Antigen-binding proteins targeting cldn18.2 and uses thereof

Abstract

The present application relates to a kind of targeting CLDN18.2 antigen binding protein and its application, the antigen binding protein includes heavy chain variable region (VH) and light chain variable region (VL), the heavy chain variable region (VH) includes as shown in SEQ ID NO:10 The amino acid sequence, the light chain variable region includes as shown in SEQ ID NO:3 The amino acid sequence.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to an antigen-binding protein that targets CLDN18.2. Background Technology

[0002] CLDN18.2 (Claudin 18.2) is expressed in differentiated gastric parietal cells but not in normal tissues. Recent studies have shown that CLDN18.2 is overexpressed in over 77% of gastric cancer patients and over 80% of pancreatic cancer patients, and is also overexpressed in solid tumors such as lung, esophageal, and ovarian cancer. CLDN18.2 belongs to the tight junction protein family and controls molecular flow between lamina propria. However, in tumors, tight junctions are disrupted, and CLDN proteins lose their primary function. Because CLDN18.2 is abundant in gastric tumors, researchers estimate that half of all patients with advanced gastric cancer could be candidates for novel therapies targeting CLDN18.2 antibodies. Furthermore, this unique target is absent from any healthy tissue other than the gastric wall, thus minimizing treatment side effects. These characteristics suggest that CLDN18.2 is an ideal target for the development of therapeutic monoclonal antibodies.

[0003] Because CLDN18.2 is a membrane protein, it is difficult to obtain high-quality antibodies against the natural protein using traditional antibody screening methods. Currently, most methods employ in vivo immunization, such as DNA injection, but this requires multiple rounds of immunization in animals. There is an urgent need to obtain high-quality CLDN18.2 antibodies that have strong and safe binding affinity to the CLDN18.2 protein. Summary of the Invention

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

[0005] For protein drugs, charge heterogeneity can affect the bioactivity, safety, and stability of biological therapeutics. The antigen-binding protein targeting CLDN18.2 provided in this application exhibits good CEX performance, i.e., a low proportion of acid-base peaks, reflecting a key attribute of high-quality antibodies.

[0006] For protein drugs, the formation of aggregates is a major cause of immune responses and serious adverse events. The antigen-binding protein targeting CLDN18.2 provided in this application has good SEC performance, that is, very low aggregate content, which reflects the key attribute of a high-safety antibody.

[0007] The antigen-binding protein targeting CLDN18.2 provided in this application has a strong binding ability to the CLDN18.2-his protein. The binding of the antigen-binding protein targeting CLDN18.2 provided in this application to CLDN18.2 is specific.

[0008] This application also provides a nucleic acid molecule encoding the isolated antigen-binding protein, an expression vector, a host cell, and a method for preparing the isolated antigen-binding protein. The isolated antigen-binding protein of this application can be used for the prevention and / or treatment of CLDN18.2-related diseases and / or conditions, such as tumors and / or cancer.

[0009] On one hand, this application provides 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 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.

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

[0011] In some embodiments, the antigen-binding fragment includes Fab, Fab', Fv fragments, 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 implementations, it includes a heavy chain constant region.

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

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

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

[0018] In some embodiments, the light chain constant region is derived from the human IgG1 light chain constant region, which contains an amino acid sequence as 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 an amino acid sequence as shown in SEQ ID NO:24, and the light chain comprising an amino acid sequence as shown in SEQ ID NO:25.

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

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

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

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

[0024] In some embodiments, the acid-base peak after CPB enzyme digestion in the CEX charge heterosome 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] On the other hand, this application provides an isolated nucleic acid molecule that encodes the antigen-binding protein described in this application.

[0026] On the other hand, this application provides a carrier containing the isolated nucleic acid molecules described in this application.

[0027] In some embodiments, the vector described in this application includes a viral vector.

[0028] In some embodiments, the vector described in this application includes a retroviral vector.

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

[0030] On the other hand, this application provides a method for preparing the antigen-binding protein described in this application, the method comprising culturing cells according to the present application under conditions of antigen-binding protein expression described in this application.

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

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

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

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

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

[0036] In some embodiments, the pharmaceutical composition is used to prevent and / or treat diseases or conditions associated with upregulation of CLDN18.2 expression.

[0037] On the other hand, this application provides the use of the antigen-binding protein, nucleic acid molecule, vector, cell, conjugate, fusion protein, and / or pharmaceutical composition described herein in the prevention and / or treatment of diseases or conditions associated with the expression of CLDN18.2.

[0038] In some implementations, the diseases or conditions associated with CLDN18.2 expression include those associated with upregulation of CLDN18.2 expression.

[0039] In some embodiments, the disease or condition associated with the expression of CLDN18.2 includes tumors.

[0040] In some embodiments, the disease or condition associated with CLDN18.2 expression includes CLDN18.2-positive tumors.

[0041] In some embodiments, the diseases or conditions associated with the expression of CLDN18.2 include solid tumors and / or hematologic malignancies.

[0042] In some embodiments, the disease or condition associated with the expression of CLDN18.2 includes solid tumors.

[0043] In some embodiments, the diseases or conditions associated with the expression of CLDN18.2 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.

[0044] On the other hand, this application provides a method for preventing and / or treating diseases or conditions associated with upregulation of CLDN18.2 expression, comprising administering to a subject in need an effective amount of the antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, the cell described in this application, the conjugate described in this application, and / or the pharmaceutical composition described in this application.

[0045] On the other hand, this 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 this application, the nucleic acid molecule described in this application, the carrier described in this application, the cell described in this application, the conjugate described in this application, and / or the pharmaceutical composition described in this application.

[0046] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0047] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention 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] Figures 1A-1E The results 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] Figures 2A-2E The results shown are the analysis results of the murine antibody heavy chain (TB001 HC-0) and humanized antibody heavy chain (TB001 HC1-HC4) described in this application in the Abysis database;

[0050] Figure 3 The display shows the results of the detection of the binding activity between the antibody protein and the antigen before and after humanization as described in this application;

[0051] Figure 4 The display shows the ADCC activity detection results of the antibody protein before and after humanization as described in this application;

[0052] Figure 5 The results shown are the CDC activity detection results of the antibody protein before and after humanization as described in this application;

[0053] Figures 6A-6D The image shown is a CEX detection spectrum of the antigen-binding protein before and after humanization as described in this application;

[0054] Figure 7 The image shown is an SEC detection map of the antigen-binding protein described in this application. Detailed Implementation

[0055] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0056] Terminology Definition

[0057] In this application, the term "antigen-binding protein" generally refers to a protein with antigen-binding ability. For example, an antigen-binding protein may include an isolated antigen-binding protein. In this application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability that has been removed from its naturally occurring state. This "isolated antigen-binding protein" may include an antigen-binding portion and optionally, allow the antigen-binding portion to employ a framework or structural portion that promotes the antigen-binding portion's conformation for binding antigens. The antigen-binding protein may include, for example, an antibody-derived protein framework region (FR) or an alternative protein framework region or artificial framework region having a transplanted CDR or CDR derivative. Such frameworks include, but are not limited to, antibody-derived framework regions containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic framework regions containing, for example, biocompatible polymers. See, for example, 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; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and fragments thereof.

[0058] In this application, the term "isolated" generally refers to something obtained artificially from its natural state. If a substance or component is found in nature as an "isolated" substance, it may be due to an alteration of its natural environment, the isolation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated 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 impurities that do not affect the activity of the substance.

[0059] In this application, the term "nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide, or ribonucleotide of any length in its isolated form, or an analogue isolated from its natural environment or synthesized artificially. As used in this invention, the term "isolated nucleic acid molecule" refers to deoxyribonucleic acid or ribonucleic acid existing in isolated form. The isolated nucleic acid molecule carries and encodes a target gene, and when transferred into a host cell, it is able to express the corresponding target gene protein or nucleic acid sequence, and the expression product can perform its biological function in the host cell. The "isolated nucleic acid molecule" can be a product isolated from the natural environment or synthesized artificially. The term "isolated nucleic acid molecule" includes not only the same nucleic acid but also any base mutations, particularly a substitution that, due to a degenerate codon in a conserved amino acid substitution, results in a synonymous codon (a different codon encoding the same amino acid residue). The term "isolated nucleic acid molecule" also includes any single-stranded sequence complementary to the nucleic acid.

[0060] As used in this invention, the term "vector" generally refers to a nucleic acid delivery vehicle that can insert a nucleic acid molecule carrying a target gene into the cell and enable the target gene to be expressed. Vectors can be used to transform, transduce, or transfect host cells, allowing the target gene they carry to be expressed within the host cell. Examples of vectors include: plasmids, phage particles, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). Typically, a vector can contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection sequences, and reporter genes. Additionally, vectors may contain replication initiation site sequences. Vectors may also include components that facilitate their entry into the cell, such as viral particles, liposomes, or protein coats, but are not limited to these substances. In addition, vectors may include specific site recombination sequences and restriction endonuclease cleavage sites.

[0061] As used in this invention, the term "host cell" generally refers to a cell that has been or can be transformed by nucleic acid molecules and is capable of expressing a selected target gene on the nucleic acid molecule. This term also includes the offspring of the host cell parent cell, regardless of whether the offspring are morphologically or genetically identical to the original parent, as long as the selected gene is present, they are still included within the scope of the term "host cell" as used herein. A host includes host cells existing in isolated form, such as host cells in a culture. The term "host cell" as used in this invention also includes the internal environmental conditions of the host cell, which contains various molecules required for biological reactions, including but not limited to specific recombinases and accessory proteins. The internal environment of the host cell can also provide a reaction site and the physicochemical conditions required for the biological reaction, such as enabling the 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 foreign genes but cannot self-package into viral particles capable of replication. The aforementioned retroviruses typically possess reverse transcriptase and contain at least three genes: gag, containing genes for proteins that make up the viral core and structure; pol, containing genes for reverse transcriptase; and env, containing genes that make up the viral capsid. When a retrovirus is used as a vector, through transfection, the retroviral vector can randomly and stably integrate its own genome and the foreign genes it carries into the host cell genome; for example, it can integrate genes encoding CAR into host cells.

[0063] As used herein, the term "antibody" includes immunoglobulin molecules capable of specifically binding to a particular antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that the immunoglobulin molecule can exhibit the desired biological activity (e.g., specific binding to a particular antigen) (Miller et al. (2003) Jour. of Immunology 170:4854-4861). The native form of the antibody is typically a tetramer, consisting of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the variable regions (VL and VH) of the light and heavy chains are primarily responsible for binding to the antigen. The variable domains of the light and heavy chains each comprise three hypervariable regions (also known as "complementarity-determining regions" or "CDRs") and a framework region interrupted by the three hypervariable regions. A constant region is recognized and interacts with by the immune system. (See, for example, Janeway et al., 2001, Immunol. Biology, 5th edition, Garland Publishing, New York). Antibodies 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. Antibodies can be derived from any suitable species. In some embodiments, antibodies are human or murine; for example, antibodies can be human, humanized, or chimeric antibodies. In some embodiments, the humanized variable region of a humanized antibody may contain retained non-human residues to increase the antibody's proper binding capacity. Humanization of antibodies can prolong their biological half-life and reduce their ability to produce an adverse immune response after administration to humans.

[0064] As used herein, the term "antigen-binding fragment" generally comprises a portion of a complete antibody, wherein the fragment retains the same specific binding ability as the complete antibody, meaning that the antigen-binding fragment can bind to antigens or antigen fragments that the complete antibody can bind. Antigen-binding fragments can be generated through DNA recombination technology or enzymatic or chemical cleavage of the complete antibody. In some embodiments, antigen-binding fragments include Fab, Fab', F(ab')2, F(ab)2, Fv, and complementarity-determining region (CDR) fragments, single-chain antibodies (such as scFv), chimeric antibodies, diantibodies, and peptides, wherein at least a portion of the complete antibody is contained, such that the aforementioned molecules retain the same specific binding ability as the complete antibody.

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

[0066] In this 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 immunoglobulins. Small additions, deletions, insertions, substitutions, or modifications of amino acids within the CDR region are also permissible, as long as they retain the antibody's ability to bind to a specific antigen. Humanized antibodies may optionally contain at least a portion of the constant region of human immunoglobulins. "Humanized antibodies" retain antigen specificity similar to the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody may minimally contain a chimeric antibody with a sequence derived from a non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (receptor antibody) may be replaced with CDR region residues from a non-human species (donor antibody) (such as a mouse, rat, rabbit, or non-human primate) having the desired properties, affinity, and / or capabilities. In some cases, FR region residues in a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications not found in receptor antibodies or in donor antibodies. These modifications can be made to further improve antibody performance, such as binding affinity.

[0067] In this application, the term "CLDN18.2" generally refers to isoform 2 of the Claudin18 family of cell tight junction proteins. The CLDN18.2 described in this application may include the complete CLDN18.2 protein and its functional fragments, functional variants, homologs, and / or proteins, peptides, or polypeptides having at least one epitope shared with CLDN18.1. The amino acid sequence of CLDN18.2 (e.g., 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 this application, the term "homology" generally refers to a protein, peptide, or polypeptide that has 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 higher) sequence homology with the amino acid sequence of the protein, peptide, or polypeptide.

[0069] In this application, the term "variant" generally refers to a protein, peptide, or polypeptide whose amino acid sequence has been substituted, deleted, or added to by one or more amino acids. The functional variant may retain the biological properties of the protein or polypeptide before the modification; for example, a functional variant of CLDN18.2 can be recognized and bound by the CLDN18.2 antigen-binding protein.

[0070] In this application, the term "CLDN18.2 positive tumor cell" refers to a tumor cell that expresses the CLDN18.2 protein on its surface. To determine whether a cell expresses the CLDN18.2 protein on its surface, the expression of CLDN18.2 mRNA can be measured by methods selected from in situ hybridization and RT-PCR (including quantitative RT-PCR), or the expression of CLDN18.2 protein on the cell surface can be measured using methods such as immunohistochemistry and FACS using an antibody against the CLDN18.2 protein. In some embodiments, a CLDN18.2 positive tumor can be a mammalian tumor, such as a mouse tumor. In this application, the term "tumor" generally refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. In this application, a tumor can include tumors associated with the expression of CLDN18.2. For example, a tumor can include a CLDN18.2 positive tumor.

[0071] In this application, the term "pharmaceutical composition" generally refers to a composition for the prevention / treatment of a disease or condition. The pharmaceutical composition may comprise the antigen-binding protein described in this application, the isolated nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical compositions of this invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0072] In this application, the term "carrier" generally refers to a pharmaceutically acceptable formulation carrier, solution, or additive that enhances the properties of a formulation. Such additives are well known to those skilled in the art. The pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components 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 cytotoxicity" refers to a function mediated by Fc receptor binding and to the lysis of target cells by an antibody in the presence of effector cells, as reported herein. The ability of antibodies to induce and mediate the initial steps of ADCC is investigated by measuring their binding to cells expressing Fcγ receptors, such as recombinant cells expressing FcγRI and / or FcγRIIA, or NK cells (which essentially express FcγRIIIA). In particular, binding to FcγR on NK cells is measured.

[0074] In this application, the term "complement-dependent cytotoxicity" or CDC refers to a mechanism that induces cell death in which one or more Fc effector domains of an antibody binding to a target activate a series of enzymatic reactions that result in the formation of pores in the target cell membrane. Typically, antigen-antibody complexes, such as antibody-coated antigen-antibody complexes on target cells, bind to and activate complement component C1q, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also lead to the 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 "size exclusion chromatography" generally refers to a chromatographic technique that separates sample molecules based on their size, also known as gel chromatography, size exclusion chromatography, etc., and is a type of liquid chromatography. The gel used in SEC has a three-dimensional network structure. Large molecules are excluded when passing through the pores of this network structure, while small molecules are retained. Multiple components with different molecular weights pass through the gel bed in order of molecular weight, exhibiting a molecular sieving effect. Commonly used gels include dextran, polyacrylamide, agarose, and lipophilic gels.

[0076] In this application, the term "CEX" or "cation exchange chromatography" refers to a method for analyzing the charge isomers of monoclonal antibodies. It is a method for separating charge isomers of monoclonal antibodies based on changes in pH or salt ionic strength. Compared to IEX (ion exchange chromatography), CEX provides a higher resolution and more stable separation method. CEX can separate different isomers from antibodies in a buffer system. In practice, CEX is often used as an effective means of controlling and stabilizing the quality of antibody drug production.

[0077] In this application, the term "conjugate" generally refers to a conjugate formed by combining the other reagent (e.g., a chemotherapeutic agent, a radioactive element, a cell growth inhibitor, a cytotoxic agent, or a nucleic acid molecule) with the antigen-binding protein or a functional fragment thereof (e.g., covalently linked by a linker molecule). For example, the conjugate can deliver the other reagent to the target cell (e.g., a tumor cell) by specifically binding the antigen-binding protein to the 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 a fragment, analogue, or derivative thereof, and the amino acid sequence of a heterologous polypeptide or protein (i.e., a second polypeptide or protein or a fragment, analogue, or derivative thereof that is different from, or is not typically part of, the first polypeptide or protein or a fragment, analogue, or derivative thereof). For example, fusion proteins can be generated by in vitro recombinant techniques well known in the art.

[0079] In this application, the term "kit" generally refers to a combination of reagents and other materials. A kit is intended to contain reagents such as buffers, protein stabilizing agents, signal generation systems (e.g., fluorescence signal generation 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, a kit may also include instructions for using the reagents. Test kits can be packaged in any suitable manner, typically having components in a single container or (if necessary) multiple containers, along with instructions for performing the test. For example, a kit may also include reagents and / or materials for quality control, such as negative and / or positive control samples, and negative and / or positive control reagents. Kits can be prepared using a variety of methods known in the art. For example, positive control samples may include samples known to express the target protein, and negative control samples may include samples known not to express the target antigen.

[0080] In this application, the terms “prevention” and / or “treatment” include not only the prevention and / or treatment of a disease, but also generally include preventing the onset of the disease, slowing or reversing the course of the disease, preventing or slowing 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 with it, and / or preventing further increase in the severity of the disease and / or any symptoms associated with it, and preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects that are generally beneficial to the patient being treated.

[0081] In this application, the terms "therapeutic effective amount" or "effective amount" generally refer to the amount of the antibody of this application sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., cancer). Therapeutic effective amount is related to the disease being treated, whereby a person skilled in the art can readily determine 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 cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0083] In this application, the term "antitumor activity" refers to a reduction in the proliferation rate, viability, or metastatic activity of tumor cells. For example, antitumor activity can be demonstrated by a reduced growth rate of abnormal cells that appear during treatment, or by a stable or reduced tumor size, or by longer survival due to treatment compared to a control without treatment. This activity can be assessed using recognized in vitro or in vivo tumor models, including but not limited to xenograft models, allogeneic transplantation models, MMTV models, and other known models in the art for studying antitumor activity.

[0084] In this application, the term "comprising" or "including" generally means including the explicitly specified features, but does not exclude other elements.

[0085] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 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. Invention Details

[0087] The CDR (Complementarity Determinant Region) of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can contact antigens or antigenic epitopes. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art; see http: / / www.bioinf.org.uk / abs / index.html#kabatnum for details. Those skilled in the art can determine the CDR region using different coding systems based on the antibody's sequence and structure. The CDR region may differ when using different coding systems. In this application, the term CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants, including CDRs whose amino acid sequences have been substituted, deleted, and / or added one or more amino acids. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; also covering its homologs, which can be amino acid sequences having at least about 85% (e.g., having 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 higher) sequence homology with the amino acid sequence of the CDR.

[0088] By utilizing the light chain variable region (TB001-LC-0) and heavy chain variable region (TB001-HC-0) of the mouse 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. Through reverse mutation design of the light and heavy chain variable regions of these humanized antibodies, different light and heavy chain sequences were selected and cross-combined, ultimately yielding 16 humanized antibodies. The amino acid sequence correspondence of the variable regions for each antibody is as follows:

[0089] Table 1. Correspondence of amino acid sequences in the variable regions of each antibody.

[0090]

[0091] antigen-binding proteins

[0092] On the one hand, this application provides a CLDN18.2 antigen-binding protein, which may include a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) may contain an amino acid sequence as shown in any one of SEQ ID NO:7-10, and the light chain variable region may contain an amino acid sequence as shown in SEQ ID NO:3-6.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0110] In some embodiments, the antigen-binding protein may include a heavy chain constant region. This heavy chain constant region is derived from the 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 some embodiments, the antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from the human IgG1 heavy chain constant region, which may contain an amino acid sequence as shown in SEQ ID NO:17.

[0112] In some embodiments, the antigen-binding protein may include a light chain constant region. This light chain constant region may be derived from the IgG light chain constant region. For example, the light chain constant region may be derived from human IgG.

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

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

[0115] The antigen-binding protein may contain an antibody or its antigen-binding fragment.

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

[0117] In some embodiments, the antigen-binding protein may be a full-length antibody. The antibody described in this application may be a monoclonal antibody or a polyclonal antibody. The antibody in the antigen-binding protein described in this application may be human and / or mouse-derived. In some embodiments, the antigen-binding protein may be a humanized antibody. The antigen-binding protein described in this application may contain the amino acid sequence shown in SEQ ID NO:26.

[0118] The antigen-binding protein described in this application may comprise heavy and / or light chain sequences with one or more conserved sequence modifications. A "conserved sequence modification" refers to an amino acid modification that does not significantly affect or alter antibody binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding protein described in this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. A conserved amino acid substitution involves replacing an amino acid residue with an amino acid residue having a similar side chain. A set of amino acid residues with similar side chains is 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), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar 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 some 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 in this application may be replaced with other amino acid residues from the same side chain group. Those skilled in the art will know that some conserved sequence modifications do not cause the antigen to lose its binding ability. For example, see 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 this application, the antigen-binding protein can bind to the human CLDN18.2 protein. In some cases, the antigen-binding protein described in this application can also cross-react with CLDN18.2 in mice (e.g., rats) and / or monkeys (e.g., cynomolgus monkeys), as detected by FACS, for example. In this application, "cross-reactivity" generally refers to the ability of an antibody to react with homologous proteins from other species.

[0120] In some embodiments, the antigen-binding protein described in this application can induce an ADCC effect. In some embodiments, the antigen-binding protein described in this application cannot induce a CDC effect.

[0121] In some embodiments, the antigen-binding protein described in this application has exhibited good quality properties in SEC detection.

[0122] The production of antibody therapeutics can be extremely challenging due to various chemical degradation processes and aggregation phenomena. Antibodies derived from mammalian cell cultures may contain large amounts of dimers, trimers, and other higher-order aggregates. Aggregates in antibody therapeutics can lead to inaccurate drug dosage and / or unexpected immune responses, thereby affecting drug safety. The consequences of immune responses to therapeutic protein products can include life-threatening complications such as serious adverse events and allergic reactions. Therefore, monitoring antibody aggregation is crucial for safety and quality assurance.

[0123] The antigen-binding protein described in this application has an aggregate content of less than or equal to 5.0% and a monomer content of greater than or equal to 95.0% as detected by SEC.

[0124] In some embodiments, the antigen-binding protein described in this application has exhibited good quality properties in CEX detection.

[0125] Charge isomers are a key quality attribute of recombinant protein drugs, especially antibody drugs, and have a significant impact on antibody stability, solubility, immunogenicity, in vivo biological activity, and pharmacokinetic activity. CEX is a highly sensitive and specific analytical method for antibody charge isomers, and an effective means of controlling antibody production quality. CEX detection is used to examine the charge heterogeneity of monoclonal antibody drugs. Acidic variants (acidic peaks) are mainly caused by oxidation, sialic acid formation, deamidation, lysine glycosylation, and disulfide bond mismatches, while basic variants (basic peaks) are mainly caused by incomplete C-terminal lysine cleavage and amidation. The presence of variants with incomplete C-terminal lysine cleavage can be detected by carboxypeptidase B digestion; a concentration of other acidic and basic peaks not exceeding 30.0% indicates a low proportion of charge heterogeneity.

[0126] In some embodiments, the antigen-binding protein described in this application exhibits an alkaline peak below 30.0% after CPB (carboxypeptidase B) cleavage in the CEX charge heterosome detection.

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

[0128] In some embodiments, the antigen-binding protein described in this application may be a separated antigen-binding protein.

[0129] On the one hand, this application also provides a conjugate comprising the antigen-binding protein.

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

[0131] Nucleic acids, vectors and cells

[0132] On the one hand, this application also provides isolated nucleic acid molecules that encode the antigen-binding protein described in this application.

[0133] This application provides one or more nucleic acid molecules that can encode the antigen-binding protein or a functional fragment thereof described in this application. For example, each of the one or more nucleic acid molecules can encode the complete antigen-binding protein or a portion thereof.

[0134] The nucleic acid molecules described in this application may be isolated. For example, they may be produced or synthesized by: (i) in vitro amplification, such as by polymerase chain reaction (PCR), (ii) by clonal recombination, (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthesis, such as by chemical synthesis. The isolated nucleic acid may be a nucleic acid molecule prepared by recombinant DNA technology. In this application, nucleic acids encoding the antibody and its antigen-binding fragment may be prepared by a variety of methods known in the art, including but not limited to, restriction fragment manipulation or overlapping extension PCR using synthetic oligonucleotides, as detailed 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] On the other hand, this application provides a vector comprising the isolated nucleic acid molecules described above.

[0136] This application provides one or more vectors containing one or more nucleic acid molecules as described in this application. Each vector may contain one or more of the nucleic acid molecules described herein. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may 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 and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation. The expression control sequence may be a tunable element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but typically includes 5' non-transcriptional sequences and 5' and 3' non-translational sequences, respectively, involved in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcriptional expression control sequence may contain a promoter region, which may contain a promoter sequence for transcriptionally controlling functionally linked nucleic acids. The expression control sequence may also include enhancer sequences or upstream activator sequences. In this application, suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerases, human U6RNA promoters, CMV promoters, and EF1a promoters. One or more nucleic acid molecules described in this application may be operatively linked to the expression control element. The vector may include a viral vector, such as a retroviral vector.

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

[0138] Reagent test kit

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

[0140] In some embodiments, the kit may contain the antigen-binding protein described in this application, wherein the concentration of the antigen-binding protein is adjusted according to actual use. In some embodiments, the kit can be used in a variety of assays, including but not limited to immunohistochemical assays, enzyme-linked immunosorbent assays, protein / peptide microarray assays, Western blotting assays, microbead immunoassays, and / or microfluidic immunoassays. In some embodiments, the kit may also include an instruction manual.

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

[0142] Pharmaceutical Composition

[0143] On the other hand, this application provides a pharmaceutical composition that may comprise the antigen-binding protein, the isolated nucleic acid molecule, the carrier, the cell, the fusion protein, and / or the conjugate, as well as a pharmaceutically acceptable carrier.

[0144] The pharmaceutically acceptable carriers described in this application may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents compatible with drug administration, and are generally safe and non-toxic. The acceptable components of the composition are preferably non-toxic to the recipient at the doses and concentrations used. The pharmaceutical compositions of this invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions. In some embodiments, the pharmaceutical compositions may also comprise suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives.

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

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

[0147] use

[0148] On the other hand, this application provides the use of the antigen-binding protein described in this application, the isolated nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, the fusion protein described in this application, and / or the conjugate described in this application in the preparation of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is used for the prevention and / or treatment of diseases or conditions associated with the expression of CLDN18.2. On the other hand, this application provides the use of the antigen-binding protein described in this application, the isolated nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, the fusion protein described in this application, the conjugate described in this application, and / or the pharmaceutical composition in the prevention and / or treatment of diseases or conditions associated with the expression of CLDN18.2.

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

[0150] The diseases or conditions associated with CLDN18.2 expression described in this application may include diseases or conditions associated with the upregulation of CLDN18.2 expression. The diseases or conditions associated with CLDN18.2 expression described in this application may include tumors associated with CLDN18.2 expression. For example, tumors associated with CLDN18.2 expression described in this application may include CLDN18.2-positive tumors. For example, CLDN18.2-positive tumors contain tumor cells expressing CLDN18.2. The diseases or conditions associated with CLDN18.2 expression described in this application may include solid tumors and / or hematologic malignancies associated with CLDN18.2 expression. The diseases or conditions associated with CLDN18.2 expression described in this application may 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.

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

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

[0153] Preparation method

[0154] On the one hand, this application provides a method for preparing the cells, comprising introducing the isolated nucleic acid molecules and / or the carrier into the cells.

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

[0156] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the antigen-binding protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0157] Example

[0158] Example 1: Humanization of mouse antibodies

[0159] 1.1 Humanization

[0160] By comparing the germline gene pools of the heavy and light chain variable regions of human antibodies with the IMGT human antibody database, germline genes of the heavy and light chain variable regions with high homology to murine antibodies were selected as templates. The CDRs of the murine antibodies were then transplanted into the corresponding human templates, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Depending on the needs, key amino acids in the backbone sequence were mutated, for example, reverting to the amino acids corresponding to the murine antibodies, to maintain the original affinity and improve the physicochemical properties of the antibody molecule, thus obtaining humanized monoclonal antibodies. The CDR amino acid residues of the antibodies were determined and annotated using the Kabat numbering system.

[0161] The humanized light chain templates for the murine antibody were IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates were IGHV1-2*06 and IGHJ4*01, and IGHV3-66*01 and IGHJ4*01, respectively. The CDR of the murine antibody was transplanted into these humanized templates to obtain the corresponding humanized versions. Depending on the needs, key amino acids in the FR region sequence of the humanized antibody were mutated in different ways, such as reverting to the amino acids corresponding to the murine antibody, to maintain the original affinity and improve the physicochemical properties of the antibody molecule. The degree of humanization of the antibody light and heavy chains before and after humanization was analyzed by Abysis, and the results are shown in Figures 1 and 2. Compared with the murine antibody light and heavy chains (TB001 LC-0 and TB001 HC-0), the H-Score values ​​of the humanized antibody light and heavy chains were significantly improved. A higher H-Score value indicates a better 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 variable region of the murine antibody is as follows:

[0164] The amino acid sequence of VL0 is shown in SEQ ID NO: 1, and the amino acid sequence of VH0 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 amino acid sequence of VH1 is shown in SEQ ID NO: 7, the amino acid sequence of VH2 is shown in SEQ ID NO: 8, the amino acid sequence of VH3 is shown in SEQ ID NO: 9, and the amino acid sequence of VH4 is shown in SEQ ID NO: 10.

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

[0169] The alignment results of the light and heavy chain sequences before and after humanization using IMGT analysis are shown in the table below. After combining four humanized light chains with four humanized heavy chains, 16 humanized antibodies were obtained. Humanized sequences were artificially synthesized and sequenced for accuracy, and then recombinantly cloned into the vector pcDNA3.4. The vector was transfected into CHO K1 cells for transient expression, and the cells were then cultured under suitable conditions to express the antibodies. The humanized antibody sequences were then isolated and purified.

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

[0171] Table 2 Comparison results of the variable regions of light chains

[0172]

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

[0174]

[0175]

[0176] 1.2 Orthogonal Combination of Humanized Antibodies

[0177] This invention selects different light and heavy chain sequences for cross-combination from the reversion mutation design of the variable regions of the aforementioned humanized antibody light and heavy chains, ultimately obtaining a variety of humanized antibodies. The specific steps are as follows: After adding the corresponding humanized antibody constant region to the variable region sequence of the humanized antibody, the corresponding target gene sequence is obtained through reverse translation using molecular biology software. The synthesized target gene is cloned downstream of the CMV promoter of an animal cell expression vector, using the SV40 terminator as the transcription terminator to obtain an expression vector capable of expression in animal cells. After obtaining the antibody expression vector, the four humanized sequences of each antibody light chain are orthogonally combined with the four heavy chain sequences to obtain 16 sets of target humanized antibody sequences. The amino acid sequences of the variable regions of each antibody are as follows:

[0178] Table 4. Amino acid sequences corresponding to the antibody variable region.

[0179] Fv 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] Simultaneously, the variable region sequence of the murine antibody was combined with the same constant region sequence of the human antibody, and the corresponding target gene sequence was obtained through reverse translation using molecular biology software. After synthesis, the target gene was cloned downstream of the CMV promoter of an animal cell expression vector, using the SV40 terminator as the transcription terminator to obtain an expression vector capable of expression in animal cells. The light and heavy chains of the human-mouse chimeric antibody, formed by combining the murine antibody variable region with the human antibody constant region, were then combined to obtain a control human-mouse chimeric antibody.

[0181] Example 2: Expression and Detection of Humanized Antibodies

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

[0183] 2.1 Octet-Titer Detection

[0184] This embodiment uses real-time reading of the thickness of the membrane formed after the target protein binds to the sensor. A real-time response monitoring spectrum is generated based on the shift value of the interference spectrum, and data analysis is performed using software. The sample signal value is fitted to a standard curve to obtain the sample titer. First, the reference standard is diluted with PBST solution to a final concentration of 50 μg / mL. Second, the diluted reference standard is added to column 1 of a 96-well plate, 200 μl / well. Then, PBST buffer is added to column 12 of the 96-well plate, 200 μl / well; and 10 mM glycine regeneration solution is added to column 11 of the 96-well plate, 200 μl / well. Samples are then added to columns 2-10 of the 96-well plate as needed, 200 μl / well. Finally, the 96-well plate is placed in an Octet instrument, and plate placement and detection are performed on Octet BLIDiscovery 12.2 software according to the sample loading table. The results are shown in Table 6.

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

[0186] This embodiment tested the binding activity of the antibody supernatant expressing the humanized sequence for the CLDN18.2 protein. The results showed that the humanized antibody supernatant has antigen-binding activity.

[0187] 1. Dilute human CLDN18.2-his antigen (catalog number: ACRO#CL2-H5587) to 1 μg / mL with coating buffer. After mixing, add 100 μl / well to a 96-well microplate and incubate overnight at 2-8°C.

[0188] 2. Washing: Discard the liquid in the 96-well microplate, add 0.05% PBST washing buffer, 300 μl / well, shake off the washing buffer, repeat this step, wash the plate 5 times. After washing, invert the 96-well microplate onto clean absorbent paper and pat off any remaining washing buffer.

[0189] Add 300 μl of 3.3% BSA-PBS to each well of a 96-well microplate and incubate at 37°C for 120 min. Then perform the blocking procedure.

[0190] 4. As in step 2, wash the plate 5 times.

[0191] 5. Dilute the sample concentration to 5000 ng / mL, and then perform serial dilutions according to Table 5. Add 100 μL / well of the serially diluted sample to a 96-well microplate and incubate at 37°C for 60 min.

[0192] Table 5 Gradient Dilution Table

[0193]

[0194]

[0195] 6. As in step 2, wash the plate 5 times.

[0196] 7. Dilute the secondary antibody IgG Fc Cross-Adsorbed Secondary Antibody (catalog number: 31413) 40,000 times with dilution buffer, add 100 μl / well to a 96-well microplate, and incubate at 37°C for 60 min.

[0197] 8. As in step 2, wash the plate 5 times.

[0198] 9. Add 100 μl / well of TMB colorimetric solution (catalog number: PR1200) and incubate at room temperature for 10 min.

[0199] 10. Add the stop solution at 50 μl / well, following the order in which the colorimetric reagents were added.

[0200] 11. Arrange the plates in the software according to the sample addition order, place the 96-well microplate into the microplate reader, and take readings 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 corresponding humanized antibodies and 1 human-mouse chimeric antibody were obtained for each murine antibody variable region sequence. The results are shown in Table 6.

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

[0204]

[0205]

[0206] Example 3: Bioactivity assay of the purified humanized antibody protein

[0207] 3.1 ELISA was used to detect the antigen-binding activity of the purified humanized antibody proteins before and after humanization.

[0208] The binding ability of purified humanized antibody proteins to human CLDN18.2-his protein was compared using ELISA. One day in advance, coat the plates. Dilute CLDN18.2-His to 1ug / ml using 1xELISA coating buffer. Add 100ul / well to each ELISA plate and coat overnight at 4°C. Remove the plate and allow it to equilibrate to room temperature. Prepare PBST (0.05% Tween-20 in 1xPBS) and blocking buffer (2% BSA in PBST). Discard the supernatant, add 300ul / well of PBST to wash the plate, repeating 6 times. Add 300ul / well of blocking buffer and block at 37°C for 2 hours. Prepare dilution buffer (0.5% BSA in PBST) and dilute the purified humanized antibody protein to 1ug / ml using the dilution buffer. Perform a 5-fold serial dilution, resulting in 8 concentration points. After blocking, wash the plate 6 times with PBST, add 100ul / well of the diluted antibody or humanized antibody supernatant, and incubate at 37°C for 2 hours. Dilute the detection antibody Goat anti-human IgG 1:10000 with the dilution buffer. Fc(HRP); After incubation, wash the plate 6 times with PBST, add 100 μL of detection antibody per well, and incubate at 37°C for 1 hour; After detection antibody incubation, wash the plate 6 times with PBST, add 100 μL of 1x TMB per well, and incubate at room temperature in the dark for about 3 minutes; Add 50 μL of ELISA stop solution per well to stop the color development, and read the OD450 value using a multi-mode microplate reader.

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

[0210] 3.2 ADCC and CDC activities were detected in the purified humanized antibody proteins before and after purification.

[0211] The ADCC activity of antibodies before and after humanization was compared using the Reporter Gene Assay. 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 results are as follows: Figure 4 As shown, there was no significant difference in ADCC activity of the antibody proteins 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 CDC effect of antibodies before and after humanization was compared using the LDH method. Commercially available normal human serum complement was used to detect the CDC effect of the antibody protein on KATOⅢ-human CLDN18.2 cells. The results are as follows: Figure 5 As shown, no CDC activity was detected in any of the antibody proteins.

[0213] Example 4: Physicochemical property analysis of the purified humanized antibody protein

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

[0215] After detecting the biological activity of humanized antibody proteins, three molecules, huB-ZG056-01, huB-ZG059-04, and huB-ZG063-08, were screened out. These three humanized and mouse-derived molecules were detected using CPB digestion and non-enzymatic digestion methods (CEX method). High-performance liquid chromatography (HPLC) was performed using a Waters Arc Premier cation exchange column (Thermo MabPac WCX-10, 4×250mm); the mobile phases were A (20mM MES pH 6.3) and B (20mM MES + 200mM NaCl pH 6.3); the flow rate was 0.6 min / ml; and the detection wavelength was 280 nm. The digested samples were added to 1 mg / ml carboxypeptidase B solution at a ratio of 100:2 (v / v), mixed, briefly centrifuged, and then incubated at 37℃ for 60 minutes. Gradient elution was used, with salt ion concentrations ranging from 0% to 100% over 3–30 min. The results are shown in Figure 6 and Table 7.

[0216] Table 7 CEX Detection Results

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

[0218] The huB-ZG056-01 molecule showed relatively high levels of basic peaks before and after enzyme digestion, with concentrations of 36.6% and 28.8% respectively, indicating that the predominantly basic peak was not in a lysine-deficient protein. The basic peaks of huB-ZG059-04 and huB-ZG063-08 molecules decreased significantly after enzyme digestion, indicating that the predominantly basic peak was in a lysine-deficient protein. Both molecules' basic peaks decreased to below 10.0% after digestion, suggesting that huB-ZG059-04 and huB-ZG063-08 have low charge variant ratios and good CEX performance. Furthermore, the results indicate that the humanized antigen-binding protein exhibits significantly improved CEX performance and a marked reduction in its acidic peak.

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

[0220] Based on the CEX detection results, the aggregation of huB-ZG059-04 and huB-ZG063-08 molecules was assessed by SEC. High-performance liquid chromatography (Waters Arc Premier) was used, with a TSKgel-G3000SW gel column (suitable for separating proteins with molecular weights of 10–500 kD) as the packing material. 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 detection results are as follows: Figure 7 And as 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 both huB-ZG059-04 and huB-ZG063-08 molecules is greater than 98.0%, and the aggregate content is low, below 1.8%. Therefore, the SEC results for huB-ZG059-04 and huB-ZG063-08 molecules are good. Compared with the murine antibody, the antibody derived by this applicant exhibits superior quality.

Claims

1. A CLDN18.2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 10 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO:

3.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment comprises a Fab, Fab’, Fv fragment, F(ab’)2, scFv, and / or di-scFv.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a monoclonal antibody.

4. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain constant region.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the heavy chain constant region is derived from a human IgG heavy chain constant region.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO:

17.

7. The antibody or antigen-binding fragment thereof of claim 1, comprising a light chain constant region.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the light chain constant region is derived from a human IgG1 light chain constant region comprising an amino acid sequence as set forth in SEQ ID NO:

16.

9. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 24 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO:

25.

10. The antibody or antigen-binding fragment thereof of claim 1, comprising an amino acid sequence as set forth in SEQ ID NO:

26.

11. The antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the CLDN18.2 is of human origin.

12. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-11.

13. A vector comprising the isolated nucleic acid molecule of claim 12.

14. The vector of claim 13, comprising a viral vector.

15. The vector of any one of claims 13-14, comprising a retroviral vector.

16. A cell comprising and / or expressing the antibody or antigen-binding fragment thereof of any one of claims 1-11, the isolated nucleic acid molecule of claim 12, and / or the vector of any one of claims 13-15.

17. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-11, the method comprising culturing the cell of claim 16 under conditions in which the antibody or antigen-binding fragment thereof of any one of claims 1-11 is expressed.

18. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-11, the isolated nucleic acid molecule of claim 12, the vector of any one of claims 13-15, and / or the cell of claim 16.

19. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-11, the isolated nucleic acid molecule of claim 12, the vector of any one of claims 13-15, and / or the cell of claim 16, and a pharmaceutically acceptable carrier.

20. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-11, the isolated nucleic acid molecule of claim 12, the vector of any one of claims 13-15, and / or the cell of claim 16 for the manufacture of a pharmaceutical composition for the treatment of a tumor selected from the group consisting of: esophageal cancer, ovarian cancer, lung cancer, pancreatic cancer, and / or gastric cancer.

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