Binding part for binding claudin 18.2 and use thereof

Abstract

A binding part for binding with Claudin 18.2 and a use thereof, in particular a fully human antibody that binds with the Claudin 18.2 protein and a use thereof. The antibody specifically binds to Claudin 18.2 without binding to Claudin 18.1, and the provided fully human antibody has high binding activity, low immunogenicity, and high safety.

Description

Claudin 18.2 binding moiety and its application

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application number CN202311281157.1 and application date September 28, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention belongs to the fields of tumor immunotherapy and molecular immunology, in particular to the field of antibodies, and specifically relates to a fully human antibody that binds to Claudin 18.2. Background Art

[0004] The claudins family is widely distributed in various epithelial tissues, with different tissues expressing different types of claudins. Claudin 18.2 is an adhesion molecule that forms tight junctions between cells, forming a barrier that regulates cell permeability. The human claudin 18.2 gene is located on chromosome 3, 3q22.3. Its first exon has two alleles, resulting in two splice variants of claudin 18.2: claudin 18.1 and claudin 18.2. These two variants differ in their N-terminal amino acid sequences and extracellular antigenic epitopes, thus preventing immune cross-reactivity. It is currently believed that claudin 18.2 is primarily regulated by a hypomethylated CpG island promoter within its coding sequence and by the transcription factor cAMP-response element blinding protein (CREB). Claudin 18.1 is primarily expressed in the lungs and rarely in other tissues. Claudin 18.2, on the other hand, is expressed in gastric epithelial cells, with particularly high expression in tissues such as gastric cancer, pancreatic cancer, and esophageal cancer, but rarely in lung tissue. The high tissue specificity of both proteins provides the molecular basis for Claudin 18.2's emergence as a therapeutic target.

[0005] During malignant transformation, cell polarity changes, exposing Claudin 18.2 to the surface of tumor cells. This allows for antibody targeting, making it an ideal target for monoclonal antibody development, particularly for solid tumor immunotherapy. Claudin 18.2 can serve as a potential specific marker for tumor diagnosis and treatment. Abnormal activation and overexpression of Claudin 18.2 have been observed in various types of malignant tumors: approximately 70% of gastric cancer patients have Claudin 18.2 overexpression in their primary tumors, while this rate is 50% for pancreatic cancer and 30% for esophageal cancer.

[0006] Currently, multiple immunotherapy strategies targeting Claudin 18.2 have been developed, including monoclonal antibodies (mAbs), bispecific antibodies (BsAbs), chimeric antigen receptor T cell (CAR-T) therapy, and antibody-drug conjugates (ADCs). Among them, the most advanced drug, Zolbetuximab, is a mouse chimeric monoclonal antibody with a human immunoglobulin 1 constant region. However, the development of antibodies against Claudin 18.2 still faces technical difficulties: First, from the perspective of the protein itself, Claudin 18.2 has four transmembrane domains, two extracellular loops, and the NH2 and COOH ends are located intracellularly. The extracellular loops of multi-transmembrane proteins are not independent domains. If only the extracellular segments are present, they cannot maintain a correct conformation and require the binding of the transmembrane segments to maintain the correct conformation. This greatly increases the difficulty of protein expression. Therefore, finding antigenic protein materials that can fully display the two extracellular loops of the protein for subsequent antibody selection is a fundamental part of the research. Secondly, Claudin 18.1 and Claudin 18.2 are both composed of 261 amino acids and have a high degree of structural consistency. Claudin 18.1 and Claudin 18.2 only differ by 7 amino acid residues in the sequence of approximately 50 amino acids in the extracellular loop ECL1, so it is crucial to obtain antibodies that specifically bind to Claudin 18.2. It is well known that humanized or fully human antibodies have low immunogenicity and are the direction of future antibody development. However, due to technical difficulties, no humanized or fully human antibodies that specifically bind to Claudin 18.2 have been reported to be approved for marketing. Therefore, it is necessary to develop a humanized or fully human antibody that specifically binds to Claudin 18.2.

[0007] Summary of the Invention

[0008] In a first aspect, the present invention provides a binding moiety that specifically binds to Claudin 18.2, comprising

[0009] A heavy chain variable region (VH), wherein the VH comprises: VH CDR1, VH CDR2 and VH CDR3; wherein the VH CDR1, CDR2 and CDR3 respectively comprise:

[0010] (1) the amino acid sequences shown in SEQ ID NOs: 2, 3, and 4;

[0011] (2) the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12; or

[0012] (3) the amino acid sequences shown in SEQ ID NOs: 28, 3 and 29,

[0013] Alternatively, a variant comprising up to 3 amino acid substitutions in said CDRs; and

[0014] (b) a light chain variable region (VL), wherein the VL comprises: VL CDR1, VL CDR2 and VL CDR3; wherein the VL CDR1, CDR2 and CDR3 respectively comprise:

[0015] (1) the amino acid sequence shown in SEQ ID NO: 6, EVT, and the amino acid sequence shown in SEQ ID NO: 8;

[0016] (2) the amino acid sequence shown in SEQ ID NO: 14, KVS, and the amino acid sequence shown in SEQ ID NO: 16;

[0017] (3) the amino acid sequence shown in SEQ ID NO: 19, EVS, and the amino acid sequence shown in SEQ ID NO: 21;

[0018] (4) the amino acid sequence set forth in SEQ ID NO: 24, the DTS, and the amino acid sequence set forth in SEQ ID NO: 26;

[0019] (5) the amino acid sequence shown in SEQ ID NO: 31, SNN, and the amino acid sequence shown in SEQ ID NO: 33;

[0020] (6) the amino acid sequence set forth in SEQ ID NO:36, AAS, and the amino acid sequence set forth in SEQ ID NO:38;

[0021] (7) the amino acid sequence shown in SEQ ID NO:40, GAS, and the amino acid sequence shown in SEQ ID NO:42; or

[0022] (8) the amino acid sequence shown in SEQ ID NO: 45, WAS, and the amino acid sequence shown in SEQ ID NO: 47,

[0023] Alternatively, it is a variant comprising up to 3 amino acid substitutions in said CDRs.

[0024] In some embodiments, wherein

[0025] The VH CDR1, CDR2 and CDR3 include the amino acid sequences shown in SEQ ID NOs: 2, 3 and 4, respectively, and the VL CDR1, CDR2 and CDR3 include the amino acid sequence shown in SEQ ID NO: 6, EVT and the amino acid sequence shown in SEQ ID NO: 8, respectively;

[0026] The VH CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and the VL CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 19, EVS, and the amino acid sequence of SEQ ID NO: 21, respectively;

[0027] The VH CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and the VL CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 24, the DTS, and the amino acid sequence of SEQ ID NO: 26, respectively;

[0028] The VH CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and the VL CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 36, AAS, and the amino acid sequence of SEQ ID NO: 38, respectively;

[0029] The VH CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and the VL CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 40, GAS, and the amino acid sequence of SEQ ID NO: 42, respectively;

[0030] The VH CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively; and the VL CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 14, KVS, and the amino acid sequence of SEQ ID NO: 16, respectively;

[0031] The VH CDR1, CDR2 and CDR3 include the amino acid sequences shown in SEQ ID NOs: 28, 3 and 29, respectively; and the VL CDR1, CDR2 and CDR3 include the amino acid sequence shown in SEQ ID NO: 31, SNN and the amino acid sequence shown in SEQ ID NO: 33, respectively; or

[0032] The VH CDR1, CDR2 and CDR3 include the amino acid sequences shown in SEQ ID NOs: 28, 3 and 29, respectively; and the VL CDR1, CDR2 and CDR3 include the amino acid sequence shown in SEQ ID NO: 45, WAS and the amino acid sequence shown in SEQ ID NO: 47, respectively.

[0033] In some embodiments, the VL is a Kappa light chain variable region or a Lambda light chain variable region.

[0034] In some embodiments, the VH comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 9, 17, 22, 27, 34, and 43; or the VL comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 13, 18, 23, 30, 35, 39, and 44.

[0035] In some embodiments, the VH and VL each comprise:

[0036] (1) the amino acid sequences shown in SEQ ID NOs: 1 and 5;

[0037] (2) the amino acid sequences shown in SEQ ID NOs: 9 and 13;

[0038] (3) the amino acid sequences shown in SEQ ID NOs: 17 and 18;

[0039] (4) the amino acid sequences shown in SEQ ID NOs: 22 and 23;

[0040] (5) the amino acid sequences shown in SEQ ID NOs: 27 and 30;

[0041] (6) the amino acid sequences shown in SEQ ID NOs: 34 and 35;

[0042] (7) the amino acid sequences shown in SEQ ID NOs: 34 and 39; or

[0043] (8) The amino acid sequences shown in SEQ ID NOs: 43 and 44.

[0044] In some embodiments, the binding portion is sdab, HcAb, Fab, Fab', F(ab')2, Fv, scFv, (scFv)2 or a full-length antibody.

[0045] In some embodiments, the binding portion is a mouse, chimeric, humanized or fully human binding portion.

[0046] In some embodiments, the binding portion is a fully human antibody.

[0047] In some embodiments, the binding portion is a fully human full-length antibody.

[0048] In a second aspect, the present invention provides a multispecific antibody or a binding fragment thereof, which comprises the binding portion described in the first aspect.

[0049] In a third aspect, the present invention provides a chimeric antigen receptor comprising:

[0050] (a) an extracellular antigen-binding domain, which comprises the binding portion of the first aspect or the multispecific antibody or binding fragment thereof of the second aspect;

[0051] (b) a transmembrane domain; and

[0052] (c) Intracellular signaling domain.

[0053] In a fourth aspect, the present invention provides a nucleic acid encoding the binding portion of the first aspect, the multispecific antibody or binding fragment thereof according to the second aspect, or the chimeric antigen receptor of the third aspect.

[0054] In a fifth aspect, the present invention provides a host cell comprising the nucleic acid described in the fourth aspect.

[0055] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the binding portion described in the first aspect, the multispecific antibody or its binding fragment described in the second aspect, the chimeric antigen receptor described in the third aspect or the host cell described in the fourth aspect and a pharmaceutically acceptable carrier.

[0056] In a seventh aspect, the present invention provides a method for treating a disease associated with Claudin 18.2 expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to the sixth aspect.

[0057] In some embodiments, the disease associated with Claudin 18.2 expression is gastric cancer, pancreatic cancer, esophageal cancer, breast cancer, colon cancer, liver cancer, ovarian cancer or lung cancer.

[0058] In an eighth aspect, the present invention provides use of the binding portion described in the first aspect, the multispecific antibody or binding fragment thereof described in the second aspect, the chimeric antigen receptor described in the third aspect, the nucleic acid described in the fourth aspect, or the host cell described in the fifth aspect in the preparation of a medicament for treating diseases related to Claudin 18.2 expression.

[0059] In some embodiments, the disease associated with Claudin 18.2 expression is gastric cancer, pancreatic cancer, esophageal cancer, breast cancer, colon cancer, liver cancer, ovarian cancer or lung cancer.

[0060] Beneficial effects of the present invention:

[0061] The amino acid sequence of a fully human antibody is 100% human and does not need to go through the humanization process. Compared with commonly used mouse antibodies or chimeric antibodies, it has low rejection reaction, good safety, and high drug success rate, and is the development trend of therapeutic antibodies. The 8 fully human antibodies targeting Claudin 18.2 provided by the present invention have undergone multiple screenings by Nanjing Pengbo's antibody production platform. Specifically: liquid phase panning was performed using a Claudin 18.2 protein close to its natural conformation; cell screening was performed using a Claudin 18.2 overexpressing cell line; and panning was performed using Claudin 18.1 as a negative cell. Compared with the transgenic mouse platform, this antibody production platform is based on the screening of a fully human natural library and has obtained better candidate molecules. It has low cost, no licensing fees for transgenic mice, and a cycle shortened by 1-2 months, which significantly accelerates the process of fully human antibody discovery and increases the probability of screening suitable molecules. The antibody binding to Claudin 18.2 provided by the present invention has high binding activity to Claudin 18.2, and EC 50 The affinity KD value is less than 10 nM, and even less than 0.1 nM. The affinity is higher than that of IMAB362. The antibody AHP33029 of the present invention has a CDC activity that is better than that of IMAB362. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1. ELISA EC analysis showing the interaction between fully human antibodies and human Claudin 18.2 protein 50 Test results.

[0063] Figure 2. FACS EC analysis of fully human antibodies and HEK293 cells overexpressing human Claudin 18.2 protein. 50 Test results.

[0064] Figure 3. FACS EC analysis of fully human antibodies and HEK293 cells overexpressing mouse Claudin 18.2 protein. 50 Test results.

[0065] Figure 4. FACS EC analysis of fully human antibodies and HEK293 cells overexpressing human Claudin 18.1 protein. 50 Test results.

[0066] Figure 5. Sensorgram showing the affinity determination of a fully human antibody to human Claudin 18.2 protein.

[0067] Figure 6 shows the results of epitope detection of fully human antibodies based on IMAB362.

[0068] Figure 7. Shows the in vitro CDC assay results of a fully human antibody targeting human Claudin 18.2.

[0069] Figure 8 shows the in vitro ADCC test results of a fully human antibody targeting human Claudin 18.2. DETAILED DESCRIPTION

[0070] The present invention relates to an antibody that binds to a Claudin 18.2 target and its application. The embodiments of the present invention are described in detail below with reference to the examples. Unless otherwise specified, the technical and scientific terms used in the present invention have the same meanings as those commonly understood by ordinary technicians in the field to which the present invention belongs.

[0071] As used herein, the terms "a" and "an" refer to one or more than one (ie, at least one) grammatical object of the article. For example, "antibody" refers to one antibody or more than one antibody.

[0072] As used herein, the term "binding moiety" refers to a molecule or portion of a molecule that binds to a target molecule (e.g., Claudin 18.2). A binding moiety may include a protein, peptide, nucleic acid, carbohydrate, lipid, or small molecular weight compound. In some embodiments, a binding moiety includes an antibody. In some embodiments, a binding moiety includes an antigen-binding fragment of an antibody. In some embodiments, a binding moiety includes a small molecular weight component. A binding moiety may also be an antibody or an antigen-binding fragment thereof. In some embodiments, a binding moiety includes a ligand-binding domain of a receptor. In some embodiments, a binding moiety includes the extracellular domain of a transmembrane receptor. A binding moiety may also be a ligand-binding domain of a receptor or the extracellular domain of a transmembrane receptor. A binding moiety may be monovalent, meaning that it contains one binding site that specifically interacts with a target molecule. A binding moiety may also be bivalent, meaning that it contains two binding sites that specifically interact with a target molecule. A binding moiety may also be multivalent, meaning that it contains multiple binding sites that specifically interact with a target molecule. A bivalent binding moiety or a multivalent binding moiety may interact with one or more epitopes on a single target molecule. A bivalent binding moiety or a multivalent binding moiety may also interact with two or more target molecules.

[0073] As used herein, the term "binding affinity" generally refers to the strength of the sum of the non-covalent interactions between the binding moiety and the target molecule. The binding of the binding moiety to the target molecule is a reversible process, and the affinity of the binding is usually reported as the equilibrium dissociation constant (KD). KD is the ratio of the dissociation rate (koff or kd) to the association rate (kon or ka). The lower the KD of the binding pair, the higher the affinity. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure. Specific illustrative embodiments include the following. In one embodiment, "KD" or "KD value" can be measured by an analysis known in the art, such as by a binding analysis. KD can be measured in a radiolabeled antigen binding assay (RIA) (Chen et al., (1999) J. Mol Biol 293: 865-881). KD or KD value can also be measured by Biacore surface plasmon resonance analysis using, for example, a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Piscataway, NJ), or biolayer interferometry using, for example, an Octet QK384 system (ForteBio, Menlo Park, CA). When a target molecule containing multiple epitopes contacts a binding moiety containing multiple binding sites that bind to the target molecule, the interaction of the binding molecule with the target molecule at one site will increase the probability of a reaction at a second site. The force of such multiple interactions between a multivalent antibody and an antigen is called avidity. For example, high avidity can compensate for low affinity, as is sometimes found for pentameric IgM antibodies, which may have lower affinity than IgG, but the high avidity of IgM due to its multivalency enables it to effectively bind the antigen. In some embodiments, the binding moieties provided herein bind to Claudin 18.2 with a KD value of less than 10 nM; in some embodiments, the binding moieties described herein bind to Claudin 18.2 with a KD value of less than 5 nM; in some embodiments, the binding moieties described herein bind to Claudin 18.2 with a KD value of less than 1 nM.

[0074] As used herein, the terms "targeting", "specific binding", "binding" mean that a polypeptide or molecule interacts with an epitope, protein or target molecule more frequently, more rapidly, for a longer duration, with greater affinity or with a certain combination thereof, compared to alternative substances including related and unrelated proteins. The binding portion (e.g., an antibody) that specifically binds to a target molecule (e.g., an antigen) can be identified, for example, by immunoassay, ELISA, SPR (e.g., Biacore) or other techniques known to those skilled in the art. Typically, the specific reaction will be at least twice the background signal or noise, and may be greater than 10 times the background signal. For discussion of antibody specificity, see, for example, Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York, pp. 332-336. The affinity of the binding portion of a specifically binding target molecule to the target molecule may be higher than its affinity for other different molecules. In some embodiments, a binding portion that specifically binds to a target molecule may bind to the target molecule with an affinity that is at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater than its affinity for another different molecule. In some embodiments, a binding portion that specifically binds to a particular target molecule binds to another different molecule with such low affinity that binding cannot be detected using an assay described herein or known in the art. In some embodiments, "specific binding" means, for example, that the binding portion has a KD of about 0.1 mM or less for the target molecule. In some embodiments, "specific binding" means that the polypeptide or molecule has a KD of about 10 μM or less, or about 1 μM or less for the target molecule. In some embodiments, "specific binding" means that the polypeptide or molecule has a KD of about 0.1 μM or less, or about 1 nM or less for the target molecule. Due to sequence identity between homologous proteins in different species, specific binding can include polypeptides or molecules that recognize proteins or targets in more than one species. Similarly, due to homology within certain regions of polypeptide sequences of different proteins, specific binding can include polypeptides or molecules that recognize more than one protein or target. It will be understood that in some embodiments, a binding moiety that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (but can include) exclusive binding, i.e., binding to a single target. Thus, in some embodiments, a binding moiety can specifically bind to more than one target. For example, in some cases, an antibody may include two identical antigen binding sites, each of which specifically binds to the same epitope on two or more proteins.In certain alternative embodiments, the antibody can be bispecific and comprise at least two antigen-binding sites with different specificities. In some embodiments, the binding moiety specifically binds to human Claudin 18.2. In some embodiments, the binding moiety specifically binds to mouse Claudin 18.2. In some embodiments, the binding moiety specifically binds to cynomolgus monkey Claudin 18.2.

[0075] As used herein, the term "binding portion" means a portion capable of binding to an antigen, including antibodies and binding fragments thereof. As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target via at least one antigen binding site, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, wherein the antigen binding site is generally within the variable region of the immunoglobulin molecule. As used herein, the term encompasses full-length polyclonal antibodies, full-length monoclonal antibodies, single-chain Fv (scFv) antibodies, light chain antibodies (LCAbs), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, fusion proteins comprising an antigen binding site of an antibody, and any other modified immunoglobulin molecule (e.g., dual variable domain immunoglobulin molecules) comprising an antigen binding site, as long as the antibody exhibits the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the designations of their heavy chain constant domains, which are designated α, δ, ε, γ, and μ, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or bound to other molecules, including but not limited to toxins and radioactive isotopes. Unless expressly indicated otherwise, the term "antibody," as used herein, includes "antigen-binding fragments" of intact antibodies.

[0076] In some embodiments, the antibodies described herein are monoclonal antibodies. In some embodiments, the antibodies described herein are fully human monoclonal antibodies. In some embodiments, the antibodies described herein are fully human full-length antibodies.

[0077] The term "antigen-binding fragment" as used in conjunction with an antibody refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibody molecules (e.g., scFv), light chain antibodies (LCAb), heavy chain antibodies (HcAb), disulfide-linked scFv (dsscFv), bispecific antibodies, trispecific antibodies, tetraspecific antibodies, minibodies, dual variable domain antibodies (DVD), and multispecific antibodies formed from antibody fragments.

[0078] As used herein, the term "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. In general, the variable regions of the heavy and light chains are each composed of four framework regions (FRs) and three complementarity determining regions (CDRs) (also known as "hypervariable regions"). The CDRs in each chain are held together tightly by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody. Regarding the determination of CDRs, there are at least two techniques: (1) methods based on cross-species sequence variability (Kabat et al., 1991, Sequences of Proteins of Immunological Interest (5th ed.) Bethesda, MD: National Institutes of Health); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., 1997, Journal of Mol. Biol., 273(4):927-48). Furthermore, a combination of these two methods is used in the art and can be used to determine CDRs. In some embodiments, the binding moiety of the present invention comprises a heavy chain variable region that is an IgG1 heavy chain variable region. In some embodiments, the binding moiety of the present invention comprises a light chain variable region that is a kappa light chain variable region. In some embodiments, the light chain variable region is a lambda light chain variable region. In some embodiments, the kappa light chain variable region sequence comprises the amino acid sequence set forth in SEQ ID NO: 13, 35, 39, or 44. In some embodiments, the lambda light chain variable region sequence comprises the amino acid sequence set forth in SEQ ID NO: 5, 18, 23, or 30.

[0079] The term "single-chain variable fragment" or "scFv" refers to a fusion protein of the heavy and light chain variable regions of an immunoglobulin, linked to a short linker peptide of ten to twenty-five amino acids. The linker is typically rich in glycine for flexibility and serine or threonine for solubility. ScFvs retain the specificity of the original immunoglobulin. ScFvs can be linked by linkers of varying lengths to form bi-scFvs, bispecific antibodies, tri-scFvs, trispecific antibodies, or tetraspecific antibodies, which can exhibit specificity for one or more antigens.

[0080] The term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that is transplanted into immune effector cells (usually T cells) and enhances T cell function. The new generation of CARs includes an extracellular binding domain, which includes scFv, as well as a hinge region, a transmembrane domain, and an intracellular signaling domain (primarily the CD3-ζ cytoplasmic domain (which is the primary transmitter of T cell activation signals) plus one or more costimulatory domains). CARs can further add factors that enhance T cell expansion, persistence, and anti-tumor activity, such as cytokines and costimulatory ligands.

[0081] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to those of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and / or binding ability, while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in the species.

[0082] As used herein, the term "fully human antibody" refers to an antibody produced by human cells and prepared using any technique known in the art or an antibody having an amino acid sequence corresponding to an antibody produced by humans.

[0083] The terms "polypeptide," "peptide," and "protein," as used interchangeably herein, refer to polymers of amino acids of any length, which may be linear or branched. They may contain non-natural or modified amino acids, or be interrupted by non-amino acids. Polypeptides, peptides, or proteins may also be modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.

[0084] The terms "polynucleotide" and "nucleic acid," as used interchangeably herein, refer to polymers of nucleotides of any length and include DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

[0085] As used herein, the term "consistent" or "consistency" percentage in the context of two or more nucleic acids or polypeptides refers to that when compared and aligned (optionally introducing gaps) for maximum consistency, two or more sequences or subsequences are identical or have a specified percentage of identical nucleotides or amino acid residues, without considering any conservative amino acid substitutions as part of sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain the alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin software packages, and variations thereof. In some embodiments, two nucleic acids or polypeptides of the present invention are substantially identical, meaning that when compared and aligned for maximum consistency, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, there is consistency in the region where the length of the amino acid sequence is at least about 10 residues, at least about 20 residues, at least about 40 to 60 residues, at least about 60 to 80 residues, or any integer value therebetween. In some embodiments, there is consistency in the region where the length of the amino acid sequence is at least about 10 residues, at least about 20 residues, at least about 40 to 60 residues, at least about 60 to 80 residues, or any integer value therebetween. In some embodiments, there is consistency in the region where the length of the amino acid sequence is longer than 60 to 80 residues (e.g., at least about 80 to 100 residues), and in some embodiments, the sequence is substantially consistent over the total length of the compared sequence (e.g., the coding region of a target protein or antibody). In some embodiments, there is consistency in the region where the length of the nucleotide sequence is at least about 10 bases, at least about 20 bases, at least about 40 to 60 bases, at least about 60 to 80 bases, or any integer value therebetween. In some embodiments, there is consistency in the region where the length of the nucleotide sequence is longer than 60 to 80 bases (e.g., at least about 80 to 1000 bases or more), and in some embodiments, the sequence is substantially consistent over the total length of the compared sequence (e.g., the nucleotide sequence encoding the protein of interest).

[0086] As used herein, the term "amino acid substitution" refers to the replacement of an amino acid residue with another amino acid residue in a polypeptide sequence. A "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue containing a side chain with similar chemical characteristics. Families of amino acid residues with similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), polar uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, replacing tyrosine with phenylalanine is a conservative substitution. In general, conservative substitutions in the sequences of the polypeptides, soluble proteins and / or antibodies of the present disclosure do not eliminate binding of the polypeptides, soluble proteins or antibodies containing the amino acid sequence to the target binding site. Methods for identifying conservative substitutions of amino acids that do not eliminate binding are well known in the art.

[0087] As used herein with respect to a binding portion (e.g., an antibody) comprising a polypeptide having a particular sequence characteristic ("reference binding portion"), the term "variant" refers to a different binding portion having a polypeptide comprising one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid sequence substitutions, deletions, and / or additions compared to the reference binding portion. Anti-Claudin 18.2 binding portion variants or anti-Claudin 18.2 antibody variants retain at least specific binding to Claudin 18.2. In some embodiments, a binding portion variant can be generated by one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1 to about 3) changes in the amino acid sequence of the reference binding portion. Furthermore, for example, variants of anti-Claudin 18.2 antibodies can be generated by one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1 to about 3) amino acid changes from the amino acid sequence of a reference anti-Claudin 18.2 antibody. The changes in the amino acid sequence can be amino acid substitutions. In some embodiments, the changes in the amino acid sequence can be conservative amino acid substitutions. In some embodiments, variant anti-Claudin 18.2 binding portions or anti-Claudin 18.2 antibody variants can be generated by one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1 to about 3) amino acid substitutions in a VH or VL region or subregion (e.g., one or more CDRs). In some embodiments, variant anti-Claudin 18.2 binding portions or anti-Claudin 18.2 antibody variants can be generated by one, at most two, at most three, at most four, or at most five amino acid substitutions in each of the VH or VL regions. In some embodiments, anti-Claudin 18.2 binding portion variants or anti-Claudin 18.2 antibody variants can be generated by one, at most two, at most three, at most four, or at most five amino acid substitutions in each of the CDR regions.

[0088] The term "vector" refers to a material for carrying or comprising a nucleic acid sequence (comprising, for example, so that the nucleic acid sequence is introduced into a host cell). Suitable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include operable selection sequences or markers that are stably incorporated into the chromosome of the host cell. In addition, the vector may include one or more selectable marker genes and appropriate expression control sequences. The selectable marker gene that may be included may, for example, provide resistance to antibiotics or toxins, supplement nutritional deficiencies, or provide key nutrients that are not present in the culture medium. The expression control sequence may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like as known in the art. When two or more nucleic acid molecules are to be co-expressed (for example, antibody heavy chain and light chain or antibody VH and VL), the two nucleic acid molecules may, for example, be inserted into a single expression vector or into separate expression vectors. For single vector expression, the encoding nucleic acid may be operably linked to a common expression control sequence or to different expression control sequences, for example, an inducible promoter and a constitutive promoter. Methods well known in the art may be used to confirm that the nucleic acid molecules are introduced into the host cell. Those skilled in the art will understand that nucleic acid molecules are expressed in sufficient amounts to produce a desired product (e.g., an anti-Claudin 18.2 antibody as described herein), and further understand that expression levels can be optimized using methods well known in the art to obtain sufficient expression.

[0089] As defined herein, a "subject" is any animal (e.g., a mammal) that is the recipient of a particular treatment, including but not limited to humans, non-human primates, canines, felines, rodents, and the like. In some embodiments, the subject is a human. A "subject" may be a patient suffering from a particular disease. In some embodiments, the subject is a patient suffering from a cancer or tumor that expresses Claudin 18.2.

[0090] The term "treating," as used herein in conjunction with a disease or condition, or a subject suffering from a disease or condition, refers to the act of inhibiting, eliminating, alleviating, and / or ameliorating the symptoms, severity of symptoms, and / or frequency of symptoms associated with the disease or condition being treated. When used with reference to a cancer or tumor, the term "treating" refers to the act of lessening the severity of a cancer or tumor, or delaying or slowing the progression of a cancer or tumor, including (a) inhibiting the growth or arresting the development of a cancer or tumor, or (b) causing regression of a cancer or tumor, or (c) delaying, ameliorating, or minimizing one or more symptoms associated with the presence of a cancer or tumor.

[0091] As used herein, the term "administering" refers to the act of delivering or causing a therapeutic agent or pharmaceutical composition to be delivered to the body of an individual by any method described herein or otherwise known in the art. The therapeutic agent can be a compound, a polypeptide, a cell, or a population of cells. Administering a therapeutic agent or pharmaceutical composition comprises prescribing the therapeutic agent or pharmaceutical composition to be delivered into the patient's body. Exemplary forms of administration include oral dosage forms, such as tablets, capsules, syrups, suspensions; injectable dosage forms, such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP); transdermal dosage forms, including creams, gels, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

[0092] As used herein, the term "therapeutically effective amount" refers to an amount of a compound, polypeptide, cell, formulation, substance, or composition as described herein sufficient to provide a therapeutic benefit in the treatment of a disease or condition or to delay or minimize one or more symptoms associated with the disease or condition. The disease or condition may be a cancer or tumor that expresses Claudin 18.2.

[0093] As used herein, the term "carrier" includes "pharmaceutically acceptable carriers," excipients, or stabilizers that are nontoxic to cells or mammals exposed to the carrier at the doses and concentrations employed. The term "carrier" may also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Compositions comprising a pharmaceutical compound may contain a prophylactically or therapeutically effective amount of, for example, an anti-β-klotho antibody in isolated or purified form and a suitable amount of a carrier so as to provide a form suitable for administration to an individual (e.g., a patient). The formulation should be appropriate for the mode of administration.

[0094] "Claudin 18.2," "Claudin 18.2," or "CLDN18.2" are used herein to refer to the type 2 isoform of Claudin 18, a member of the Claudin family of cell surface proteins. Claudin is an important component of tight cell junctions, which form a paracellular barrier that controls the flow of molecules between cells. Different claudins are expressed on different tissues, and their altered functions have been associated with the development of cancer in these tissues. In normal tissue, expression of Claudin-18.2 is restricted to epithelial cells of the gastric mucosa. Claudin 18.2 expression is maintained after malignant transformation of gastric cancer and its metastases. Ectopic activation of Claudin 18.2 has also been found in pancreatic, esophageal, ovarian, and lung tumors.

[0095] The antibody may be Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0096] In some embodiments, the Claudin 18.2 binding portion comprises an antibody. In some embodiments, the Claudin 18.2 binding portion comprises an antigen-binding fragment of an antibody. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0097] In some embodiments, the claudin 18.2 binding portion comprises a Fab. In some embodiments, the antibody is a Fab'. In some embodiments, the claudin 18.2 binding portion comprises a F(ab')2. In some embodiments, the claudin 18.2 binding portion comprises an Fv. In some embodiments, the claudin 18.2 binding portion comprises an scFv. In some embodiments, the claudin 18.2 binding portion comprises a disulfide-linked scFv [(scFv)2]. In some embodiments, the claudin 18.2 binding portion comprises a bispecific antibody (dAb).

[0098] In some embodiments, the Claudin 18.2 binding portion comprises a recombinant antibody. In some embodiments, the Claudin 18.2 binding portion comprises a monoclonal antibody. In some embodiments, the Claudin 18.2 binding portion comprises a polyclonal antibody. In some embodiments, the Claudin 18.2 binding portion comprises a chimeric antibody. In some embodiments, the Claudin 18.2 binding portion comprises a humanized antibody. In some embodiments, the Claudin 18.2 binding portion comprises a human antibody.

[0099] In some embodiments, the Claudin 18.2 binding moiety comprises a bispecific binding moiety. In some embodiments, the Claudin 18.2 binding moiety comprises a multispecific binding moiety.

[0100] In some embodiments, an anti-claudin 18.2 antibody binds to claudin 18.2 with an affinity that is at least 20-fold greater than the affinity with which the antibody binds to claudin 18.1. In some embodiments, an anti-claudin 18.2 antibody binds to claudin 18.2 with an affinity that is at least 50-fold greater than the affinity with which the antibody binds to claudin 18.1. In some embodiments, an anti-claudin 18.2 antibody binds to claudin 18.2 with an affinity that is at least 100-fold greater than the affinity with which the antibody binds to claudin 18.1. In some embodiments, the anti-claudin 18.2 antibody does not detectably bind to claudin 18.1.

[0101] The CDRs of antibodies are defined by a variety of methods / systems by those skilled in the art. These systems and / or definitions have been developed and improved over many years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and is commonly used. The Chothia definition is based on the position of the structural loop region. The IMGT system is based on sequence variability and position within the structure of the variable domain. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analysis of available antibody crystal structures. An exemplary system is a combination of Kabat and Chothia. Software programs (e.g., abYsis) for analyzing antibody sequences and determining CDRs are available and known to those skilled in the art.

[0102] The specific CDR sequences defined herein are generally based on the Kabat definition. However, it will be understood that reference to one or more heavy chain CDRs and / or one or more light chain CDRs of a particular antibody will encompass all CDR definitions as known to those skilled in the art.

[0103] In some embodiments, the Claudin 18.2 binding portion is an anti-Claudin 18.2 antibody that includes one, two, three, four, five, and / or six CDRs of any of the antibodies described herein.

[0104] In some embodiments, the Claudin 18.2 binding moiety is a human antibody. Human antibodies can be prepared using various techniques known in the art. In some embodiments, human antibodies are produced by immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are produced by lymphocytes isolated from immune individuals. In any case, cells that produce antibodies against the target antigen can be produced and isolated. In some embodiments, human antibodies are selected from a phage library, wherein the phage library expresses human antibodies. Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable region gene repertoires from unimmunized donors. The technology for the production and use of antibody phage libraries is well known in the art. Once antibodies are identified, affinity maturation strategies known in the art (including but not limited to chain shuffling and site-directed mutagenesis) can be used to produce higher affinity human antibodies. In some embodiments, human antibodies are produced in transgenic mice containing human immunoglobulin loci. After immunization, these mice are able to produce a full spectrum of human antibodies in the absence of endogenous immunoglobulin production.

[0105] Unless otherwise stated, the methods and materials of the embodiments described below are conventional products that can be purchased on the market. Those skilled in the art will understand that the methods and materials described below are merely exemplary and should not be considered as limiting the scope of the present invention.

[0106] Example 1: Obtaining positive clones of human Claudin 18.2 fully human antibody

[0107] Using a fully human natural library to screen for lead antibody molecules specifically targeting human Claudin 18.2:

[0108] Using a fully human naive library developed by Pengbo Bio, two screening strategies were employed. The first involved four rounds of liquid-phase panning (liquid-cell-liquid-cell) using biotinylated Claudin 18.2 protein and cell panning using the positive HEK293 / Human Claudin 18.2 cell line and the negative HEK293 / Human Claudin 18.1 cell line, respectively. The second involved three rounds of cell panning using the positive HEK293 / Human Claudin 18.2 cell line and the negative HEK293 / Human Claudin 18.1 cell line to obtain phage library eluates. The neutralized phage panning eluate was added to a culture of TG1 bacteria in the logarithmic growth phase, mixed, and incubated at 37°C for 45 minutes. After sufficient infection, the culture was serially diluted, plated on agar plates with the relevant resistance, and incubated inverted at 37°C overnight. The next day, prepare a 96-well sterile deep-well plate with 0.5 ml of 2YT medium (with 0.2% w / v glucose and 0.1 mg / ml ampicillin) dispensed into each well. Use a sterile pipette to pick a single colony from the plate and incubate it in the plate with shaking at 37°C, 220 rpm, and overnight (16-18 hours). The next day, transfer 0.05 ml of the overnight culture to a freshly prepared sterile deep-well plate (with 0.45 ml of 2YT medium containing a final concentration of 0.1 mg / ml ampicillin). Cultivate to an OD600 of approximately 0.6-0.8, then add 0.5 ml of induction medium (2YT containing 0.1 mg / ml ampicillin, 0.2 mM IPTG, 0.2% glycine, 0.04% Triton X-100, and 0.4% glycerol) and incubate in a shaker at 30°C, 220 rpm, and overnight (16-18 hours). The overnight expressed bacterial solution was centrifuged at 4000 rpm for 10 minutes to obtain the soluble expression supernatant, which was used to detect the binding of the selected monoclonal clones to the positive cell HEK293 / Human Claudin 18.2 cell line and the negative cell HEK293 / Human Claudin 18.1 cell line (Nanjing Pengbo) by FACS to obtain positive antibody clones.

[0109] FACS assay: FACS binding assays were used to evaluate the ability of antibodies in the supernatant to bind to human Claudin 18.2 and Claudin 18.1 antigens expressed on the cell membranes of HEK293 / Human Claudin 18.2 and HEK293 / Human Claudin 18.1 cells. HEK293 / Human Claudin 18.2 and HEK293 / Human Claudin 18.1 cells were harvested and washed three times with PBS. 1×105 The cells were incubated with 50 μl of the supernatant at 4°C for 40 minutes. The cells were then washed three times with PBS and 100 μl of Alexa Fluor was added to the well plate. 647-labeled goat anti-human IgG F(ab')2 fragment antibody (Jackson, 109-605-006) was incubated for 30 minutes at 4°C. Finally, the cells were washed three times with PBS, resuspended in PBS, and the fluorescence signal of the secondary antibody was read using FACS Calibur (BD).

[0110] Example 2: Variable region sequencing of positive clones and recombinant production of monoclonal antibodies

[0111] Based on the FACS test results, the corresponding bacterial liquid of the positive clones was selected for culture, plasmid extraction, PCR and single clone Sanger sequencing. Finally, 8 positive clones were obtained. The corresponding relationship between the clones and their sequence numbers is shown in Table 1.

[0112] Table 1. Positive clones and their corresponding sequence numbers

[0113] DNA fragments containing codon-optimized heavy chain, kappa light chain variable region, and lambda light chain variable region were synthesized and inserted into the huIgG1-opt_pcDNA3.4, hukappa-opt_pcDNA3.4, and hulambda-opt_pcDNA3.4 expression vectors, respectively. The three vectors were synthesized by Nanjing Pengbo to form expression plasmids.

[0114] The above plasmids were transfected into CHO-S cells with the heavy and light chain plasmids according to the corresponding clone numbers. After 7 days of culture in a shake flask at 37°C, the supernatant was collected for antibody purification. Prior to purification, the tubing and Protein A column were depyrogenated with 0.2M NaOH. The column was re-equilibrated with a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0). The harvested cell culture supernatant was then diluted 1:1 with 2× the above buffer and sterilized by filtration. The filtered supernatant was incubated with the Protein A column at room temperature for 2 hours, and the column was washed with 1× the above buffer. IgG was eluted with sterile 0.1M sodium citrate (pH 3.5). The eluate was collected and neutralized with one-ninth volume of sterile 1M Tris-HCl (pH 9.0). Under sterile conditions, the product buffer was exchanged into PBS (pH 7.4) to remove any elution buffer and the sample was concentrated. After concentration, the antibodies were quantified by OD280nm using an extinction coefficient Ec of 1.43 (0.1%).

[0115] The purified antibodies were analyzed by SDS-PAGE using a BioRad electrophoresis system with a 10% precast gel (GenScript). The gel was stained with Estain 2.0 (GenScript) and the molecular size and purity were estimated by comparing the stained bands with Protein Ladder (GenScript).

[0116] Example 3: ELISA Binding Detection of Fully Human Antibodies to Human Claudin 18.2 Protein

[0117] Indirect ELISA was used to evaluate the binding ability of the above-mentioned purified antibodies to the recombinant protein of human Claudin 18.2 (GenScript, Z03504). The human Claudin 18.2 protein was diluted to 1 μg / ml with carbonate buffer, 100 μl / well was added to the ELISA plate, and coated at 4°C for 16 hours. The plate was washed with PBST (0.05% Tween), and 300 μl / well of PBS containing 3% skim milk was added and blocked at 37°C for 1 hour. The blocking solution was then discarded, and 100 μl of purified antibody diluted 3 times from the first well at 15 μg / ml (about 100 nM) was added, for a total of 11 test concentration gradients. PBST (0.05% Tween) was added to the last well and incubated in a 37°C oven for 1 hour. The plate was washed 3 times with PBST, and 100 μl / well of mouse anti-human IgG Fc fragment conjugated with horseradish peroxidase (GenScript, A01854) was added and incubated at 37°C for 0.5 hours. The plate was washed 6 times with PBST, and then TMB colorimetric solution (GenScript) was added and incubated at room temperature in the dark for 10 minutes. The reaction was terminated by adding 50 μl of 1M HCl stop solution (Sigma), and the plate was read at 450 nm using a microplate reader. The ELISA binding results of 8 positive recombinant antibodies to human Claudin 18.2 protein are shown in Figure 1. The specific EC values ​​of each antibody are as follows: 50 As shown in Table 2 below: Compared with the Yang Shen antibody zotuximab IMAB362 (HuIgG1), the binding ability of these tested antibodies to the human Claudin 18.2 antigen protein is significantly better than that of the Yang Shen antibody.

[0118] Table 2. ELISA EC of purified antibodies and human Claudin 18.2 related proteins 50 Detection

[0119] Example 4: FACS EC analysis of fully human antibodies against HEK293 cell lines expressing human, mouse Claudin 18.2, and human Claudin 18.1 50

[0120] HEK-293 cell lines expressing human, mouse, and human Claudin 18.2 were collected and washed three times with PBS. 1×10 5 Add 100 μl of purified antibody at 45 μg / ml (about 300 nM) to each test cell and 3-fold gradient dilution to a total of 11 test concentration gradients, and incubate at 4°C for 45 minutes. Then wash the cells 3 times with PBS, add 100 μl of FITC-labeled goat anti-human IgG F(ab')2 fragment antibody, and incubate at 4°C for 30 minutes. Finally, wash the cells 3 times with PBS and read the signal with FACS BD Calibur. As shown in Figure 2-4, the antibody binds to HEK-293 cells expressing human and mouse Claudin 18.2, but does not bind to HEK-293 cells expressing human Claudin 18.1. Specific FACS EC values ​​of the antibody 50 As shown in Table 3 below: Compared with the Yangshen antibody IMAB362, the test antibody has species cross-reactivity with mouse Claudin 18.2, and its binding ability to HEK-293 cells expressing human and mouse Claudin 18.2 is stronger or equivalent than that of the Yangshen antibody, and it has no binding to HEK-293 cells expressing human Claudin 18.1.

[0121] Table 3. FACS EC of fully human antibodies against Claudin 18.2 and Claudin 18.1 associated cells 50 Detection

[0122] Example 5: SPR Binding Detection of Fully Human Antibodies to Human Claudin 18.2 Protein

[0123] The affinity of the purified antibody to the human Claudin 18.2 protein was determined using the surface plasmon resonance (SPR) biosensor Biacore 8K (Cytiva). The antibody was immobilized on the sensor chip by Fc capture. The human Claudin 18.2 protein was used as the analyte. The dissociation (kd) and association (ka) rate constants were obtained using the Biacore 8K evaluation software, and the equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka. The results of the SPR affinity determination are shown in Table 4 below, and the related sensor diagram is shown in Figure 5. The test antibody and the Yangshen antibody IMAB362 have comparable affinity levels to the human Claudin 18.2 antigen protein, and the equilibrium dissociation constant (KD) is within 10 -8 within the range.

[0124] Table 4. Affinity determination of humanized fully human antibodies and human Claudin 18.2 protein

[0125] Example 6: Identification of the binding epitope of a fully human antibody to human Claudin 18.2 protein

[0126] Indirect ELISA was used to evaluate whether the binding epitope of the above-mentioned purified antibodies to the human Claudin 18.2 protein was the same as that of the Yangshen antibody IMAB362. The ELISA plate was coated with 1 μg / ml of recombinant human Claudin 18.2 protein using 100 μl / well of CBS and coated overnight at 4°C. The plate was washed with PBST (0.05% Tween) and blocked with 300 μl / well of PBS containing 3% skim milk at 37°C for 1 hour. The blocking solution was then discarded, and 50 μl of 30 μg / ml (about 200 nM) of purified antibody was added to the first well, and diluted in a 3-fold gradient for a total of 11 test concentration gradients, and incubated at 37°C for 1 hour. Then 50 μl of 0.72 μg / ml biotinylated Yangshen antibody was added and incubated at 37°C for 1 hour. The plate was washed 3 times with PBST, and 100 μl / well of horseradish peroxidase-conjugated streptavidin antibody (Kinsher) was added and incubated at 37°C for 0.5 hours. The plate was washed 6 times with PBST, and then TMB colorimetric solution (Kinsher) was added and incubated at room temperature in the dark for 10 minutes. The reaction was terminated by adding 50 μl of 1M HCl stop solution (Sigma). The plate was read at 450 nm using a microplate reader. The results of the binding epitope detection experiment of the fully human antibody and the human Claudin 18.2 protein are shown in Figure 6. The specific IC 50 As shown in Table 5 below: The binding epitope of the test antibody to the human Claudin 18.2 protein is the same as that of the Yangshen antibody IMAB362.

[0127] Table 5. ELISA IC values ​​of purified antibodies and human Claudin 18.2-related proteins 50 Detection

[0128] Example 7: In vitro CDC experiment of fully human antibodies targeting human Claudin 18.2

[0129] Adjust the CHO-K1 / human Claudin 18.2 cell suspension to an appropriate plating density (5000 cells / well) using CDC buffer. Add the target cell suspension to the appropriate 384-well assay plate. Immediately add the control working solution, sample working solution, and CDC buffer, mix thoroughly, and incubate in a cell culture incubator (37°C, 5% CO2) for approximately 30 minutes. Dilute and mix the NHSCs in CDC buffer to prepare a working solution (at a concentration 4 times the final assay concentration). After incubation, add the NHSC working solution to the appropriate wells of the 384-well assay plate. Mix thoroughly, transfer to a cell culture incubator, and incubate at 37°C, 5% CO2 for approximately 4 hours. Remove the Cell Titer-Glo Luminescent working solution and allow it to return to room temperature before removing the 384-well assay plate. As needed, add 30 μl of Cell Titer-Glo Luminescent working solution to each well, mix thoroughly, and incubate at room temperature for 5-10 minutes. Luminescence signals were read using a microplate reader, and the percentage of cell lysis caused by CDC was calculated. The results showed that the CDC activity of the tested molecules was superior to or comparable to that of Yang Shen, with AHP33029 showing the highest CDC activity. Detailed results are shown in Table 6 and Figure 7.

[0130] The calculation formula is as follows:

[0131] Cell lysis % = 100% × (1-(RLU Sample -RLU NHSC ) / (RLU cell+NHSC -RLU NHSC ))

[0132] Note: Sample group = target cells + test sample + NHSC working solution, used to detect sample-mediated complement killing signals; Cell + NHSC group = target cells + NHSC working solution, used to detect the signal level under the condition of natural lysis of all target cells;

[0133] NHSC group = only NHSC working solution, used to detect the background signal value of NHSC;

[0134] Table 6. In vitro CDC test results of fully human antibodies targeting human Claudin 18.2

[0135] Example 8: In vitro ADCC experiment of fully human antibodies targeting human Claudin 18.2

[0136] Remove cells from the CO2 incubator and observe them under an inverted microscope. Then, transfer them to a biosafety cabinet that has been UV-irradiated for at least 30 minutes. Discard the culture medium and wash the cells once with 4-6 ml of DPBS. Add 1-2 ml of Accutase to digest the cells. Once the cells become round and detach from the wall, gently shake them until they naturally fall off. Stop the digestion with 4-6 ml of complete culture medium. Collect the cell suspension into a centrifuge tube and centrifuge at 125 × g for 5 minutes. Discard the supernatant and resuspend the cells in complete culture medium. Mix thoroughly and count the cells. Prepare a target cell suspension of the appropriate volume and density using complete culture medium based on the number of samples. Transfer the target cell suspension (CHO-K1 / Claudin 18.2) to the appropriate wells of a 96-well assay plate. During transfer, be sure to pipette the target cells to ensure a uniform cell suspension. Transfer different concentrations of control / sample working solution to the appropriate wells of the assay plate according to the experimental layout and incubate at room temperature for 30 minutes. Collect the effector cells GS-J2C / CD16A 158V and resuspend the effector cells in the experimental buffer, mix and count, and use the experimental buffer to prepare a volume of effector cell suspension with a certain density according to the number of samples. Transfer a certain effector cell suspension to the corresponding wells of the 96-well experimental plate. Pay attention to blowing the cells during the transfer process to keep the cell suspension in a uniform state. Transfer the experimental plate to a carbon dioxide incubator (37°C, 5% CO2) and incubate for 6 hours. After the incubation of the experimental plate is completed, take out the experimental plate and equilibrate to room temperature; according to the experimental arrangement, add a certain volume of fluorescent detection reagent to each well and incubate at room temperature in the dark for 5 to 10 minutes; then use an enzyme reader to read the chemiluminescence signal value. The results show that the activity of the ADCC reporter gene experiment of the test molecule is better than or equivalent to that of Yang Shen. Detailed results are shown in Table 7 and Figure 8.

[0137] The calculation formula is as follows:

[0138] Y=Bottom+(Top-Bottom) / (1+10^((LogEC 50 -X)×HillSlope))

[0139] X = independent variable

[0140] Bottom = Minimum dose response

[0141] HillSlope = curvature parameter hillslope

[0142] LogEC 50 = half-dose concentration EC 50

[0143] Top = Maximum dose response

[0144] Table 7. In vitro ADCC test results of fully human antibodies targeting human Claudin 18.2

[0145] The amino acid and nucleotide sequence information mentioned in this article are as follows:

[0146] AHP29153-VH fully human antibody variable region amino acid sequence: SEQ ID NO: 1

[0147] AHP29153-VH CDR1 region amino acid sequence: SEQ ID NO: 2

[0148] AHP29153-VH CDR2 region amino acid sequence: SEQ ID NO: 3

[0149] AHP29153-VH CDR3 region amino acid sequence: SEQ ID NO: 4

[0150] AHP29153-VL fully human antibody variable region amino acid sequence: SEQ ID NO: 5

[0151] AHP29153-VL CDR1 region amino acid sequence: SEQ ID NO: 6

[0152] AHP29153-VL CDR2 region amino acid sequence: SEQ ID NO: 7

[0153] AHP29153-VL CDR3 region amino acid sequence: SEQ ID NO: 8

[0154] AHP30826-VH fully human antibody variable region amino acid sequence: SEQ ID NO: 9

[0155] AHP30826-VH CDR1 region amino acid sequence: SEQ ID NO: 10

[0156] AHP30826-VH CDR2 region amino acid sequence: SEQ ID NO: 11

[0157] AHP30826-VH CDR3 region amino acid sequence: SEQ ID NO: 12

[0158] AHP30826-VL fully human antibody variable region amino acid sequence: SEQ ID NO: 13

[0159] AHP30826-VL CDR1 region amino acid sequence: SEQ ID NO: 14

[0160] AHP30826-VL CDR2 region amino acid sequence: SEQ ID NO: 15

[0161] AHP30826-VL CDR3 region amino acid sequence: SEQ ID NO: 16

[0162] AHP31548-VH fully human antibody variable region amino acid sequence: SEQ ID NO: 17

[0163] AHP31548-VH CDR1 region amino acid sequence: SEQ ID NO: 2

[0164] AHP31548-VH CDR2 region amino acid sequence: SEQ ID NO: 3

[0165] AHP31548-VH CDR3 region amino acid sequence: SEQ ID NO: 4

[0166] AHP31548-VL fully human antibody variable region amino acid sequence: SEQ ID NO: 18

[0167] AHP31548-VL CDR1 region amino acid sequence: SEQ ID NO: 19

[0168] AHP31548-VL CDR2 region amino acid sequence: SEQ ID NO: 20

[0169] AHP31548-VL CDR3 region amino acid sequence: SEQ ID NO: 21

[0170] AHP32913-VH fully human antibody variable region amino acid sequence: SEQ ID NO: 22

[0171] AHP32913-VH CDR1 region amino acid sequence: SEQ ID NO: 2

[0172] AHP32913-VH CDR2 region amino acid sequence: SEQ ID NO: 3

[0173] AHP32913-VH CDR3 region amino acid sequence: SEQ ID NO: 4

[0174] AHP32913-VL fully human antibody variable region amino acid sequence: SEQ ID NO: 23

[0175] AHP32913-VL CDR1 region amino acid sequence: SEQ ID NO: 24

[0176] AHP32913-VL CDR2 region amino acid sequence: SEQ ID NO: 25

[0177] AHP32913-VL CDR3 region amino acid sequence: SEQ ID NO: 26

[0178] AHP32919-VH fully human antibody variable region amino acid sequence: SEQ ID NO: 27

[0179] AHP32919-VH CDR1 region amino acid sequence: SEQ ID NO: 28

[0180] AHP32919-VH CDR2 region amino acid sequence: SEQ ID NO: 3

[0181] AHP32919-VH CDR3 region amino acid sequence: SEQ ID NO: 29

[0182] AHP32919-VL fully human antibody variable region amino acid sequence: SEQ ID NO: 30

[0183] AHP32919-VL CDR1 region amino acid sequence: SEQ ID NO: 31

[0184] AHP32919-VL CDR2 region amino acid sequence: SEQ ID NO: 32

[0185] AHP32919-VL CDR3 region amino acid sequence: SEQ ID NO: 33

[0186] AHP32982-VH fully human antibody variable region amino acid sequence: SEQ ID NO: 34

[0187] AHP32982-VH CDR1 region amino acid sequence: SEQ ID NO: 2

[0188] AHP32982-VH CDR2 region amino acid sequence: SEQ ID NO: 3

[0189] AHP32982-VH CDR3 region amino acid sequence: SEQ ID NO: 4

[0190] AHP32982-VL fully human antibody variable region amino acid sequence: SEQ ID NO: 35

[0191] AHP32982-VL CDR1 region amino acid sequence: SEQ ID NO: 36

[0192] AHP32982-VL CDR2 region amino acid sequence: SEQ ID NO: 37

[0193] AHP32982-VL CDR3 region amino acid sequence: SEQ ID NO: 38

[0194] AHP33007-VH fully human antibody variable region amino acid sequence: SEQ ID NO: 34

[0195] AHP33007-VH CDR1 region amino acid sequence: SEQ ID NO: 2

[0196] AHP33007-VH CDR2 region amino acid sequence: SEQ ID NO: 3

[0197] AHP33007-VH CDR3 region amino acid sequence: SEQ ID NO: 4

[0198] AHP33007-VL fully human antibody variable region amino acid sequence: SEQ ID NO: 39

[0199] AHP33007-VL CDR1 region amino acid sequence: SEQ ID NO: 40

[0200] AHP33007-VL CDR2 region amino acid sequence: SEQ ID NO: 41

[0201] AHP33007-VL CDR3 region amino acid sequence: SEQ ID NO: 42

[0202] AHP33029-VH fully human antibody variable region amino acid sequence: SEQ ID NO: 43

[0203] AHP33029-VH CDR1 region amino acid sequence: SEQ ID NO: 28

[0204] AHP33029-VH CDR2 region amino acid sequence: SEQ ID NO: 3

[0205] AHP33029-VH CDR3 region amino acid sequence: SEQ ID NO: 29

[0206] AHP33029-VL fully human antibody variable region amino acid sequence: SEQ ID NO: 44

[0207] AHP33029-VL CDR1 region amino acid sequence: SEQ ID NO: 45

[0208] AHP33029-VL CDR2 region amino acid sequence: SEQ ID NO: 46

[0209] AHP33029-VL CDR3 region amino acid sequence: SEQ ID NO: 47

Claims

1. A binding moiety that specifically binds to Claudin 18.2, comprising A heavy chain variable region (VH), the VH comprising: VH CDR1, VH CDR2 and VH CDR3; wherein, the VH CDR1, CDR2 and CDR3 respectively include: (1) the amino acid sequences shown in SEQ ID NOs: 2, 3 and 4; (2) the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12; or (3) the amino acid sequences shown in SEQ ID NOs: 28, 3 and 29, Alternatively, a variant comprising up to 3 amino acid substitutions in said CDRs; and (b) a light chain variable region (VL), wherein the VL comprises: VL CDR1, VL CDR2 and VL CDR3; wherein the VL CDR1, CDR2 and CDR3 respectively comprise: (1) the amino acid sequence shown in SEQ ID NO:6, EVT and the amino acid sequence shown in SEQ ID NO:8; (2) the amino acid sequence shown in SEQ ID NO:14, KVS and the amino acid sequence shown in SEQ ID NO:16; (3) the amino acid sequence shown in SEQ ID NO: 19, EVS, and the amino acid sequence shown in SEQ ID NO: 21; (4) the amino acid sequence shown in SEQ ID NO:24, DTS and the amino acid sequence shown in SEQ ID NO:26; (5) the amino acid sequence shown in SEQ ID NO:31, SNN and the amino acid sequence shown in SEQ ID NO:33; (6) the amino acid sequence shown in SEQ ID NO:36, AAS, and the amino acid sequence shown in SEQ ID NO:38; (7) the amino acid sequence shown in SEQ ID NO:40, GAS, and the amino acid sequence shown in SEQ ID NO:42; or (8) the amino acid sequence shown in SEQ ID NO:45, WAS, and the amino acid sequence shown in SEQ ID NO:47, Alternatively, it may be a variant comprising up to 3 amino acid substitutions in said CDRs.

2. The binding moiety according to claim 1, wherein The VH CDR1, CDR2 and CDR3 include the amino acid sequences shown in SEQ ID NOs: 2, 3 and 4, respectively, and the VL CDR1, CDR2 and CDR3 include the amino acid sequence shown in SEQ ID NO: 6, EVT and the amino acid sequence shown in SEQ ID NO: 8, respectively; The VH CDR1, CDR2 and CDR3 include the amino acid sequences shown in SEQ ID NOs: 2, 3 and 4, respectively, and the VL CDR1, CDR2 and CDR3 include the amino acid sequence shown in SEQ ID NO: 19, EVS and the amino acid sequence shown in SEQ ID NO: 21, respectively; The VH CDR1, CDR2 and CDR3 include the amino acid sequences shown in SEQ ID NOs: 2, 3 and 4, respectively, and the VL CDR1, CDR2 and CDR3 include the amino acid sequence shown in SEQ ID NO: 24, the DTS and the amino acid sequence shown in SEQ ID NO: 26, respectively; The VH CDR1, CDR2 and CDR3 include the amino acid sequences shown in SEQ ID NOs: 2, 3 and 4, respectively, and the VL CDR1, CDR2 and CDR3 include the amino acid sequence shown in SEQ ID NO: 36, AAS and the amino acid sequence shown in SEQ ID NO: 38, respectively; The VH CDR1, CDR2 and CDR3 include the amino acid sequences shown in SEQ ID NOs: 2, 3 and 4, respectively, and the VL CDR1, CDR2 and CDR3 include the amino acid sequence shown in SEQ ID NO: 40, GAS and the amino acid sequence shown in SEQ ID NO: 42, respectively; The VH CDR1, CDR2 and CDR3 include the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12, respectively, and the VL CDR1, CDR2 and CDR3 include the amino acid sequence shown in SEQ ID NO: 14, KVS and the amino acid sequence shown in SEQ ID NO: 16, respectively; The VH CDR1, CDR2 and CDR3 include the amino acid sequences shown in SEQ ID NOs: 28, 3 and 29, respectively; and the VL CDR1, CDR2 and CDR3 include the amino acid sequence shown in SEQ ID NO: 31, SNN and the amino acid sequence shown in SEQ ID NO: 33, respectively; or The VH CDR1, CDR2 and CDR3 include the amino acid sequences shown in SEQ ID NOs: 28, 3 and 29, respectively; and the VL CDR1, CDR2 and CDR3 include the amino acid sequence shown in SEQ ID NO: 45, WAS and the amino acid sequence shown in SEQ ID NO: 47, respectively.

3. The binding moiety according to claim 1 or 2, wherein the VL is a Kappa light chain variable region or a Lambda light chain variable region.

4. A binding portion according to claim 3, wherein the VH comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 9, 17, 22, 27, 34 and 43; or the VL comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 13, 18, 23, 30, 35, 39 and 44.

5. The binding moiety of claim 4, wherein the VH and VL each comprise: (1) the amino acid sequences shown in SEQ ID NOs: 1 and 5; (2) the amino acid sequences shown in SEQ ID NOs: 9 and 13; (3) the amino acid sequences shown in SEQ ID NOs: 17 and 18; (4) the amino acid sequences shown in SEQ ID NOs: 22 and 23; (5) the amino acid sequences shown in SEQ ID NOs: 27 and 30; (6) the amino acid sequences shown in SEQ ID NOs: 34 and 35; (7) the amino acid sequences shown in SEQ ID NOs: 34 and 39; or (8) The amino acid sequences shown in SEQ ID NOs: 43 and 44.

6. The binding moiety according to any one of claims 1 to 5, which is a sdab, HcAb, Fab, Fab', F(ab')2, Fv, scFv, (scFv)2 or a full length antibody.

7. The binding portion of claim 1 which is mouse, chimeric, humanized or fully human.

8. The binding moiety according to claim 7, which is of fully human origin.

9. The binding portion of claim 6, which is a fully human full-length antibody.

10. A multispecific antibody or binding fragment thereof, comprising the binding portion of any one of claims 1-9.

11. A chimeric antigen receptor comprising: (a) an extracellular antigen binding domain, which comprises a binding portion according to any one of claims 1 to 9 or a multispecific antibody or a binding fragment thereof according to claim 10; (b) a transmembrane domain; and (c) Intracellular signaling domain.

12. A nucleic acid encoding the binding moiety of any one of claims 1-9, the multispecific antibody or binding fragment thereof of claim 10, or the chimeric antigen receptor of claim 11.

13. A host cell comprising the nucleic acid according to claim 12.

14. A pharmaceutical composition comprising the binding moiety according to any one of claims 1 to 9, the multispecific antibody or binding fragment thereof according to claim 10, the chimeric antigen receptor according to claim 11 or the host cell according to claim 13 and a pharmaceutically acceptable carrier.

15. A method for treating a Claudin 18.2 expression-related disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 14 to the subject.

16. The method according to claim 15, wherein the Claudin 18.2 expression-related disease is gastric cancer, pancreatic cancer, esophageal cancer, breast cancer, colon cancer, liver cancer, ovarian cancer or lung cancer.

17. Use of the binding portion according to any one of claims 1 to 9, the multispecific antibody or binding fragment thereof according to claim 10, the chimeric antigen receptor according to claim 11, the nucleic acid according to claim 12 or the host cell according to claim 13 in the preparation of a medicament for treating a disease associated with Claudin 18.2 expression.

18. The use according to claim 17, wherein the disease associated with Claudin 18.2 expression is gastric cancer, pancreatic cancer, esophageal cancer, breast cancer, colon cancer, liver cancer, ovarian cancer or lung cancer.

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