Antigen binding protein targeting CLDN18.2 and application thereof

Abstract

The invention relates to a targeted CLDN18.2 antigen binding protein and application thereof, the antigen binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprises an amino acid sequence as shown in SEQ ID NO: 10, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 3.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an antigen binding protein targeting CLDN18.2. Background Art

[0002] CLDN18.2 (Claudin 18.2) is expressed in differentiated gastric parietal cells and is not expressed in normal tissues. Recent studies have shown that CLDN18.2 is overexpressed in more than 77% of gastric cancer patients and more than 80% of pancreatic cancer patients. In addition, it is overexpressed in solid tumors such as lung cancer, esophageal cancer, and ovarian cancer. CLDN18.2 belongs to the family of tight junction proteins, which can control the flow of molecules between layer cells. However, in tumors, tight junctions are destroyed and CLDN proteins lose their main role. Because CLDN18.2 is present in large quantities in gastric tumors, researchers estimate that half of all advanced gastric cancer patients can be candidates for new therapies targeting CLDN18.2 antibodies. In addition, this unique target does not exist in any healthy tissue except the gastric wall, thereby minimizing treatment side effects. These characteristics indicate that CLDN18.2 is an ideal target for the development of therapeutic monoclonal antibodies.

[0003] Since CLDN18.2 is a membrane protein, it is difficult to obtain high-quality antibodies against natural proteins using traditional antibody screening methods. Currently, most methods use in vivo immunization, such as DNA injection, but multiple rounds of immunization are required in animals. At this stage, it is urgent to obtain high-quality CLDN18.2 antibodies that have strong binding ability to the CLDN18.2 protein and are safe. Summary of the invention

[0004] The present application provides an antigen binding protein capable of targeting and binding to CLDN18.2, which exhibits one or more desired functional properties, such as strong binding ability to CLDN18.2-his protein with a humanized antibody protein, no significant difference in ADCC activity of the antibody protein before and after humanization, good CEX performance and / or good SEC performance.

[0005] For protein drugs, charge heterogeneity can affect the biological activity, safety and stability of biotherapeutic drugs. The antigen binding protein targeted to CLDN18.2 provided in the present application has a good CEX performance, that is, the ratio of acid-base peaks is low, which reflects the key attributes of high-quality antibodies.

[0006] For protein drugs, the formation of aggregates is an important cause of immune response and serious adverse events. The antigen-binding protein targeted to CLDN18.2 provided in the present application has good SEC performance, that is, very low aggregate content, which reflects the key attribute of high-safety antibodies.

[0007] The antigen binding protein targeted to CLDN18.2 provided in the present application has a strong binding ability to the CLDN18.2-his protein. The antigen binding protein targeted to CLDN18.2 provided in the present application has specific binding to CLDN18.2.

[0008] The present application also provides nucleic acid molecules encoding the isolated antigen-binding protein, expression vectors, host cells, and methods for preparing the isolated antigen-binding protein. The isolated antigen-binding protein described in the present application can be used to prevent and / or treat CLDN18.2-related diseases and / or conditions, such as tumors and / or cancers.

[0009] On the one hand, the present application provides a CLDN18.2 antigen binding protein, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3.

[0010] In some embodiments, it comprises an antibody or an antigen-binding fragment thereof.

[0011] In some embodiments, the antigen binding fragment comprises Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv and / or di-scFv.

[0012] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0013] In some embodiments, the antibody is a humanized antibody.

[0014] In some embodiments, it comprises a heavy chain constant region.

[0015] In some embodiments, the heavy chain constant region is derived from a human IgG heavy chain constant region.

[0016] In some embodiments, the heavy chain constant region is derived from a human IgG1 heavy chain constant region, which comprises the amino acid sequence shown in SEQ ID NO:17.

[0017] In some embodiments, the antigen binding protein comprises a light chain constant region.

[0018] In some embodiments, the light chain constant region is derived from a human IgG1 light chain constant region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0019] In some embodiments, the antigen binding protein comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO:24, and the light chain comprising the amino acid sequence shown in SEQ ID NO:25.

[0020] In some embodiments, the CLDN18.2 protein is of human origin.

[0021] In some embodiments, the antigen binding protein has ADCC activity.

[0022] In some embodiments, the monomer amount of the antigen binding protein detected by SEC is greater than or equal to 95.0%.

[0023] In some embodiments, the aggregate amount of the antigen binding protein detected by SEC is less than or equal to 5.0%.

[0024] In some embodiments, the acid-base peak after CPB enzyme cleavage in the CEX charge heterogeneity detection of the antigen-binding protein is less than 30.0%. In some embodiments, the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:26.

[0025] In another aspect, the present application provides an isolated nucleic acid molecule encoding the antigen binding protein described in the present application.

[0026] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule described in the present application.

[0027] In some embodiments, the vectors described herein include viral vectors.

[0028] In some embodiments, the vectors described herein comprise retroviral vectors.

[0029] On the other hand, the present application provides a cell, which contains and / or expresses the antigen binding protein described in the present application, the nucleic acid molecule described in the present application, and / or the vector described in the present application.

[0030] On the other hand, the present application provides a method for preparing the antigen-binding protein described in the present application, the method comprising culturing the cell described in the present application under conditions in which the antigen-binding protein described in the present application is expressed.

[0031] On the other hand, the present application provides a fusion protein comprising the antigen binding protein described in the present application.

[0032] On the other hand, the present application provides a conjugate comprising the antigen-binding protein described in the present application.

[0033] On the other hand, the present application provides a kit comprising the antigen binding protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, the fusion protein described in the present application and / or the conjugate described in the present application.

[0034] On the other hand, the present application provides a pharmaceutical composition comprising the antigen binding protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, the fusion protein described in the present application and / or the conjugate described in the present application, and a pharmaceutically acceptable carrier.

[0035] On the other hand, the present application provides a use of the antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, the fusion protein described in the present application and / or the conjugate described in the present application in the preparation of a pharmaceutical composition.

[0036] In some embodiments, the pharmaceutical composition is used to prevent and / or treat a disease or condition associated with up-regulated expression of CLDN18.2.

[0037] On the other hand, the present application provides an antigen binding protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, the conjugate described in the present application, the fusion protein described in the present application and / or the pharmaceutical composition described in the present application for preventing and / or treating diseases or conditions related to the expression of CLDN18.2.

[0038] In some embodiments, the disease or disorder associated with the expression of CLDN18.2 includes a disease or disorder associated with upregulated expression of CLDN18.2.

[0039] In some embodiments, the disease or disorder associated with the expression of CLDN18.2 comprises a tumor.

[0040] In some embodiments, the disease or disorder associated with the expression of CLDN18.2 comprises a CLDN18.2-positive tumor.

[0041] In some embodiments, the disease or condition associated with the expression of CLDN18.2 includes solid tumors and / or hematological tumors.

[0042] In some embodiments, the disease or condition associated with the expression of CLDN18.2 comprises a solid tumor.

[0043] In some embodiments, the disease or condition associated with the expression of CLDN18.2 includes gastric cancer, gastroesophageal junction cancer, gastroesophageal cancer, esophageal adenocarcinoma, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, head and neck cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, bile duct cancer, bladder cancer and / or leukemia.

[0044] On the other hand, the present application provides a method for preventing and / or treating a disease or condition associated with up-regulation of CLDN18.2 expression, comprising administering to a subject in need thereof an effective amount of the antigen binding protein described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, the conjugate described herein and / or the pharmaceutical composition described herein.

[0045] On the other hand, the present application provides a method for regulating the expression of CLDN18.2 in a subject, comprising administering to the subject the antigen binding protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, the conjugate described in the present application and / or the pharmaceutical composition described in the present application.

[0046] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0048] Figure 1A-1E Shown are the analysis results of the murine antibody light chain (TB001 LC-0) and the humanized antibody light chain (TB001 LC1-LC4) described in this application in the Abysis database;

[0049] Figure 2A-2E Shown are the analysis results of the murine antibody heavy chain (TB001 HC-0) and the humanized antibody heavy chain (TB001 HC1-HC4) described in the present application in the Abysis database;

[0050] Figure 3 What is shown is the result of the antibody protein and antigen binding activity test before and after humanization described in this application;

[0051] Figure 4 Shown are the ADCC activity test results of the antibody protein before and after humanization described in this application;

[0052] Figure 5 Shown are the results of CDC activity testing of antibody proteins before and after humanization described in this application;

[0053] Figure 6A-6D Shown is the CEX detection profile of the antigen binding protein before and after humanization described in the present application;

[0054] Figure 7 Shown is the SEC detection spectrum of the antigen binding protein described in this application. DETAILED DESCRIPTION

[0055] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0056] Definition of terms

[0057] In the present application, the term "antigen binding protein" generally refers to a protein with antigen binding ability. For example, antigen binding proteins can include separated antigen binding proteins. In the present application, the term "separated antigen binding protein" generally refers to a protein with antigen binding ability that has been separated from its naturally occurring state. The "separated antigen binding protein" can include a portion that binds to an antigen and, optionally, allows the antigen binding portion to adopt a framework or framework portion that promotes the conformation of the antigen binding portion to bind to the antigen. Antigen binding proteins can include, for example, antibody-derived protein framework regions (FRs) or alternative protein framework regions or artificial framework regions with transplanted CDRs or CDR derivatives. Such frameworks include, but are not limited to, antibody-derived framework regions that include, for example, mutations introduced to stabilize the three-dimensional structure of antigen binding proteins and fully synthetic framework regions that include, for example, biocompatible polymers. See, e.g., Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Examples of antigen binding proteins include, but are not limited to, human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv, IgD antibodies; IgE antibodies; Ig M antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and fragments thereof.

[0058] In this application, the term "isolated" generally refers to an artificially obtained substance from a natural state. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0059] In the present application, the term "nucleic acid molecule" generally refers to nucleotides, deoxyribonucleotides or ribonucleotides in an isolated form of any length, or analogs isolated from their natural environment or artificially synthesized. The term "isolated nucleic acid molecule" used in the present invention refers to deoxyribonucleic acid or ribonucleic acid in an isolated form. The isolated nucleic acid molecule carries and encodes the target gene, and when it is transferred into a host cell, it can express the corresponding target gene protein or nucleic acid sequence, and the expression product can play its biological function in the host cell. The "isolated nucleic acid molecule" can be a product isolated from a natural environment or artificially synthesized. The term "isolated nucleic acid molecule" includes not only the same nucleic acid, but also any base mutations, which especially include a replacement, which results in the generation of synonymous codons (different codons encoding the same amino acid residue) due to the degenerate code in the conservative amino acid replacement. The term "isolated nucleic acid molecule" also includes any single-stranded sequence complementary to the nucleic acid.

[0060] The term "vector" used in the present invention generally refers to a nucleic acid delivery vehicle into which a nucleic acid molecule carrying a target gene can be inserted and the target gene can be expressed. The vector can transform, transduce or transfect the host cell so that the target gene it carries can be expressed in the host cell. For example, the vector includes: plasmid, phagemid, cosmid, artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as λ phage or M13 phage and animal virus, etc. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). Generally speaking, a vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, screening sequences and reporter genes. In addition, the vector may also contain a replication initiation site sequence. The vector may also include components that assist it in entering the cell, such as virus particles, liposomes or protein shells, but not only these substances. In addition, the vector may also include specific site recombination sequences and restriction endonuclease cleavage sites.

[0061] The term "host cell" used in the present invention generally refers to a cell that has been or can be transformed by a nucleic acid molecule and can express a target gene selected on the nucleic acid molecule. The term also includes the offspring of the host cell mother cell, regardless of whether the offspring is completely identical to the original parent in morphology or genetic structure, as long as the selected gene exists, it is still included in the scope of the term "host cell" used herein. The host includes host cells in isolated form, such as host cells in culture. The term "host cell" used in the present invention also includes the environmental conditions inside the host cell. The molecules required for various biological reactions in the internal environment of the host cell include but are not limited to specific recombinases and accessory proteins. The internal environment of the host cell can also provide a reaction site for the biological reaction and the physical and chemical conditions required for the reaction, such as being able to achieve targeted binding and recombination of specific recombinases and specific recombination sites.

[0062] In this application, the term "retroviral vector" generally refers to a vector that can clone and express exogenous genes, but cannot self-package into viral particles with proliferation ability. The retroviruses mostly have reverse transcriptase, and retroviruses contain at least three genes: gag, which contains genes for proteins that make up the center and structure of the virus; pol, which contains genes for reverse transcriptase and env, which contains genes that make up the viral shell. When a retrovirus is used as a vector, through transfection, the retroviral vector can randomly and stably integrate its own genome and the exogenous genes it carries into the host cell genome, for example, the gene encoding CAR can be integrated into the host cell.

[0063] The term "antibody" used in this application includes immunoglobulin molecules that can specifically bind to a specific antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as the immunoglobulin molecules can exhibit the desired biological activity (such as specific binding to a specific antigen) (Miller et al (2003) Jour. of Immunology 170: 4854-4861). The natural form of the antibody is usually a tetramer and is composed of two identical immunoglobulin chain pairs, each pair having a light chain and a heavy chain. In each pair, the light chain and heavy chain variable regions (VL and VH) are mainly responsible for binding to the antigen. The light chain and heavy chain variable domains respectively include three hypervariable regions (also called "complementary determining regions" or "CDRs") and a framework region interrupted by the three hypervariable regions. The constant region can be recognized by the immune system and interact with it. (See, e.g., Janeway et al., 2001, Immunol. Biology, 5th edition, Garland Publishing, New York). The antibody can be of any type (e.g., IgG, IgE, IgM, IgD or IgA), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2) or any subclass of the above antibodies. The antibody can be derived from any suitable species. In some embodiments, the antibody is human or murine, for example, the antibody can be a human, humanized or chimeric antibody. In some embodiments, the human variable region framework region of a humanized antibody can contain some non-human residues to increase the correct binding ability of the antibody. After humanization, the antibody can prolong its biological half-life and reduce its ability to produce adverse immune responses after administration to the human body.

[0064] The term "antigen binding fragment" as used in this application generally comprises a portion of an intact antibody, wherein the fragment retains the same specific binding ability as the intact antibody, i.e., the antigen binding fragment can bind to an antigen or antigen fragment that the intact antibody can bind to. Antigen binding fragments can be produced by DNA recombinant technology or enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen binding fragments include Fab, Fab', F(ab')2, F(ab)2, Fv, and complementary determining region (CDR) fragments, single-chain antibodies (such as scFv), chimeric antibodies, diabodies, and polypeptides, wherein at least a portion of an intact antibody is included, so that the above-mentioned molecules retain the same specific binding ability as the intact antibody.

[0065] The term "antigen" as used herein generally refers to a molecule or molecule fragment that can be specifically bound by an antibody. In some embodiments, an antigen can be used to produce an antibody that can bind to the antigen in an animal. An antigen can have one or more epitopes that can interact with different antibodies. The antigen described herein can include CLDN18.2.

[0066] In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulin. In the CDR region, small additions, deletions, insertions, replacements or modifications of amino acids may also be permitted, as long as they still retain the ability of the antibody to bind to a specific antigen. Humanized antibodies may optionally include at least a portion of a human immunoglobulin constant region. "Humanized antibodies" retain antigenic specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody may minimally include a chimeric antibody derived from a sequence of a non-human immunoglobulin. In some cases, the CDR region residues in a human immunoglobulin (receptor antibody) may be replaced with CDR region residues of a non-human species (donor antibody) (such as mouse, rat, rabbit or non-human primate) with desired properties, affinity and / or ability. In some cases, the FR region residues of a human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity.

[0067] In the present application, the term "CLDN18.2" generally refers to subtype 2 of the cell tight junction protein family Claudin18. The CLDN18.2 described in the present application may include a complete CLDN18.2 protein and its functional fragments, functional variants, homologs and / or proteins, peptides or polypeptides having at least one common epitope with CLDN18.1. The amino acid sequence of CLDN18.2 (eg, human CLDN18.2) is known in the art. For example, the human CLDN18.2 nucleotide sequence can be shown under GeneBank accession number NM_001002026.3. For example, the mouse CLDN18.2 nucleotide sequence can be shown under GeneBank accession number NM_001194921.1. For example, the cynomolgus monkey CLDN18.2 nucleotide sequence can be shown under GeneBank accession number XM_001114708.4.

[0068] In the present application, the "homolog" generally refers to a protein, peptide or polypeptide having at least about 85% (for example, at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein, peptide or polypeptide.

[0069] In the present application, the "variant" generally refers to a protein, peptide or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein, peptide or polypeptide. The functional variant can retain the biological properties of the protein or polypeptide before the change, such as the functional variant of CLDN18.2 can be recognized and bound by the CLDN18.2 antigen binding protein.

[0070] In the present application, the term "CLDN18.2 positive tumor cell" refers to a tumor cell expressing CLDN18.2 protein on its surface. In order to determine whether a cell expresses CLDN18.2 protein on the surface, the expression of CLDN18.2 mRNA can be determined by a method selected from in situ hybridization and RT-PCR (including quantitative RT-PCR), or antibodies against CLDN18.2 protein can be used to measure CLDN18.2 protein expression on the cell surface in methods such as immunohistochemistry, FACS, etc. In some embodiments, the CLDN18.2 positive tumor can be a tumor planted in a mammal, such as a tumor planted in a mouse. In the present application, the term "tumor" generally refers to or describes a physiological condition in a mammal that is generally characterized by unregulated cell growth. In the present application, a tumor may include a tumor associated with the expression of CLDN18.2. For example, a tumor may include a CLDN18.2 positive tumor.

[0071] In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise an antigen binding protein as described herein, an isolated nucleic acid molecule as described herein, a vector as described herein, and / or a cell as described herein, and optionally a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition of the present invention includes, but is not limited to, liquid, frozen, and lyophilized compositions.

[0072] In the present application, the term "carrier" generally refers to a pharmaceutically acceptable formulation carrier, solution or additive that enhances the properties of the formulation. Such additives are well known to those skilled in the art. The pharmaceutical composition may also include a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used.

[0073] In this application, the term "ADCC" or "antibody-dependent cellular cytotoxicity" is a function mediated by Fc receptor binding and refers to the lysis of target cells by antibodies as reported herein in the presence of effector cells. The ability of antibodies to induce the initial step of mediating ADCC is investigated by measuring their binding to cells expressing Fcγ receptors, such as cells recombinantly expressing FcγRI and / or FcγRIIA, or NK cells (essentially expressing FcγRIIIA). In particular, the binding to FcγR on NK cells is measured.

[0074] In this application, the term "complement-dependent cytotoxicity" or CDC refers to a mechanism of inducing cell death in which the Fc effector domain(s) of an antibody that binds to a target activates a series of enzymatic reactions that cause holes to form in the target cell membrane. Typically, an antigen-antibody complex, such as an antigen-antibody complex on an antibody-coated target cell, binds and activates complement component C1q, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also lead to deposition of complement components on the surface of target cells, which facilitates ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0075] In this application, the term "SEC" or "exclusion chromatography" generally refers to a chromatography technique that separates sample molecules according to their size, also known as gel chromatography, molecular exclusion chromatography, size exclusion chromatography, etc., and is a type of liquid chromatography. The gel used in SEC itself has a three-dimensional network structure. Macromolecules are excluded when passing through the pores on this network structure, and small molecules are retained when passing through. When passing through the gel bed, various components of different molecular weights pass through in order according to their molecular weight, showing a molecular sieving effect. In general, commonly used gels can include dextran, polyacrylamide, agarose, and lipophilic gel.

[0076] In this application, the term "CEX" or "cation exchange chromatography" is a method for analyzing monoclonal antibody charge isomers. It is a method for separating monoclonal antibody charge isomers based on changing pH or salt ion strength. Compared with IEX (ion exchange chromatography), CEX provides a high resolution and more stable separation method. The CEX can separate different isomers from antibodies in a buffer system. In actual situations, CEX is usually used as an effective means to control and stabilize the quality of antibody drug production.

[0077] In the present application, the term "conjugate" generally refers to a conjugate formed by conjugating (e.g., covalently linked via a linker molecule) the other agent (e.g., a chemotherapeutic agent, a radioactive element, a cell growth inhibitor, a cytotoxic agent, or a nucleic acid molecule, etc.) to the antigen-binding protein or its functional fragment. For example, the conjugate can deliver the other agent to a target cell (e.g., a tumor cell) by specifically binding the antigen-binding protein to an antigen on the target cell.

[0078] In this application, the term "fusion protein" generally refers to a polypeptide or protein containing the amino acid sequence of a first polypeptide or protein or its fragment, analog or derivative and the amino acid sequence of a heterologous polypeptide or protein (i.e., a second polypeptide or protein or its fragment, analog or derivative that is different from the first polypeptide or protein or its fragment, analog or derivative, or is generally not a part of the first polypeptide or protein or its fragment, analog or derivative). For example, a fusion protein can be produced by in vitro recombinant techniques well known in the art.

[0079] In the present application, the term "kit" generally refers to a combination of reagents and other materials. The kit is expected to include reagents, such as buffers, protein stabilizing reagents, signal generating systems (e.g., fluorescent signal generating systems), antibodies, control proteins, and test containers (e.g., microtiter plates, etc.). The term "kit" is not intended to be limited to a specific combination of reagents and / or other materials. For example, the kit also includes instructions for using the reagents. The test kit can be packaged in any suitable manner, typically having components in a single container or (if necessary) in multiple containers and a sheet of instructions for testing. For example, the kit may also include reagents and / or materials for quality control, such as negative and / or positive quality control samples, negative and / or positive control reagents. The kit can be prepared by a variety of methods known in the art. For example, a positive quality control sample may include a sample known to express a target protein, and a negative quality control sample may include a sample known not to express a target antigen.

[0080] In the present application, the terms "prevention" and / or "treatment" include not only preventing and / or treating a disease, but also generally include preventing the onset of the disease, slowing down or reversing the progression of the disease, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated therewith, and / or preventing further increase in the severity of the disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects that are generally beneficial to the treated patient.

[0081] In the present application, the term "therapeutically effective amount" or "effective amount" generally refers to an amount of the present invention's antibody sufficient to prevent or alleviate symptoms associated with a disease or disorder (e.g., cancer). The therapeutically effective amount is related to the disease being treated, wherein those skilled in the art can easily discern the actual effective amount.

[0082] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat or monkey.

[0083] In the present application, the term "anti-tumor activity" refers to a decrease in the proliferation rate, viability or metastatic activity of tumor cells. For example, anti-tumor activity can be shown by a decrease in the growth rate of abnormal cells that occur during treatment, or a stable or reduced tumor size, or a longer survival period due to treatment compared to a control without treatment. This activity can be evaluated using recognized in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models known in the art for studying anti-tumor activity.

[0084] In this application, the term "comprises", "comprising" or "including" generally means including the features explicitly stated, but not excluding other elements.

[0085] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. DETAILED DESCRIPTION OF THE INVENTION

[0087] The CDR of an antibody is also called the complementarity determining region, which is a part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. The antibody CDR can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat / Chothia, etc. These coding systems are known in the art, and can be found at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In the present application, the CDR covers the CDR sequence obtained by any CDR division method; it also covers its variants, which include the amino acid sequence of the CDR after substitution, deletion and / or addition of one or more amino acids. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs may be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the CDR.

[0088] From the light chain variable region (TB001-LC-0) and heavy chain variable region (TB001-HC-0) of the murine CLDN18.2 antibody chB-ZG055-00, four humanized light chain variable regions (TB001-LC-1, TB001-LC-2, TB001-LC-3 and TB001-LC-4) and four humanized heavy chain variable regions (TB001-HC-1, TB001-HC-2, TB001-HC-3 and TB001-HC-4) were obtained. From the back mutation design of the light chain and heavy chain variable regions of the above humanized antibodies, different light chain and heavy chain sequences were selected for cross-combination, and finally 16 humanized antibodies were obtained. The corresponding relationship of the amino acid sequence of the variable region of each antibody is as follows:

[0089] Table 1 Correspondence of amino acid sequences of variable regions of various antibodies

[0090]

[0091] Antigen Binding Protein

[0092] On the one hand, the present application provides a CLDN18.2 antigen binding protein, which may comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 7-10, and the light chain variable region may comprise an amino acid sequence as shown in SEQ ID NOs: 3-6.

[0093] The antigen-binding protein described in the present application may comprise a heavy chain variable region (VH) and a light chain variable region (VL) selected from any one of the following groups:

[0094] 1) The heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 3;

[0095] 2) the heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 4;

[0096] 3) the heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 5;

[0097] 4) the heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 6;

[0098] 5) the heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 3;

[0099] 6) the heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 4;

[0100] 7) The heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 5;

[0101] 8) The heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO:8, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO:6.

[0102] 9) the heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 3;

[0103] 10) The heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 4;

[0104] 11) The heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 5;

[0105] 12) The heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 6;

[0106] 13) The heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 3;

[0107] 14) the heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 4;

[0108] 15) The heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 5;

[0109] 16) The heavy chain variable region (VH) may comprise the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 6.

[0110] In certain embodiments, the antigen-binding protein may comprise a heavy chain constant region. The heavy chain constant region is derived from an IgG heavy chain constant region. For example, the heavy chain constant region may be derived from human IgG. The heavy chain constant region may be derived from human IgG1, IgG2, IgG3, and IgG4.

[0111] In certain embodiments, the antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region, which may comprise an amino acid sequence as shown in SEQ ID NO:17.

[0112] In certain embodiments, the antigen-binding protein may comprise a light chain constant region. The light chain constant region may be derived from an IgG light chain constant region. For example, the light chain constant region may be derived from human IgG.

[0113] In certain embodiments, the light chain constant region may be derived from a human IgG1 light chain constant region, which may comprise the amino acid sequence shown in SEQ ID NO:16.

[0114] The antigen binding protein described in the present application may comprise a heavy chain with an amino acid sequence as shown in SEQ ID NO:24 and / or a light chain with an amino acid sequence as shown in SEQ ID NO:25.

[0115] The antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.

[0116] The antigen binding protein, wherein the antigen binding fragment may comprise Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv and / or di-scFv.

[0117] In certain embodiments, the antigen binding protein may be a full-length antibody. The antibody described in the present application may be a monoclonal antibody or a polyclonal antibody. The antigen binding protein described in the present application, wherein the antibody may be human and / or murine. In certain embodiments, the antigen binding protein may be a humanized antibody. The antigen binding protein described in the present application may comprise an amino acid sequence as shown in SEQ ID NO: 26.

[0118] The antigen-binding proteins described herein may include heavy and / or light chain sequences with which there are one or more conservative sequence modifications. The so-called "conservative sequence modification" refers to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the separated antigen-binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are amino acid residues that are replaced with amino acid residues with similar side chains. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the heavy chain variable region and / or light chain variable region of the isolated antigen-binding protein described herein can be replaced with other amino acid residues of the same side chain group. Those skilled in the art know that some conservative sequence modifications will not abolish antigen binding. For details, see, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0119] In the present application, the antigen binding protein can bind to human CLDN18.2 protein. In some cases, the antigen binding protein described in the present application can also cross-react with CLDN18.2 of mice (e.g., mice) and / or monkeys (e.g., cynomolgus monkeys). For example, detected by FACS. In the present application, "cross-reaction" generally refers to the ability of an antibody to react with homologous proteins from other species.

[0120] In some embodiments, the antigen binding proteins described in the present application are capable of inducing an ADCC effect. In some embodiments, the antigen binding proteins described in the present application are not capable of inducing a CDC effect.

[0121] In some embodiments, the antigen binding proteins described herein exhibit good quality attributes in SEC assays.

[0122] Antibodies and Related ProductsThe production of antibody therapeutics can be very challenging due to various chemical degradation processes and aggregation phenomena. Antibodies produced in mammalian cell culture may contain a large number of dimers, trimers and other higher-order aggregates. Aggregates in antibody therapeutics can lead to inaccurate drug dosing and / or unexpected immune responses, thus affecting the safety of the drug. The consequences of immune responses to therapeutic protein products can range from significant impacts to life-threatening complications such as serious adverse events and allergic reactions. Therefore, monitoring antibody aggregation is very important for safety and quality assurance.

[0123] The antigen binding protein described in the present application may have a polymer amount less than or equal to 5.0% as detected by SEC, and a monomer amount greater than or equal to 95.0% as detected by SEC.

[0124] In some embodiments, the antigen binding proteins described herein exhibit good quality attributes in CEX testing.

[0125] Charge isomers are key quality attributes of recombinant protein drugs, especially antibody drugs, and have an important impact on the stability, solubility, immunogenicity, in vivo biological activity and pharmacokinetic activity of antibodies. CEX is a highly sensitive and specific analytical method for antibody charge isomers, and is an effective means to control the quality of antibody production. CEX detection is to examine the charge heterogeneity of monoclonal antibody drugs. Acidic variants (acidic peaks) are mainly caused by oxidation, sialic acid, deamidation, lysine glycation, disulfide bond mismatching, etc., and alkaline variants (alkaline peaks) are mainly caused by incomplete removal of C-terminal lysine, amidation, etc. Carboxypeptidase B digestion can be used to detect the presence of variants with incomplete removal of C-terminal lysine; when the content of other acidic and alkaline peaks does not exceed 30.0%, it indicates that the charge heterogeneity ratio is low.

[0126] In some embodiments, in the CEX charge heterogeneity detection, the antigen-binding protein described in the present application can have a basic peak lower than 30.0% after being cleaved by CPB enzyme (carboxypeptidase B).

[0127] The physical / chemical properties and / or biological activity of the CLDN18.2 antigen binding proteins described in the present application can be identified, screened or characterized by various assays known in the art. In certain embodiments, for example, the antigen binding activity of the antigen binding proteins or fusion proteins of the present application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.

[0128] In some embodiments, the antigen binding protein described herein may be an isolated antigen binding protein.

[0129] In one aspect, the present application also provides a conjugate comprising the antigen binding protein.

[0130] On the one hand, the present application also provides a fusion protein comprising the antigen binding protein.

[0131] Nucleic acids, vectors and cells

[0132] In one aspect, the present application also provides an isolated nucleic acid molecule encoding the antigen binding protein described in the present application.

[0133] The present application provides one or more nucleic acid molecules, which can encode the antigen-binding protein or its functional fragment described in the present application. For example, each of the one or more nucleic acid molecules can encode a complete antigen-binding protein or a part of a functional fragment thereof.

[0134] The nucleic acid molecules described in the present application can be isolated. For example, they can be produced or synthesized by the following methods: (i) amplified in vitro, such as produced by polymerase chain reaction (PCR) amplification, (ii) produced by cloning recombination, (iii) purified, such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis. The isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA technology. In the present application, nucleic acids encoding the antibodies and their antigen-binding fragments can be prepared by a variety of methods known in the art, including but not limited to, using restriction fragment manipulation or overlapping extension PCR using synthetic oligonucleotides, and specific operations can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.

[0135] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule described above.

[0136] The present application provides one or more vectors, which contain one or more nucleic acid molecules described in the present application. Each vector may contain one or more of the nucleic acid molecules. In addition, the vector may also contain other genes, such as marker genes that allow the vector to be selected in appropriate host cells and under appropriate conditions. In addition, the vector may also contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art, for example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. The expression control sequence may be an adjustable element. The specific structure of the expression control sequence may vary according to the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences that participate in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcription control functionally connected to a nucleic acid. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6 RNA promoter, CMV promoter and EF1a promoter. One or more nucleic acid molecules described in the present application may be operably linked to the expression control element. The vector may include a viral vector, such as a retroviral vector.

[0137] On the other hand, the present application provides a cell, which may contain and / or express the above-mentioned antigen binding protein, the above-mentioned isolated nucleic acid molecule, and / or the above-mentioned vector. The nucleic acid molecule described in the present application can be introduced into the cell by methods known in the art, such as electroporation, liposome transfection, viral infection, etc. In some embodiments, the cell provided in the present application can be human.

[0138] Reagent test kit

[0139] On the other hand, the present application provides a kit, which may contain the antigen binding protein described in the present application, the isolated nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, the fusion protein described in the present application, and / or the conjugate described in the present application.

[0140] In some embodiments, the kit may include the antigen binding protein described in the present application, wherein the concentration of the antigen binding protein is adjusted according to the actual use. In some embodiments, the kit can be used in a variety of detection assays, including but not limited to immunohistochemistry, enzyme-linked immunosorbent assay, protein / peptide chip assay, immunoblot assay, microbead immunoassay and / or microfluidic immunoassay. In some embodiments, the kit may also include instructions for use.

[0141] In certain embodiments, the kit can be used to detect CLDN18.2 in a sample. In certain embodiments, the kit can be used to diagnose a disease or condition associated with CLDN18.2 expression.

[0142] Pharmaceutical composition

[0143] On the other hand, the present application provides a pharmaceutical composition, which may include the antigen binding protein, the isolated nucleic acid molecule, the vector, the cell, the fusion protein, and / or the conjugate, and a pharmaceutically acceptable carrier.

[0144] Pharmaceutically acceptable carriers described herein can include any and all solvents, dispersion media, coatings, isotonic agents and absorption delay agents compatible with drug administration, usually safe and nontoxic. The acceptable components of compositions are preferably nontoxic to recipients at used dosages and concentrations. Pharmaceutical compositions of the present invention include but are not limited to liquid, freezing and lyophilized compositions. In certain embodiments, the pharmaceutical composition can also include one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable preparations of preservatives.

[0145] In some embodiments, the pharmaceutical composition may include instructions for use.

[0146] In the present application, the pharmaceutical composition may be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal administration and / or administration via a subcutaneous depot.

[0147] use

[0148] On the other hand, the present application provides the antigen binding protein described in the present application, the isolated nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, the fusion protein described in the present application, and / or the conjugate described in the present application, for use in the preparation of a pharmaceutical composition. In certain embodiments, the pharmaceutical composition is used to prevent and / or treat a disease or condition associated with the expression of CLDN18.2. On the other hand, the present application provides the antigen binding protein, the isolated nucleic acid molecule, the vector, the cell, the fusion protein described in the present application, the conjugate described in the present application, and / or the pharmaceutical composition for use in the prevention and / or treatment of a disease or condition associated with the expression of CLDN18.2.

[0149] On the other hand, the present application provides a method for preventing and / or treating a disease or condition associated with the expression of CLDN18.2, comprising administering to a subject in need thereof an effective amount of the antigen binding protein described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, the fusion protein described herein, the conjugate described herein, and / or the pharmaceutical composition described herein.

[0150] The diseases or conditions associated with the expression of CLDN18.2 described herein may include diseases or conditions associated with upregulated expression of CLDN18.2. The diseases or conditions associated with the expression of CLDN18.2 described herein may include tumors associated with the expression of CLDN18.2. For example, the tumors associated with the expression of CLDN18.2 described herein may include CLDN18.2-positive tumors. For example, the CLDN18.2-positive tumors contain tumor cells expressing CLDN18.2. The diseases or conditions associated with the expression of CLDN18.2 described herein may include solid tumors and / or hematological tumors associated with the expression of CLDN18.2. The diseases or conditions associated with the expression of CLDN18.2 described herein may include gastric cancer, gastroesophageal junction cancer, gastroesophageal cancer, esophageal adenocarcinoma, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, head and neck cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, bile duct cancer, bladder cancer and / or leukemia.

[0151] On the other hand, the present application provides a use of the antigen-binding protein described in the present application, the isolated nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, the fusion protein described in the present application, the conjugate described in the present application and / or the kit described in the present application in the preparation of a kit.

[0152] On the other hand, the present application provides a method for detecting CLDN18.2 in a sample, which comprises administering the antigen-binding protein described herein, the isolated nucleic acid molecule described herein, the vector described herein, the cell described herein, the fusion protein described herein, the conjugate described herein and / or the kit described herein.

[0153] Preparation method

[0154] On the one hand, the present application provides a method for preparing the cell, comprising introducing the isolated nucleic acid molecule and / or the vector into the cell.

[0155] On the one hand, the present application provides a method for preparing the antigen-binding protein, which comprises culturing the cells under conditions where the antigen-binding protein is expressed.

[0156] Without intending to be bound by any theory, the following examples are merely intended to illustrate the antigen-binding proteins, preparation methods and uses of the present application, and are not intended to limit the scope of the present invention.

[0157] Example

[0158] Example 1 Humanization of murine antibodies

[0159] 1.1 Humanization

[0160] By comparing the IMGT human antibody heavy and light chain variable region germline gene database, the heavy chain and light chain variable region germline genes with high homology to the mouse antibody are selected as templates, and the CDRs of the mouse antibody are transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. According to needs, the key amino acids in the framework sequence are mutated differently, such as back-mutated to the amino acids corresponding to the mouse antibody, to maintain the original affinity and improve the physical and chemical properties of the antibody molecule, that is, to obtain a humanized monoclonal antibody. The CDR amino acid residues of the antibody are determined and annotated by the Kabat numbering system.

[0161] The humanized light chain templates of mouse antibodies are IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-2*06 and IGHJ4*01, as well as IGHV3-66*01 and IGHJ4*01. The CDRs of mouse antibodies are transplanted into their humanized templates to obtain the corresponding humanized versions. According to needs, the key amino acids in the FR region sequence of the humanized antibody are mutated differently, such as backmutated to the amino acids corresponding to the mouse antibody, so as to maintain the original affinity and improve the physical and chemical properties of the antibody molecule. Before and after humanization, the degree of humanization of the antibody light and heavy chains was analyzed by Abysis, and the results are shown in Figures 1 and 2. Compared with the light and heavy chains of mouse antibodies (TB001 LC-0 and TB001 HC-0), the H-Score values ​​of the light and heavy chains of the humanized antibodies are greatly improved. The higher the H-Score value, the better the degree of humanization.

[0162] From this analysis, four humanized light chain variable regions (VL1, VL2, VL3 and VL4) and four humanized heavy chain variable regions (VH1, VH2, VH3 and VH4) were obtained.

[0163] The specific sequence of the mouse antibody variable region is as follows:

[0164] The VL0 amino acid sequence is shown in SEQ ID NO:1, and the VH0 amino acid sequence is shown in SEQ ID NO:2.

[0165] The specific sequence of the humanized antibody is as follows:

[0166] The amino acid sequence of VL1 is shown in SEQ ID NO:3, the amino acid sequence of VL2 is shown in SEQ ID NO:4, the amino acid sequence of VL3 is shown in SEQ ID NO:5, and the amino acid sequence of VL4 is shown in SEQ ID NO:6.

[0167] The VH1 amino acid sequence is shown in SEQ ID NO:7, the VH2 amino acid sequence is shown in SEQ ID NO:8, the VH3 amino acid sequence is shown in SEQ ID NO:9, and the VH4 amino acid sequence is shown in SEQ ID NO:10.

[0168] The amino acid sequence of the humanized light chain template IGKV4-1*01 is shown in SEQ ID NO: 11, the amino acid sequence of the humanized light chain template IGKJ2*01 is shown in SEQ ID NO: 12, the amino acid sequence of the humanized heavy chain template IGHV1-2*06 is shown in SEQ ID NO: 13, the amino acid sequence of the humanized heavy chain template IGHV3-66*01 is shown in SEQ ID NO: 14, the amino acid sequence of the humanized heavy chain template IGHJ4*01 is shown in SEQ ID NO: 15, the amino acid sequence of the humanized IgG1 light chain constant region is shown in SEQ ID NO: 16, and the amino acid sequence of the humanized IgG1 heavy chain constant region is shown in SEQ ID NO: 17.

[0169] The results of the IMGT analysis and comparison of the light and heavy chain sequences before and after humanization are shown in the following table. After combining 4 humanized light chains with 4 humanized heavy chains, 16 humanized antibodies were obtained. The humanized sequences were artificially synthesized and sequenced for accuracy, and then recombinantly cloned into the vector pcDNA3.4. The vectors were transfected into CHO K1 cells for transient expression, and then the cells were cultured under appropriate conditions to express the antibodies. After separation and purification, the humanized antibody sequences were obtained.

[0170] The comparison results of murine antibody light chain variable region (VL0) and humanized antibody light chain variable region (VL1-VL4) in the IMGT database (the comparison templates are humanized light chain template IGKV4-1*01 and humanized light chain template IGKJ2*01) and the comparison results of murine antibody heavy chain variable region (VH0) and humanized antibody light chain variable region (VH1-VH4) in the IMGT database are shown in the following table (the comparison templates of TB001 HC-0, TB001 HC-1, TB001 HC-2 and TB001 HC-3 are humanized heavy chain template IGHV1-2*06 and humanized heavy chain template IGHJ4*01, and the comparison template of TB001 HC-4 is humanized heavy chain template IGHV3-66*01 and humanized heavy chain template IGHJ4*01).

[0171] Table 2 Comparison results of light chain variable regions

[0172]

[0173] Table 3 Comparison results of heavy chain variable regions

[0174]

[0175]

[0176] 1.2 Orthogonal combination of humanized antibodies

[0177] The present invention selects different light chain and heavy chain sequences from the above-mentioned back mutation designs of the light chain and heavy chain variable regions of the humanized antibodies for cross-combination, and finally obtains a variety of humanized antibodies. The specific steps are as follows: After adding the corresponding human antibody constant region to the humanized antibody variable region sequence, the corresponding target gene sequence is obtained by reverse translation through molecular biology software. After synthesizing the target gene, clone it into the downstream of the CMV promoter of the animal cell expression vector, and use the SV40 terminator as the transcription terminator to obtain an expression vector that can be expressed in animal cells. After obtaining the antibody expression vector, the 4 humanized sequences of each antibody light chain are orthogonally combined with the 4 heavy chain sequences to obtain 16 groups of target humanized antibody sequences. The variable region amino acid sequences of each antibody are as follows:

[0178] Table 4 Amino acid sequences corresponding to antibody variable regions

[0179] F VH1 VH2 VH3 VH4 VL1 huB-ZG056-01 huB-ZG057-02 huB-ZG058-03 huB-ZG059-04 VL2 huB-ZG060-05 huB-ZG061-06 huB-ZG062-07 huB-ZG063-08 VL3 huB-ZG064-09 huB-ZG065-10 huB-ZG066-11 huB-ZG067-12 VL4 huB-ZG068-13 huB-ZG069-14 huB-ZG070-15 huB-ZG071-16

[0180] At the same time, the variable region sequence of the mouse antibody was added with the same constant region sequence of the human antibody, and the corresponding target gene sequence was obtained by reverse translation through molecular biology software. After the target gene was synthesized, it was cloned into the downstream of the CMV promoter of the animal cell expression vector, and the SV40 terminator was used as the transcription terminator to obtain an expression vector that can be expressed in animal cells. The light chain and heavy chain of the human-mouse chimeric antibody that combined the variable region of the mouse antibody with the constant region of the human antibody were combined to obtain a control human-mouse chimeric antibody.

[0181] Example 2 Expression and detection of humanized antibodies

[0182] The expression vector in Example 1 was used on the ExpiCHO-S commercial transient transfection kit platform to express the antibody. After 7 days, the cell culture supernatant was harvested and the antibody concentration in the supernatant was quantified using a biofilm interferometry protein-A biosensor (Octet, ForteBio). Different concentration gradients were set according to the antibody titer in the supernatant, and the antigen affinity was detected using the ELISA method.

[0183] 2.1 Octet-Titer Detection

[0184] In this embodiment, the thickness of the film layer formed after the target protein binds to the sensor is read in real time, and a real-time reaction monitoring spectrum is made through the displacement value of the interference spectrum, and the data is analyzed by software, and the signal value of the sample is fitted with the standard curve to obtain the Titer of the sample. First, the reference sample is diluted with PBST solution at a final concentration of 50μg / mL; secondly, the diluted reference sample is added to the first column of the 96-well plate, 200μl / well; then, the buffer PBST is added to the 12th column of the 96-well plate, 200μl / well, and the regeneration solution 10mM glycine solution is added to the 11th column of the 96-well plate, 200μl / well; and the samples are respectively drawn and added to the 2nd-10th columns of the 96-well plate as needed, 200μl / well; finally, the 96-well plate is placed in the Octet instrument, and the plate layout detection is performed on the Octet BLIDiscovery 12.2 software according to the sample addition table, and the results are shown in Table 6.

[0185] 2.2 Binding activity of antibody supernatant with specific antigen

[0186] This example tests the binding activity of the antibody supernatant expressed by the humanized sequence to the CLDN18.2 protein. The results show that the humanized antibody supernatant has binding activity to the antigen.

[0187] 1. Take the coating buffer and dilute the human CLDN18.2-his antigen (Cat. No.: ACRO#CL2-H5587) to 1μg / mL. After mixing, add 100μl / well to a 96-well ELISA plate and coat overnight at 2-8°.

[0188] 2. Washing: discard the liquid in the 96-well ELISA plate, add 0.05% PBST washing solution, 300μl / well, shake off the washing solution, repeat this step, wash the plate 5 times, after washing, turn the 96-well ELISA plate upside down on clean absorbent paper, and tap off the residual washing solution.

[0189] 3.3% BSA-PBS was added to 96-well ELISA plate at 300 μl / well and incubated at 37° for 120 min.

[0190] 4. Wash the plate 5 times as in step 2.

[0191] 5. Dilute the sample to 5000 ng / mL, and then perform gradient dilution according to Table 5. Add 100 μl / well of the gradient diluted sample to a 96-well ELISA plate and incubate at 37° for 60 min.

[0192] Table 5 Gradient dilution table

[0193]

[0194]

[0195] 6. Wash the plate 5 times as in step 2.

[0196] 7. Dilute the secondary antibody IgG Fc Cross-Adsorbed Secondary Antibody (Cat. No.: 31413) 40,000 times with diluent, add 100 μl / well to a 96-well ELISA plate, and incubate at 37° for 60 min.

[0197] 8. Wash the plate 5 times as in step 2.

[0198] 9. Add 100 μl / well TMB colorimetric solution (Cat. No.: PR1200) and incubate at room temperature for 10 min.

[0199] 10. Add stop solution at 50 μl / well in the order of adding color development solution.

[0200] 11. Arrange the plates in the software according to the order of sample addition, place the 96-well ELISA plate into the ELISA reader, and read the results at a detection wavelength of 450nm and a reference wavelength of 650nm.

[0201] 2.3 Antibody protein purification

[0202] Then, protein A affinity chromatography was used for purification, and 16 humanized antibodies and 1 human-mouse chimeric antibody were obtained for each mouse antibody variable region sequence. The results are shown in Table 6.

[0203] Table 6 Humanized antibody supernatant titer, antigen affinity test and protein harvest after purification

[0204]

[0205]

[0206] Example 3: Biological activity detection of purified humanized antibody protein

[0207] 3.1 ELISA antigen binding activity detection of purified humanized antibody proteins

[0208] The ELISA method was used to compare the binding ability of the purified antibody protein before and after humanization with human CLDN18.2-his protein. Coat the plate one day in advance, dilute CLDN18.2-His to 1ug / ml with 1xELISA coating solution, take the ELISA enzyme-labeled plate, add 100ul / well, and coat overnight at 4℃; take out the plate, equilibrate to room temperature, and prepare PBST (0.05% Tween-20in 1xPBS) and blocking solution (2% BSA in PBST); discard the supernatant, add 300ul / well PBST to wash the plate, and repeat 6 times; add 300ul / well blocking solution, block at 37℃ for 2 hours; prepare diluent (0.5% BSA in PBST), and use the diluent to dilute the humanized antibody purified protein to 1ug / ml, and perform 5-fold gradient dilution, with a total of 8 concentration points; after blocking, wash the plate 6 times with PBST, add 100ul / well of diluted antibody or humanized antibody supernatant, and incubate at 37℃ for 2 hours; dilute the detection antibody Goat anti human IgG with diluent 1:10000 Fc (HRP); after incubation, wash the plate 6 times with PBST, add 100ul / well of detection antibody, and incubate at 37°C for 1 hour; after incubation with detection antibody, wash the plate 6 times with PBST, add 100ul / well of 1x TMB, and develop color at room temperature in the dark for about 3 minutes; add 50ul / well ELISA stop solution to stop color development, and read the OD450 value with a multifunctional microplate reader.

[0209] Test results such as Figure 3 As shown, the humanized antibody protein has a strong binding to human CLDN18.2-his protein, and the EC50 values ​​of huB-ZG056-01, huB-ZG059-04, huB-ZG063-08, and huB-ZG067-12 are 0.864ng / ml, 1.734ng / ml, 1.075ng / ml, and 2.12ng / ml, respectively. The binding ability is equivalent to that of the chimeric antibody chB-ZG055-00, and its EC50 is 1.624ng / ml.

[0210] 3.2 ADCC and CDC activity detection of purified humanized antibody proteins

[0211] The ADCC effect of the antibody before and after humanization was compared using Reporter Gene Assay. The ADCC bioassay Effector Cell V variant cells were used as effector cells, and NCI N87-human CLDN18.2 cells were used as target cells. The test results are shown in Figure 4 As shown, there was no significant difference in the ADCC activity of the antibody protein before and after humanization. The EC50 values ​​of chB-ZG055-00, huB-ZG056-01, huB-ZG059-04, huB-ZG063-08, and huB-ZG067-12 were 1.279 nM, 1.443 nM, 1.395 nM, 1.137 nM, and 1.178 nM, respectively.

[0212] The LDH method was used to compare the CDC effects of the antibodies before and after humanization. Commercial normal human serum complement was used to detect the CDC effects of the antibody protein on KATOⅢ-human CLDN18.2 cells. The test results are as follows Figure 5 As shown, no CDC activity was detected for all antibody proteins.

[0213] Example 4 Analysis of the physical and chemical properties of the purified humanized antibody protein

[0214] 4.1 CEX charge variant analysis of antibodies before and after humanization

[0215] After biological activity detection of humanized antibody proteins, three molecules, huB-ZG056-01, huB-ZG059-04, and huB-ZG063-08, were screened out. The three humanized molecules and mouse molecules were detected by CPB digestion and non-enzymatic CEX methods. High-performance liquid chromatography (Waters Arc Premier) was performed on a cation exchange column (Thermo MabPac WCX-10, 4×250mm); A (20mM MES pH 6.3) and B (20mM MES+200mM NaCl pH6.3) were used as mobile phases; the flow rate was 0.6min / ml; and the detection wavelength was 280nm. The digested samples were mixed with 1mg / ml carboxypeptidase B solution at a ratio of 100:2 (v / v), centrifuged instantly, and digested at 37℃ for 60 minutes. Gradient elution was used with a salt ion concentration of 0% to 100% within 3 to 30 min. The results are shown in FIG6 and Table 7.

[0216] Table 7 CEX test results

[0217] Sample name Acidic peak% Main peak% Basic peak% Mouse molecule 47.8 43.5 8.7 Mouse molecule-enzyme digestion 48.5 45.8 5.8 huB-ZG056-01 7.0 56.4 36.6 huB-ZG056-01-digestion 7.4 63.8 28.8 huB-ZG059-04 13.3 68.9 17.9 huB-ZG059-04-digestion 13.0 81.2 5.7 huB-ZG063-08 10.7 70.2 19.0 huB-ZG063-08-digestion 10.9 82.8 6.3

[0218] For the huB-ZG056-01 molecule, the alkaline peak contents before and after enzyme digestion were 36.6% and 28.8%, respectively, which were relatively high, indicating that the main alkaline peak was not a lysine-deficient protein; the alkaline peaks of the two molecules of huB-ZG059-04 and huB-ZG063-08 decreased significantly after enzyme digestion, indicating that the main alkaline peak was a lysine-deficient protein, and the alkaline peaks after enzyme digestion all dropped below 10.0%, so the charge variant ratios of the two molecules of huB-ZG059-04 and huB-ZG063-08 were low, and the CEX performance was good. In addition, the results showed that the CEX performance of the humanized antigen-binding protein was significantly improved, and its acidic peak was significantly reduced.

[0219] 4.2 SEC analysis of antibodies before and after humanization

[0220] According to the CEX test results, SEC was performed on huB-ZG059-04 and huB-ZG063-08 molecules to evaluate their aggregation. High-performance liquid chromatography (Waters Arc Premier) was used. The chromatographic column was filled with a chromatographic gel suitable for separating proteins with a molecular weight of 10 to 500 kD (TSKgel-G3000SW gel chromatography column); the mobile phase was 50 mmol / L phosphate buffer + 300 mmol / L sodium chloride buffer (pH 6.8±0.1); the flow rate was 0.8 min / ml; the detection wavelength was 280 nm; and isocratic elution was used. The test results are as follows Figure 7 And shown in Table 8.

[0221] Table 8 SEC test results

[0222] Sample name Fragment (%) monomer(%) Polymer(%) huB-ZG059-04 0.2 98.0 1.8 huB-ZG063-08 0.3 98.2 1.5

[0223] In summary, the main peak content of huB-ZG059-04 and huB-ZG063-08 molecules is greater than 98.0%, and the aggregate content is less than 1.8%, so the SEC results of huB-ZG059-04 and huB-ZG063-08 molecules are good. Compared with mouse antibodies, the humanized antibodies of this application show better quality.

Claims

1. A CLDN18.2 antigen binding protein, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

3.

2. The antigen binding protein of claim 1, comprising an antibody or an antigen binding fragment thereof.

3. The antigen-binding protein according to any one of claims 2, wherein the antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv and / or di-scFv. The antigen-binding protein according to claim 2 , wherein the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody. The antigen binding protein according to claim 1 , comprising a heavy chain constant region. The antigen binding protein according to claim 5 , wherein the heavy chain constant region is derived from a human IgG heavy chain constant region.

7. The antigen-binding protein according to claim 6, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

17. The antigen binding protein of claim 1 , comprising a light chain constant region. 9 . The antigen-binding protein according to claim 8 , wherein the light chain constant region is derived from a human IgG1 light chain constant region, which comprises the amino acid sequence shown in SEQ ID NO:

16.

10. The antigen-binding protein according to claim 1, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

25. The antigen-binding protein according to claim 1 , comprising the amino acid sequence shown in SEQ ID NO:

26.

12. The antigen binding protein according to any one of claims 1 to 11, wherein the CLDN18.2 is of human origin.

13. An isolated nucleic acid molecule encoding the antigen binding protein of any one of claims 1-12.

14. A vector comprising the isolated nucleic acid molecule of claim 13.

15. The vector of claim 14, which comprises a viral vector.

16. The vector according to any one of claims 14-15, which comprises a retroviral vector.

17. A cell comprising and / or expressing the antigen binding protein of any one of claims 1 to 12, the isolated nucleic acid molecule of claim 13, and / or the vector of any one of claims 14 to 16.

18. A method for preparing the antigen binding protein of any one of claims 1-12, the method comprising culturing the cell of claim 17 under conditions where the antigen binding protein of any one of claims 1-12 is expressed.

19. A fusion protein comprising the antigen binding protein according to any one of claims 1 to 12.

20. A conjugate comprising the antigen binding protein according to any one of claims 1 to 12.

21. A kit comprising the antigen binding protein of any one of claims 1-12, the isolated nucleic acid molecule of claim 13, the vector of any one of claims 14-16, the cell of any one of claim 17, the fusion protein of claim 19, and / or the conjugate of claim 20.

22. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1-12, the isolated nucleic acid molecule of claim 13, the vector of any one of claims 14-16, the cell of claim 17, the fusion protein of claim 19, and / or the conjugate of claim 20, and a pharmaceutically acceptable carrier.

23. Use of the antigen binding protein of any one of claims 1-12, the isolated nucleic acid molecule of claim 13, the vector of any one of claims 14-16, the cell of claim 17, the fusion protein of claim 19, and / or the conjugate of claim 20 in the preparation of a pharmaceutical composition.

24. The use according to claim 23, wherein the pharmaceutical composition is used to treat tumors.

25. The use according to claim 23, wherein the pharmaceutical composition is used to treat CLDN18.2-positive tumors.

26. The use according to claim 25, wherein the CLDN18.2-positive tumors include gastric cancer, gastroesophageal junction cancer, gastroesophageal cancer, esophageal adenocarcinoma, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, bile duct cancer, bladder cancer and / or leukemia.

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