Sequence ID 36: 5111D12 BSM VL Amino Acid Sequence DPVLTQSPSFLSLTVGQRVTINC Q ASQSISSYLA WYQQKPGQPPKLLIY RASTLAS GVPSRFSGSGSGTEFTLTISSLQSDDFATYYC QCTDYGYNYLGA FGGGTRVEIK > Sequence ID 37: 5194G6 Rabbit VH Nucleic Acid Sequence CAGGAGCAGCTGAAGGAGTCCGGGGGAGGCCTGGTCCAGCCTGGGGGATCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCTTCAGTAGCGTCTACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGCAGTTATACGAGTGATGGTAGCGCTTACTACGCGAACTGGGCGACAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACGAGTCTGACAGACGCGGACACGGCCACCTATTTCTGTGCGAGAGATAGGACTGATAGTCGTGATTATTTTTTTGACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCGAGC > Sequence ID 38: 5194G6 Rabbit VH Amino Acid Sequence QEQLKESGGGLVQPGGSLTLTCKASGFSFS SVYYMC WVRQAPGKGLEWIG CSYTSDGSAYYANWATG RFTISKTSSTTVTLQMTSLTDADTATYFCAR DRTDSRDYFFDL WGPGTLVTVSS > Sequence ID 39: 5194G6 Rabbit VL Nucleic Acid Sequence GCCTATGATATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCAGCATCAGTTGTCAGTCCAGTCAGAATGTTTATAGTAACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCTTCTGGGGTCTCATCGCGATTCAGAGGCAGTGGATCCGGGACACAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTACTATTGTCAACAGGGTTATAAATATGGTAATGTTAATAATCTTTTCGGCGGAGGGACCGAGGTGGAGGTCAAA > Sequence ID 40: 5194G6 Rabbit VL Amino Acid Sequence AYDMTQTPASVEAAVGGTVSISC QSSQNVYSNYLS WYQQKPGQPPKLLIY RASTLAS GVSSRFRGSGSGTQFTLTISGVECADAATYYC QQGYKYGNVNNL FGGGTEVEVK > Sequence ID 41: 5194G6 BSM VH Nucleic Acid Sequence GAGGAGCAGCTGCTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCACCGCCTCCGGCTTCACCTTCGAGTCCGTGTACTACATGTGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCTGCTCCTACACCTCCGACGGCTCCGCCTACTACGCCAACTGGGCCACCGGCAGGTTCACCATCTCCAAGACCTCCATCAACACCGTGTTCCTGCAGATGAGGTCCCTGAGGTCCGAGGACACCGCCATCTACTACTGCGCCAGGGACAGGACCGACTCCAGGGACTACTTCTTCGACCTGTGGGGCCCCGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 42: 5194G6 BSM VH Amino Acid Sequence EEQLLESGGGLVQPGGSLRLSCTASGFTFESVYYMCWVRQAPGKGLEWIGCSYTSDGSAYYANWATGRFTISKTSINTVFLQMRSLRSEDTAIYYCARDRTDSRDYFFDLWGPGTLVTVSS > Sequence ID 43: 5194G6 BSM VL Nucleic Acid Sequence GCCTACAGGATGACCCAGTCCCCCTCCTCCTTCTCCGCCTCCACCGGCCAGAGGGTGACCATCACCTGCCAGTCCTCCCAGAACGTGTACTCCAACTACCTGTCCTGGTACCAGCAGAAGCCCGGCAAGCCCCCCAAGCTGCTGATCTACAGGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCAGGGGCTCCGGCTCCGGCACCCAGTTCACCCTGACCATCTCCTGCGTGCAGTGCGACGACTTCGCCACCTACTACTGCCAGCAGGGCTACAAGTACGGCAACGTGAACAACCTGTTCGGCGGCGGCACCAGGGTGGAGATCAAG > Sequence ID 44: 5194G6 BSM VL Amino Acid Sequence AYRMTQSPSSFSASTGQRVTITC QSSQNVYSNYLS WYQQKPGKPPKLLIY RASTLAS GVPSRFRGSGSGTQFTLTISCVQCDDFATYYC QQGYKYGNVNNL FGGGTRVEIK > Sequence ID 45: 5194G6 FRS VH Nucleic Acid Sequence CAGGAGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCTCCGTGTACTACATGTGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCTGCTCCTACACCTCCGACGGCTCCGCCTACTACGCCAACTGGGCCACCGGCAGGTTCACCATCTCCAAGACCTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACAGGACCGACTCCAGGGACTACTTCTTCGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 46: 5194G6 FRS VH Amino Acid Sequence QEQLVESGGGLVQPGGSLRLSCAASGFTFSSVYYMCWVRQAPGKGLEWVGCSYTSDGSAYYANWATGRFTISKTSKNTVYLQMNSLRAEDTAVYYCARDRTDSRDYFFDLWGQGTLVTVSS > Sequence ID 47: 5194G6 FRS VL Nucleic Acid Sequence GCCTACGTGATGACCCAGTCCCCCTCCTCCCTGTCCGCCTCCGTGGGCGACAGGGTGACCATCTCCTGCCAGTCCTCCCAGAACGTGTACTCCAACTACCTGTCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACAGGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCGTGGAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCTACAAGTACGGCAACGTGAACAACCTGTTCGGCGGCGGCACCAAGGTGGAGATCAAG > Sequence ID 48: 5194G6 FRS VL Amino Acid Sequence AYVMTQSPSSLSASVGDRVTISC QSSQNVYSNYLS WYQQKPGQAPKLLIY RASTLAS GVPSRFSGSGSGTDFTLTISSVEPEDFATYYC QQGYKYGNVNNL FGGGTKVEIK > Sequence ID 49: 5194G6 GS VH Nucleic Acid Sequence CAGGAGCAGCTGCTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCTCCGTGTACTACATGTGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCTGCTCCTACACCTCCGACGGCTCCGCCTACTACGCCAACTGGGCCACCGGCAGGTTCACCATCTCCAAGACCTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACAGGACCGACTCCAGGGACTACTTCTTCGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC > Sequence ID 50: 5194G6 GS VH Amino Acid Sequence QEQLLESGGGLVQPGGSLRLSCAASGFTFSSVYYMCWVRQAPGKGLEWVGCSYTSDGSAYYANWATGRFTISKTSKNTVYLQMNSLRAEDTAVYYCARDRTDSRDYFFDLWGQGTLVTVSS > Sequence ID 51: 5194G6 GS VL Nucleic Acid Sequence GCCTACAGGATGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTGACCATCTCCTGCCAGTCCTCCCAGAACGTGTACTCCAACTACCTGTCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACAGGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCGGCGTGCAGTCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCTACAAGTACGGCAACGTGAACAACCTGTTCGGCGGCGGCACCAAGGTGGAGATCAAG > Sequence ID 52: 5194G6 GS VL Amino Acid Sequence AYRMTQSPSSVSASVGDRVTISC QSSQNVYSNYLS WYQQKPGQAPKLLIY RASTLAS GVPSRFSGSGSGTDFTLTISGVQSEDFATYYC QQGYKYGNVNNL FGGGTKVEIK > Sequence ID 53: 5119H11 Rabbit VH Nucleic Acid Sequence CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACGGTCTCTGGATTCTCCCTCAGTACCTATTCAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTTACTATGGTGAGCCCACATACTGCGCGGCCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGATCAGGTGATCGTAATGGCTACTACTTTAACATCTGGGGCCCAGGCACCCTGGTCACCGTCTCGAGC > Sequence ID 54: 5119H11 Rabbit VH Amino Acid Sequence QSVEESGGRLVTPGTPLTLTCTVSGFSLS TYSMG WVRQAPGKGLEWIG VIYYGEPTYCAAWAKG RFTISKTSSTTVDLKITSPTTEDTATYFCAR SGDRNGYYFNI WGPGTLVTVSS > Sequence ID 55: 5119H11 Rabbit VL Nucleic Acid Sequence GCCTATGATATGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTAGCTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGCCGATGCAGCCACTTACTACTGTCAACAGGTTGCTGTTATTGGTAATGTTGGGGATAATACTTTCGGCGGAGGGACCGAGGTGGAGGTCAAA > Sequence ID 56: 5119H11 Rabbit VL Amino Acid Sequence AYDMTQTPASVSAAVGGTVTIKC Q ASQSISSYLA WYQQKPGQPPKLLIY YASTLAS GVPSRFKGSGSGTEFTLTISGVQCADAATYYC QQVAVIGNVGDNT FGGGTEVEVK > Sequence ID 57: 5103F3 VH-CDRH1 GFAMG > Sequence ID 58: 5103F3 VH-CDRH2 FIDSGGGAFYATWARG > Sequence ID 59: 5103F3 VH-CDRH3 HGGNTYYYAMDP > Sequence ID 60: 5103F3 VL-CDRL1 QASQSISNYLA > Sequence ID 61: 5103F3 VL-CDRL2 SASTLAS > Sequence ID 62: 5103F3 VL-CDRL3 QQVYSVTNIDNA > Sequence ID 63: 5208H10 VH-CDRH1 TYAMS > Sequence ID 64: 5208H10 VH-CDRH2 IIDAVGRTYYASWAKS > Sequence ID 65: 5208H10 VH-CDRH3 WWDL > Sequence ID 66: 5208H10 VL-CDRL1 QSSQSVYDSNDLT > Sequence ID 67: 5208H10 VL-CDRL2 LASTLASGVPS > Sequence ID 68: 5208H10 VL-CDRL3 AAHYNSDIYT > Sequence ID 69: 5046A2 VH-CDRH1 SNGMS > Sequence ID 70: 5046A2 VH-CDRH2 FIDTDGSAYSASWAKG > Sequence ID 71: 5046A2 VH-CDRH3 AYVSGSTGYNFNI > Sequence ID 72: 5046A2 VL-CDRL1 QASQSISSYLA > Sequence ID 73: 5046A2 VL-CDRL2 KASTLAS > Sequence ID 74: 5046A2 VL-CDRL3 QQGYTIRNIDNA > Sequence ID 75: 5111D12 VH-CDRH1 SYSMG > Sequence ID 76: 5111D12 VH-CDRH2 YLNTGGSTYYASWVNG > Sequence ID 77: 5111D12 VH-CDRH3 AGGVSSNMGFNI > Sequence ID 78: 5111D12 VL-CDRL1 QASQSISSYLA > Sequence ID 79: 5111D12 VL-CDRL2 RASTLAS > Sequence ID 80: 5111D12 VL-CDRL3 QCTDYGYNYLGA > Sequence ID 81: 5119H11 VH-CDRH1 TYSMG > Sequence ID 82: 5119H11 VH-CDRH2 VIYYGEPTYCAAWAKG > Sequence ID 83: 5119H11 VH-CDRH3 SGDRNGYYFNI > Sequence ID 84: 5119H11 VL-CDRL1 QASQSISSYLA > Sequence ID 85: 5119H11 VL-CDRL2 YASTLAS > Sequence ID 86: 5119H11 VL-CDRL3 QQVAVIGNVGDNT > Sequence ID 87: Human Claudin18.2 Nucleic Acid Sequence ATGGCCGTGACCGCCTGCCAGGGCCTGGGCTTCGTGGTGAGCCTGATCGGCATCGCCGGCATCATCGCCGCCACCTGCATGGACCAGTGGAGCACCCAGGACCTGTACAACAACCCCGTGACCGCCGTGTTCAACTACCAGGGCCTGTGGAGGAGCTGCGTGAGGGAGAGCAGCGGCTTCACCGAGTGCAGGGGCTACTTCACCCTGCTGGGCCTGCCCGCCATGCTGCAGGCCGTGAGGGCCCTGATGATCGTGGGCATCGTGCTGGGCGCCATCGGCCTGCTGGTGAGCATCTTCGCCCTGAAGTGCATCAGGATCGGCAGCATGGAGGACAGCGCCAAGGCCAACATGACCCTGACCAGCGGCATCATGTTCATCGTGAGCGGCCTGTGCGCCATCGCCGGCGTGAGCGTGTTCGCCAACATGCTGGTGACCAACTTCTGGATGAGCACCGCCAACATGTACACCGGCATGGGCGGCATGGTGCAGACCGTGCAGACCAGGTACACCTTCGGCGCCGCCCTGTTCGTGGGCTGGGTGGCCGGCGGCCTGACCCTGATCGGCGGCGTGATGATGTGCATCGCCTGCAGGGGCCTGGCCCCCGAGGAGACCAACTACAAGGCCGTGAGCTACCACGCCAGCGGCCACAGCGTGGCCTACAAGCCCGGCGGCTTCAAGGCCAGCACCGGCTTCGGCAGCAACACCAAGAACAAGAAGATCTACGACGGCGGCGCCAGGACCGAGGACGAGGTGCAGAGCTACCCCAGCAAGCACGACTACGTGTGA > Sequence ID 88: Human Claudin18.1 Nucleic Acid Sequence ATGTCCACCACCACATGCCAAGTGGTGGCGTTCCTCCTGTCCATCCTGGGGCTGGCCGGCTGCATCGCGGCCACCGGGATGGACATGTGGAGCACCCAGGACCTGTACGACAACCCCGTCACCTCCGTGTTCCAGTACGAAGGGCTCTGGAGGAGCTGCGTGAGGCAGAGTTCAGGCTTCACCGAATGCAGGCCCTATTTCACCATCCTGGGACTTCCAGCCATGCTGCAGGCAGTGCGAGCCCTGATGATCGTAGGCATCGTCCTGGGTGCCATTGGCCTCCTGGTATCCATCTTTGCCCTGAAATGCATCCGCATTGGCAGCATGGAGGACTCTGCCAAAGCCAACATGACACTGACCTCCGGGATCATGTTCATTGTCTCAGGTCTTTGTGCAATTGCTGGAGTGTCTGTGTTTGCCAACATGCTGGTGACTAACTTCTGGATGTCCACAGCTAACATGTACACCGGCATGGGTGGGATGGTGCAGACTGTTCAGACCAGGTACACATTTGGTGCGGCTCTGTTCGTGGGCTGGGTCGCTGGAGGCCTCACACTAATTGGGGGTGTGATGATGTGCATCGCCTGCCGGGGCCTGGCACCAGAAGAAACCAACTACAAAGCCGTTTCTTATCATGCCTCAGGCCACAGTGTTGCCTACAAGCCTGGAGGCTTCAAGGCCAGCACTGGCTTTGGGTCCAACACCAAAAACAAGAAGATATACGATGGAGGTGCCCGCACAGAGGACGAGGTACAATCTTATCCTTCCAAGCACGACTATGTGTAA

Claims

1. An isolated monoclonal antibody (mAb) or its antigen-binding fragment having at least 90% identity with the sequence numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, and possessing binding specificity to CLDN18.

2. The isolated mAb or antigen-binding fragment according to claim 1, wherein the isolated mAb or antigen-binding fragment selectively binds to CLDN18.2 but does not bind to CLDN18.

1.

3. The isolated mAb or antigen-binding fragment thereof according to claim 1, further comprising a human framework region.

4. The isolated mAb or antigen-binding fragment thereof according to claim 1, wherein the isolated mAb is a humanized antibody, a chimeric antibody, or a recombinant antibody.

5. The isolated mAb or antigen-binding fragment thereof according to claim 1, wherein the isolated mAb is IgG.

6. The isolated mAb or its antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is an Fv, Fab, F(ab')2, scFv, or scFv2 fragment.

7. The isolated mAb or antigen-binding fragment thereof according to claim 1, wherein the isolated mAb is a bispecific antibody, a tripspecific antibody, or a multispecific antibody.

8. The isolated mAb or antigen-binding fragment thereof according to claim 1, comprising an IgG1 heavy chain having an amino acid sequence having at least 90% identity with SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, or 54.

9. The isolated mAb or antigen-binding fragment thereof according to claim 1, comprising a kappa light chain having an amino acid sequence having at least 90% identity with SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, or 56.

10. The isolated mAb or antigen-binding fragment thereof according to claim 1, comprising a variable heavy chain domain having an amino acid sequence having at least 90% identity with SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, or 54, or a variable light chain domain having an amino acid sequence having at least 90% identity with SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, or 56.

11. An isolated mAb or its antigen-binding fragment having binding specificity to CLDN18, The isolated mAb or antigen-binding fragment thereof comprises a variable heavy chain domain having complementarity-determining regions (CDRs) 1, CDR2, and CDR3 of SEQ ID NOs: 57, 58, and 59; 63, 64, and 65; 69, 70, and 71; 75, 76, and 77; or 81, 82, and 83, and a variable light chain domain having CDR1, CDR2, and CDR3 of SEQ ID NOs: 60, 61, and 62; 66, 67, and 68; 72, 73, and 74; 78, 79, and 80; or 84, 85, and 86.

12. An isolated nucleic acid encoding an isolated mAb or its antigen-binding fragment as described in claim 1, an IgG1 heavy chain as described in claim 8, a kappa light chain as described in claim 9, a variable light chain domain as described in claim 10, or a variable heavy chain domain as described in claim 11.

13. An expression vector comprising an isolated nucleic acid as described in claim 12, wherein the expression vector is expressible in a cell.

14. A host cell comprising the nucleic acid described in claim 12, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

15. A method for producing an antibody, comprising culturing the host cells described in claim 14 so that antibodies are produced.

16. An immunoconjugate comprising an isolated mAb or its antigen-binding fragment according to claim 1, conjugated to a drug unit via a linker, An immunoconjugate in which the linker comprises a covalent bond selected from an ester bond, an ether bond, an amine bond, an amide bond, a disulfide bond, an imide bond, a sulfone bond, a phosphate bond, a phosphate ester bond, a peptide bond, a hydrazone bond, or a combination thereof.

17. The immunoconjugate according to claim 16, wherein the drug unit comprises a cytotoxic agent, an immunomodulatory reagent, or a combination thereof.

18. The immunoconjugate according to claim 16, wherein the cytotoxic agent is selected from a proliferation inhibitor, a tubulin binder, a DNA intercalator, a DNA alkylating agent, an enzyme inhibitor, an immunomodulator, an antimetabolite, a chemotherapeutic agent from a class of radioisotopes, or a combination thereof.

19. The immunoconjugate according to claim 16, wherein the cytotoxic agent is selected from calicheamicin, ozogamicin, monomethyl auristatin E, emtansine, derivatives, or combinations thereof.

20. The immunoconjugate according to claim 16, wherein the immunomodulatory reagent activates or inhibits immune cells, T cells, NK cells, B cells, macrophages, or dendritic cells.

21. A pharmaceutical composition comprising an isolated mAb or its antigen-binding fragment as described in claim 1, or an immunoconjugate as described in claim 19, and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition according to claim 21, further comprising a radioisotope, a radionuclide, a toxin, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

23. The pharmaceutical composition according to claim 22, wherein the therapeutic agent comprises an anti-estrogen agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA, or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof.

24. A method for treating a subject having cancer, comprising administering to the subject an effective amount of the isolated mAb or antigen-binding fragment thereof described in claim 1.

25. The method according to claim 24, wherein the cancer comprises cells expressing claudin 18.

2.

26. The method according to claim 24, wherein the cancer includes colorectal cancer, pancreatic cancer, esophageal cancer, nasopharyngeal cancer, anal cancer, rectal cancer, gastric cancer, or bladder cancer.

27. The method according to claim 24, further comprising co-administering an effective amount of the therapeutic agent.

28. The method according to claim 24, wherein the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, an anti-estrogen agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA, or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof.

29. The method according to claim 24, wherein the subject is a human.

30. A solution comprising an isolated mAb or its antigen-binding fragment according to claim 1 in an effective concentration, wherein the solution is the target plasma.

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