CLDN18.2 Antibodies and Their Applications

Abstract

The present invention provides a CLDN18.2 antibody and its applications. Specifically, the present invention also describes nucleic acids encoding the antibody, compositions comprising the antibody, methods for preparing the antibody, and methods for treating or preventing diseases, such as cancer and / or inflammatory diseases, using the antibody.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a CLDN18.2 antibody and its application. Background Art

[0002] Gastrointestinal and pancreatic tumors pose a great threat to human life. Although treatment methods such as surgical treatment, radiotherapy, chemotherapy, and interventional treatment have certain effects on such tumors, the survival rate of patients has not been significantly improved.

[0003] Cell immunotherapy is an emerging tumor treatment method. It constructs an expression vector of a chimeric antigen receptor (CAR) through molecular biology techniques, and introduces the expression vector into immune cells isolated from the human body. After the CAR is expressed on the cell surface, the cells are amplified and cultured, and then infused back into the human body. CAR is composed of an antigen recognition domain, a hinge region, a transmembrane region, and an intracellular signaling domain connected in sequence. Immune cells expressing CAR can specifically recognize and bind to target cells and kill them by releasing specific immune factors.

[0004] Gastric cancer is one of the most common cancers in the world. In China, gastric cancer is the second most common malignant tumor and is considered one of the most difficult cancers to cure in the world. Despite the progress made in treatment options in recent years, the recurrence of gastric cancer is inevitable. The five-year survival rate of patients with advanced gastric cancer is about 5–20%, and the median overall survival period is about 10 months. With the in-depth study of the molecular mechanisms of the occurrence and development of gastric cancer, targeted therapy has become an effective treatment option for advanced cancer, and the targets mainly include EGFR, HER-2, VEGF, VEGFR, etc. As a highly specifically expressed cell surface molecule, CLDN18.2 is only expressed on differentiated gastric mucosal epithelial cells in normal tissues. Therefore, it is necessary to develop therapeutic antibodies against CLDN18.2 with greater anti-gastric cancer potential, lower toxicity, and lower drug dosage.

[0005] In summary, there is an urgent need in this field to develop a new CLDN18.2 antibody and its application. Summary of the Invention

[0006] The purpose of the present invention is to provide a new CLDN18.2 antibody and its application.

[0007] In the first aspect of the present invention, a heavy chain variable region of an antibody is provided. The heavy chain variable region includes the following three complementarity-determining regions CDR:

[0008] CDR1 shown in SEQ ID NO:2,

[0009] CDR2 shown in SEQ ID NO:3, and

[0010] The CDR3 shown in SEQ ID NO:4.

[0011] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:1.

[0012] In the second aspect of the present invention, there is provided a heavy chain of an antibody, which has the heavy chain variable region and the heavy chain constant region described in the first aspect of the present invention.

[0013] In another preferred example, the heavy chain constant region is human or murine.

[0014] In another preferred example, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO:13.

[0015] In the third aspect of the present invention, there is provided a light chain variable region of an antibody, and the light chain variable region has complementarity determining regions CDR selected from the following group:

[0016] CDR1’ shown in SEQ ID NO:6,

[0017] CDR2’ shown in SEQ ID NO:7, and

[0018] CDR3’ shown in SEQ ID NO:8.

[0019] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO:5.

[0020] In the fourth aspect of the present invention, there is provided a light chain of an antibody, and the light chain has the light chain variable region and the light chain constant region described in the third aspect of the present invention.

[0021] In another preferred example, the constant region of the light chain is human or murine.

[0022] In another preferred example, the light chain constant region has the amino acid sequence shown in SEQ ID NO:14.

[0023] In the fifth aspect of the present invention, there is provided an antibody, which has:

[0024] (1) The heavy chain variable region as described in the first aspect of the present invention; and / or

[0025] (2) The light chain variable region as described in the third aspect of the present invention.

[0026] In another preferred embodiment, the antibody has: a heavy chain as described in the second aspect of the present invention; a heavy chain as described in the second aspect of the present invention. In another preferred embodiment, the antibody described in the present invention is an antibody specifically against the CLDN18.2 protein.

[0027] In another preferred embodiment, the antibody includes: single-chain antibody, double-chain antibody, monoclonal antibody, chimeric antibody (such as human-mouse chimeric antibody), murine antibody, or humanized antibody.

[0028] In the sixth aspect of the present invention, there is provided a recombinant protein having:

[0029] (i) a sequence of a heavy-chain variable region as described in the first aspect of the present invention, a sequence of a heavy chain as described in the second aspect of the present invention, a sequence of a light-chain variable region as described in the third aspect of the present invention, a sequence of a light chain as described in the fourth aspect of the present invention, or a sequence of an antibody as described in the fifth aspect of the present invention; and

[0030] (ii) optionally, a tag sequence for assisting expression and / or purification.

[0031] In another preferred embodiment, the tag sequence includes a 6His tag.

[0032] In another preferred embodiment, the recombinant protein is specifically against the CLDN18.2 protein.

[0033] In the seventh aspect of the present invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

[0034] (1) a heavy-chain variable region as described in the first aspect of the present invention, a heavy chain as described in the second aspect of the present invention, a light-chain variable region as described in the third aspect of the present invention, a light chain as described in the fourth aspect of the present invention, or an antibody as described in the fifth aspect of the present invention; or

[0035] (2) a recombinant protein as described in the sixth aspect of the present invention.

[0036] In another preferred embodiment, the polynucleotide has the sequences shown in SEQ ID NO:9, 10, 11, 12.

[0037] In the eighth aspect of the present invention, there is provided a vector containing the polynucleotide described in the seventh aspect of the present invention.

[0038] In another preferred embodiment, the vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0039] In a ninth aspect of the present invention, there is provided a genetically engineered host cell that contains the vector described in the eighth aspect of the present invention or a polynucleotide described in the seventh aspect of the present invention integrated into its genome.

[0040] In a tenth aspect of the present invention, there is provided an immunoconjugate that contains:

[0041] (a) a heavy chain variable region as described in the first aspect of the present invention, a heavy chain as described in the second aspect of the present invention, a light chain variable region as described in the third aspect of the present invention, a light chain as described in the fourth aspect of the present invention, an antibody as described in the fifth aspect of the present invention, or a recombinant protein as described in the sixth aspect of the present invention; and

[0042] (b) a conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0043] In an eleventh aspect of the present invention, there is provided a pharmaceutical composition that contains:

[0044] (i) a heavy chain variable region as described in the first aspect of the present invention, a heavy chain as described in the second aspect of the present invention, a light chain variable region as described in the third aspect of the present invention, a light chain as described in the fourth aspect of the present invention, or an antibody as described in the fifth aspect of the present invention, a recombinant protein as described in the sixth aspect of the present invention, or an immunoconjugate as described in the tenth aspect of the present invention; and

[0045] (ii) a pharmaceutically acceptable carrier.

[0046] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0047] In another preferred embodiment, the pharmaceutical composition is used for preparing a drug for treating tumors, and the tumors are selected from the group consisting of: gastric cancer, esophageal cancer, cholangiocarcinoma, pancreatic cancer, lung cancer, ovarian cancer, and colon cancer.

[0048] In a twelfth aspect of the present invention, there is provided the use of a heavy chain variable region as described in the first aspect of the present invention, a heavy chain as described in the second aspect of the present invention, a light chain variable region as described in the third aspect of the present invention, a light chain as described in the fourth aspect of the present invention, or an antibody as described in the fifth aspect of the present invention, a recombinant protein as described in the sixth aspect of the present invention, or an immunoconjugate as described in the tenth aspect of the present invention, for preparing a medicament, a reagent, a test plate, or a kit;

[0049] The reagent, test plate, or kit is used for: detecting CLDN18.2 protein in a sample;

[0050] The medicament is used for treating or preventing tumors that express CLDN18.2 protein.

[0051] In another preferred example, the tumors include: gastric cancer, esophageal cancer, cholangiocarcinoma, pancreatic cancer, lung cancer, ovarian cancer, and colon cancer.

[0052] In another preferred example, the tumors are selected from: gastric cancer and pancreatic cancer.

[0053] In another preferred example, the reagent includes a chip and immune microparticles coated with antibodies.

[0054] In a thirteenth aspect of the present invention, a method for detecting CLDN18.2 protein in a sample is provided. The method includes the steps of:

[0055] (1) Contacting the sample with the antibody described in the fifth aspect of the present invention;

[0056] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CLDN18.2 protein in the sample.

[0057] In a fourteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided. The method includes:

[0058] (a) Culturing the host cell described in the ninth aspect of the present invention under conditions suitable for expression;

[0059] (b) Isolating the recombinant polypeptide from the culture. The recombinant polypeptide is the antibody described in the fifth aspect of the present invention or the recombinant protein described in the sixth aspect of the present invention.

[0060] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Shows the hybridoma method system of the present invention.

[0062] Figure 2 Shows the summary of hCLDN18.2 positive hybridomas.

[0063] Figure 3 Shows the FACS detection of cross-reactivity of the subcloned hybridoma supernatant. Among them, the abscissa is the fluorescence intensity, detecting the binding strength of the detection antibody and CLDN18.2; the ordinate SSC is the side scatter of the cells, detecting the complexity of the cells.

[0064] Figure 4 Shows the titer detection of the subclones.

[0065] Figure 5The flow cytometry detection chart showing the identification results of C18-16-1H1-1A8 recombinant antibody expression. Among them, FL1-H is the fluorescence intensity, detecting the binding intensity of the detection antibody and CLDN18.2; SSC-H subset is the side scatter of cells, detecting the complexity of cells.

[0066] Figure 6 Shows the construction map of the expression vector of the heavy chain.

[0067] Figure 7 Shows the construction map of the expression vector of the light chain.

[0068] Figure 8 Shows the identification result chart of the FACS binding of the antibody to 18.2-K562. Among them, IgG1 is the negative control. Detailed implementation mode

[0069] Through extensive and in-depth research and a large number of screenings, the present inventors unexpectedly discovered a monoclonal antibody against CLDN18.2. Experimental results show that this monoclonal antibody has high specificity and strong affinity and can specifically bind to CLDN18.2. Based on this, the present invention was completed.

[0070] CLDN18.2

[0071] Claudins is a protein family whose function is to maintain tight junctions that control intercellular molecular exchange. It is widely distributed in gastric, pancreatic and lung tissues and can be used for diagnosis and treatment. Claudin (CLDN) has 4 transmembrane domains and is involved in physiological processes such as the regulation of paracellular permeability and conductance in the body. Its family contains at least 24 members. CLDN18 is a member of the Claudin protein family and is encoded by the CLDN18 gene in the human body. The human CLDN18 gene has two different exon 1s, which generate two protein subtypes, CLDN18.1 and CLDN18.2, through alternative splicing after transcription. Both of these subtypes consist of 261 amino acids and only differ by 8 amino acids in the extracellular region at the N-terminus.

[0072] The CLDN18.2 subtype is a stomach-specific subtype. Under normal physiological conditions, Claudin 18.2 (CLDN18.2) is only expressed on the surface of short-lived cells in the human gastric mucosa epithelium; however, it is highly expressed in various tumors such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, and colon cancer. For example, the expression of this target exists in 50%-80% of gastric cancer patients. CLDN18.2 is usually buried in the gastric mucosa and is basically inaccessible to monoclonal antibodies in normal tissues. The occurrence of malignant tumors will lead to the disruption of tight junctions, exposing the CLDN18.2 epitope on the surface of tumor cells and making it a specific target. Therefore, CLDN18.2 confers specificity to targeted therapy. Recently, it has been found that the expression of CLDN18.2 in pancreatic cancer (50%), esophageal cancer, and lung cancer also shows the potential for diagnosing and treating other tumors.

[0073] The term

[0074] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of approximately 150,000 daltons with the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also have regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by multiple constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0075] As used herein, the term "variable" means that certain parts of the variable regions in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the variable regions of the light chain and the heavy chain. The more conserved parts in the variable regions are called framework regions (FRs). The variable regions of natural heavy chains and light chains each contain four FR regions, which are generally in a β-sheet configuration and are connected by three CDRs forming connecting loops, and in some cases can form partial β-sheet structures. The CDRs in each chain are closely juxtaposed by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cell cytotoxicity.

[0076] The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of two distinct classes (referred to as κ and λ) based on the amino acid sequence of its constant region. Immunoglobulins can be divided into different classes according to the amino acid sequence of their heavy chain constant regions. There are mainly five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

[0077] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for minor possible naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually consist of different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the property of the antibody, which is obtained from a substantially homogeneous antibody population, and should not be construed as requiring any particular method for producing the antibody.

[0078] The present invention also includes monoclonal antibodies having the corresponding amino acid sequences of the monoclonal antibodies against the CLDN18.2 protein described above, monoclonal antibodies having the variable region chains of the monoclonal antibodies against the CLDN18.2 protein, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain containing hypervariable regions (complementary determining regions, CDRs), provided that the hypervariable regions are the same as or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light chain and heavy chain of the present invention.

[0079] As is known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the monoclonal antibodies against the CLDN18.2 protein or fragments thereof. The present invention also includes cell surface markers or antigens that bind to the monoclonal antibodies against the CLDN18.2 protein or fragments thereof.

[0080] The present invention includes not only intact monoclonal antibodies but also immunologically active antibody fragments, such as Fab or (Fab')2 fragments; antibody heavy chains; and antibody light chains.

[0081] As used herein, the term "heavy chain variable region" and "V H " are used interchangeably.

[0082] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0083] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody comprises the following three complementarity determining regions CDR:

[0084] CDR1, having the amino acid sequence GYTFTSYWMH (SEQ ID NO: 2), and having the encoding nucleotide sequence ggctacactttcaccagctactggatgcac (SEQ ID NO: 15);

[0085] CDR2, having the amino acid sequence MIHPNSGSTN (SEQ ID NO: 3), and having the encoding nucleotide sequence atgattcatcctaatagtggtagtactaac (SEQ ID NO: 16);

[0086] CDR3, having the amino acid sequence GGYYGNSLDF (SEQ ID NO: 4), and having the encoding nucleotide sequence gggggctactatggtaactcccttgacttc (SEQ ID NO: 17).

[0087] In another preferred example, the amino acid sequence of the heavy chain variable region is:

[0088] MGWSYIILFLVATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVRQRPGQGLEWIGMIHPNSGSTNYNGKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARGGYYGNSLDFWGQGTSLTVSS (SEQ ID NO.: 1);

[0089] And its encoding nucleotide sequence is:

[0090] atgggatggagctatatcatcctctttttggtagcaacagctacaggtgtccactcccaggtccaactgcagcagcctggggctgagctggtaaagcctggggcttcagtgaagttgtcctgcaaggcttctggctacactttcaccagctactggatgcactgggtgaggcagaggcctggacaaggccttgagtggattggaatgattcatcctaatagtggtagtactaactacaatgggaagttcaagagcaaggccacactgactgtagacaaatcctccagcacagcctacatgcaactcagcagcctgacatctgaggactctgcggtctatttctgtgcaagagggggctactatggtaactcccttgacttctggggccaaggcacctctctcacagtctcctca(SEQ ID NO.:9)。

[0091] In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the above heavy chain variable region and heavy chain constant region, and the heavy chain constant region can be murine or human.

[0092] In another preferred example, the amino acid sequence of the heavy chain constant region (mIgG2c) is:

[0093] AKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEVLHNHLTTKTISRSLGK(SEQ ID No:13).

[0094] In another preferred example, the nucleotide sequence encoding the heavy chain constant region (mIgG2c) is as follows:

[0095]

[0096] As used herein, the term "light chain variable region" and "V L " are used interchangeably.

[0097] In a preferred embodiment of the present invention, the light chain variable region of the antibody according to the present invention has complementarity determining regions CDRs selected from the group consisting of:

[0098] CDR1’, having the amino acid sequence KSSQSLLNSGNQKNYLT (SEQ ID NO:6), and its encoding nucleotide sequence is aagtccagtcagagtctgttaaacagtggaaatcaaaagaactacttgacc (SEQ ID NO.:18);

[0099] CDR2’, having the amino acid sequence WASTRES (SEQ ID NO:7), and its encoding nucleotide sequence is tgggcatccactagggaatct (SEQ ID NO.:19);

[0100] CDR3’, having the amino acid sequence QNAYSYPFT (SEQ ID NO:8), and its encoding nucleotide sequence is cagaatgcttatagttatccattcacg (SEQ ID NO.:20).

[0101] In another preferred example, the amino acid sequence of the light chain variable region is:

[0102] MESQTQVLMSLLFWVSGTCGDIVMTQSPSSLTVTAREKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNAYSYPFTFGSGTKLEIK (SEQ ID NO.:5),

[0103] and its encoding nucleotide sequence is:

[0104] atggaatcacagactcaggtcctcatgtccctgctgttctgggtatctggtacctgtggggacattgtgatgacacagtctccatcctccctgactgtgacagcaagagagaaggtcactatgagctgcaagtccagtcagagtctgttaaacagtggaaatcaaaagaactacttgacctggtaccagcagaaaccagggcagcctcctaaactgttgatctactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctggaacagatttcactctcaccatcagcagtgtgcaggctgaagacctggcagtttattactgtcagaatgcttatagttatccattcacgttcggctcggggacaaagttggaaataaaa(SEQ ID NO.:10)。

[0105] In a preferred embodiment of the present invention, the light chain of the antibody comprises the above-mentioned light chain variable region and light chain constant region, and the light chain constant region can be murine or human.

[0106] In another preferred example, the amino acid sequence of the light chain constant region (IgK) is:

[0107] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSS TLTLTKDEYE RHNSYTCEAT HKTSTSPIVK SFNRNEC(SEQ ID No:14).

[0108] In another preferred example, the nucleotide sequence encoding the light chain constant region (IgK) is:

[0109] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG(SEQ ID No:12).

[0110] In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably, and all refer to an antibody that specifically binds to the CLDN18.2 protein, such as a protein or polypeptide having a heavy chain variable region (such as the amino acid sequence of SEQ ID NO.:1) and / or a light chain variable region (such as the amino acid sequence of SEQ ID NO.:5). They may or may not contain an initiating methionine.

[0111] In another preferred embodiment, the antibody is a murine or human-murine chimeric monoclonal antibody against the CLDN18.2 protein, and its heavy chain constant region and / or light chain constant region can be a humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is the heavy chain constant region or light chain constant region of human IgG1, IgG2, etc.

[0112] The present invention also provides other proteins or fusion expression products having the antibody of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain and a light chain containing variable regions, as long as the variable regions are the same as or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy chain and light chain of the antibody of the present invention.

[0113] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called complementarity-determining regions (CDRs), which divide the segment into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form loop structures that are brought close to each other in spatial structure by the β-sheets formed by the intervening FRs. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acids that make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.

[0114] The variable regions of the heavy and / or light chains of the antibodies of the present invention are of particular interest because at least some of them are involved in binding to the antigen. Accordingly, the present invention includes molecules having monoclonal antibody light and heavy chain variable regions with CDRs, provided that the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified herein.

[0115] The present invention includes not only intact monoclonal antibodies, but also fragments of antibodies having immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives, and analogs of the said antibodies.

[0116] As used herein, the terms "fragment", "derivative", and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0117] The antibody of the present invention refers to a polypeptide having CLDN18.2 protein-binding activity and including the above CDR regions. This term also includes variant forms of the polypeptide containing the above CDR regions that have the same function as the antibody of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when substituting amino acids with similar or close properties, the function of the protein usually will not be changed. Another example is that adding one or several amino acids at the C-terminus and / or N-terminus usually will not change the function of the protein. This term also includes active fragments and active derivatives of the antibody of the present invention.

[0118] The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the coding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using the antiserum against the antibody of the present invention.

[0119] The present invention also provides other polypeptides, such as fusion proteins containing human antibodies or their fragments. In addition to almost full-length polypeptides, the present invention also includes fragments of the antibody of the present invention. Generally, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, most preferably at least about 100 consecutive amino acids.

[0120] In the present invention, the "conservative variant of the antibody of the present invention" refers to a polypeptide in which, compared with the amino acid sequence of the antibody of the present invention, at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acids are replaced by amino acids with similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0121] Table 1

[0122]

[0123]

[0124] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or their fragments or their fusion proteins. The polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide can be the same as or a degenerate variant of the coding region sequences shown in SEQ ID NO.: 9, 10, 11, 12, 15, 16, 17, 18, 19, 20. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding an amino acid sequence identical to the polypeptide of the present invention, but having differences from the coding region sequences shown in SEQ ID NO.: 9, 10, 11, 12, 15, 16, 17, 18, 19, 20.

[0125] The polynucleotides encoding the mature polypeptides of the present invention include: coding sequences encoding only the mature polypeptides; the coding sequences of the mature polypeptides and various additional coding sequences; the coding sequences of the mature polypeptides (and optional additional coding sequences) and non-coding sequences.

[0126] The term "polynucleotide encoding a polypeptide" can be a polynucleotide including the polynucleotide encoding this polypeptide, or can also be a polynucleotide further including additional coding and / or non-coding sequences.

[0127] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at a lower ionic strength and a higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least above 90%, preferably above 95%. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides shown in SEQ ID NO.: 1 and / or SEQ ID NO.: 5.

[0128] The full-length nucleotide sequence or its fragments of the antibodies of the present invention can generally be obtained by PCR amplification, recombination or synthetic methods. A feasible method is to synthesize the relevant sequences by synthetic methods, especially when the fragment length is short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0129] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually to clone it into a vector, then transfer it into cells, and then isolate the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in an isolated form.

[0130] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0131] The present invention also relates to vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0132] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0133] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well-known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl2 method, and the steps used are well-known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0134] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. According to the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cell. When the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0135] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatments, treatment with protein precipitants (salting-out methods), centrifugation, osmotic lysis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0136] The antibodies of the present invention can be used alone or in combination or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these substances.

[0137] Detectable labels for diagnostic purposes include, but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0138] Therapeutic agents that can bind to or be conjugated with the antibodies of the present invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biological toxins (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines such as IL-2, etc. (Gillies et al., 1992, PNAS 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al., 2003, Cancer Research 63, 3202); 4. Gold nanoparticles / nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 5. Virus particles (Peng et al., 2004, Gene therapy 11, 1234); 6. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 7. Nanomagnetic particles; 8. Prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0139] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substances being formulated and the disease to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0140] The pharmaceutical composition of the present invention can be directly used to bind to the CLDN18.2 protein molecule, and thus can be used for the prevention and treatment of tumors. In addition, other therapeutic agents can also be used simultaneously.

[0141] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001 - 99 wt%, preferably 0.01 - 90 wt%, more preferably 0.1 - 80 wt%) of the monoclonal antibody (or its conjugate) of the present invention as described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram per kilogram of body weight per day - about 5 milligrams per kilogram of body weight. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0142] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 8 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight - about 1 milligram per kilogram of body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0143] Hybridoma cell line

[0144] The present invention also provides a hybridoma cell line capable of producing the monoclonal antibody against CLDN18.2 protein of the present invention; preferably, the present invention provides a hybridoma cell line with a high titer of monoclonal antibody against CLDN18.2 protein.

[0145] After obtaining the hybridoma for producing the monoclonal antibody against CLDN18.2 protein of the present invention, those skilled in the art can conveniently use this hybridoma cell line to prepare the antibody. In addition, those skilled in the art can also easily know the structure of the antibody of the present invention (such as the variable region of the heavy chain and the variable region of the light chain of the antibody), and then can prepare the monoclonal antibody of the present invention by recombinant methods.

[0146] Preparation of monoclonal antibody

[0147] The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, the antigen of the present invention can be administered to an animal to induce the production of monoclonal antibodies. For monoclonal antibodies, hybridoma technology can be utilized for preparation (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981) or can be prepared by recombinant DNA methods (U.S. Patent No. 4,816,567).

[0148] Representative myeloma cells are those that are efficiently fusible, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to the culture medium (HAT medium substrate), including myeloma cell lines, such as murine myeloma cell lines, including myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, San Diego, California, USA) and SP-2, NZ0 or X63-Ag8-653 cells (available from American Type Culture Collection, Rockville, Maryland, USA). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibodies Production Techniques and Applications, pages 51-63 (Marcel Dekker, Inc., New York, 1987)].

[0149] The medium in which the hybridoma cells grow is analyzed to detect the production of monoclonal antibodies with the desired specificity, e.g., by an in vitro binding assay such as an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). The location of the antibody-expressing cells can be detected by FACS. Then, the hybridoma clones can be subcloned by a limiting dilution step and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) pp. 59-103). Suitable media used for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown as ascites tumors in animals.

[0150] The monoclonal antibodies secreted by the subclones are appropriately isolated from the medium, ascites, or serum by conventional immunoglobulin purification processes such as, for example, the protein A-Sepharose method, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0151] The present invention provides a monoclonal antibody against the CLDN18.2 protein, particularly a monoclonal antibody against the CLDN18.2 protein. In a preferred embodiment of the present invention, the monoclonal antibody is prepared by culturing hybridoma cells. The supernatant of the hybridoma cell culture is taken, and IgG is crudely extracted by the saturated ammonium sulfate precipitation method. Then, the crudely extracted antibody is purified by an affinity chromatography column (Protein G-Sephrose).

[0152] In a preferred embodiment of the present invention, the monoclonal antibody is prepared by the method of producing monoclonal antibodies in the ascites of Balb / C mice. Approximately hybridoma cells are inoculated into the peritoneal cavity of sensitized mice, and obvious abdominal distension can be seen in about 10 days. The ascites is extracted, crudely extracted by the saturated ammonium sulfate precipitation method, and then the crudely extracted antibody is purified by an affinity chromatography column (Protein G-Sephrose).

[0153] Labeled immunoglobulin

[0154] In a preferred example of the present invention, the immunoglobulin carries a detectable label. More preferably, the label is selected from the group consisting of a colloidal gold label, a colored label, or a fluorescent label.

[0155] Colloidal gold labeling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the present invention, the monoclonal antibody against the CLDN18.2 protein is labeled with colloidal gold to obtain a colloidal gold-labeled monoclonal antibody.

[0156] The monoclonal antibody of CLDN18.2 protein of the present invention has very good specificity and high titer.

[0157] Methods and Samples

[0158] The present invention relates to a method for detecting tumors in samples lysed with cells and / or tissues. The steps of the method are generally as follows: obtaining a cell and / or tissue sample; lysing the sample in a medium; detecting the level of CLDN18.2 protein in the lysed sample. The sample used in the method of the present invention can be any sample including cells present in a cell preservation solution, as used in liquid-based cytology tests.

[0159] Kits

[0160] The present invention also provides a kit containing the antibody (or its fragment) of the present invention or the detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, an instruction manual, a buffer, etc.

[0161] The present invention further designs a detection kit for detecting the level of CLDN18.2, which includes an antibody that recognizes CLDN18.2 protein, a lysis medium for lysing the sample, general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0162] The main advantages of the present invention include:

[0163] (1) The antibody of the present invention has high specificity, strong affinity, can be prepared in large quantities, and the quality of the monoclonal antibody is easy to control.

[0164] (2) The antibody of the present invention can be used for targeted drugs, antibody-drug conjugates or multifunctional antibodies that specifically target CLDN18.2-positive tumor cells.

[0165] (3) The antibody of the present invention can be used to prepare reagents for diagnosing tumors or for preparing chimeric antigen receptor immune cells.

[0166] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0167] The experimental system of the present invention

[0168] The present invention adopts a hybridoma method system. Through DNA and cellular immunization and flow cytometry screening, positive hybridoma cells are obtained, and the screening system is stable.

[0169] The hybridoma method system is as Figure 1 shown.

[0170] Sequences related to A8 antibody

[0171] Table 2. Sequences related to A8 antibody

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] Note: The underlined part is the Leader sequence. Among them, VL is the variable region of the light chain, CL is the constant region of the light chain, CH is the constant region of the heavy chain, and VH is the variable region of the heavy chain.

[0178] Preparation of CLDN18.2 antibody in Example 1

[0179] 1. Immunize mice

[0180] 2. Serum detection

[0181] 3. Fusion

[0182] 4. Screening after fusion

[0183] 5. Subcloning and establishing a strain

[0184] 6. Antibody sequence analysis

[0185] 7. Expression verification of antibody sequence

[0186] 8. Affinity detection of antibody

[0187] Example 2 hCLDN18.2 positive hybridoma

[0188] As Figure 2 shown, the hCLDN18.2 positive hybridomas in the present invention are summarized.

[0189] Example 3 Cross-reactivity detection of hybridoma supernatant

[0190] CHO-hCLDN18.1+ hybridoma supernatant + goat anti-mouse IgGFc-FITC

[0191] Aliquot an appropriate amount of CHO-hCLDN18.1 cells into a 1.5 ml EP tube. After centrifugation, resuspend the cells in 50 μl of PBS. Add 50 μl of goat anti-mouse IgG Fc-FITC diluted 1:500 with PBS, and let it stand at 4°C for 15 minutes. After centrifugation to replace the supernatant, resuspend the cells in 200 μl of PBS and perform FACS flow analysis.

[0192] As Figure 3 shown, the cross-reactivity with 18.1 was detected by flow cytometry using CHO stable transfected cells (Cell Pool). The antibody (C18-16-1H1-1A8) is a CLDN18.2 specific antibody and does not react with CLDN18.1.

[0193] Example 4 Detection of Hybridoma Supernatant Titer

[0194] 10-fold serial dilution:

[0195] CHO-hCLDN18.2 + Hybridoma supernatant (10-fold serial dilution) + Goat anti-mouse IgGFc-FITC

[0196] Aliquot an appropriate amount of CHO-hCLDN18.2 cells into a 1.5 ml EP tube. After centrifugation, resuspend the cells in 50 μl of PBS. Add 50 μl of goat anti-mouse IgG Fc-FITC diluted 1:500 with PBS, and let it stand at 4°C for 15 minutes. After centrifugation to replace the supernatant, resuspend the cells in 200 μl of PBS and perform FACS flow analysis.

[0197] The detection of the titer of the hybridoma supernatant of the positive clone of mouse No. 16 is as Figure 4 shown. Select C18-16-1H1-1A8 (A8) for the expression verification of the antibody sequence.

[0198] The information related to A8 antibody is as follows:

[0199] Cell line C18-16-1H1-1A8

[0200] Source: Fusion of SP2 / 0 cells and C57 mouse B cells

[0201] Subtype IgG2c, kappa

[0202] Heavy chain V gene mIgHV1-64

[0203] Light chain V gene MIgKV8-19.

[0204] Example 5 Expression Verification of Antibody Sequence

[0205] Add 10 μg each of the heavy and light chain expression vectors (as Figure 6 and 7Using the calcium phosphate method, it was co-transfected into 293T cells in a 10-cm culture dish. Three days later, the supernatant was collected and verified by FACS in CHO-hCLDN18.2 cells.

[0206] The results of the flow cytometry assay are as Figure 5 shown.

[0207] The recombinant antibody C18-16-1H1-1A8 in the 293T supernatant has good activity and the correct sequence.

[0208] Activity detection of the antibody of Example 6A8

[0209] 6.1. Cell seeding: Prepare cell suspensions of 18.2-K562, 18.1-K562, and K562, and adjust the density to 1×10 6 / mL. Take 3 round-bottom 96-well plates, labeled as plate1, plate2, and plate3. Use a 100-μL pipette to add 100 μL of the suspensions of 18.2-K562, 18.1-K562, and K562 to plate1, plate2, and plate3 respectively. Centrifuge at 300 g for 5 min in a centrifuge. Discard the supernatant.

[0210] 6.2. Add antibody diluent: Dilute the antibody with FACS Buffer to 8 concentration gradients of diluents, namely 20 μg / mL, 6.667 μg / mL, 2.222 μg / mL, 0.741 μg / mL, 0.247 μg / mL, 0.082 μg / mL, 0.027 μg / mL, and 0.002 μg / mL. Use a 100-μL pipette to add the antibody diluent to 18.2-K562, 18.1-K562, and K562 respectively, 100 μL per well, and incubate at 4°C for 60 min. Wash the plate: Wash the plate 2 times with FACS Buffer.

[0211] 6.3. Add secondary antibody: Dilute the secondary antibody Anti-Human IgG FITC with FACS Buffer at 1:150, and add 100 μL per well to each well, and incubate at 4°C for 30 min. Wash the plate: Wash the plate 3 times with FACS buffer.

[0212] 6.4. Run on the machine: Detect the samples on a flow cytometer.

[0213] The experimental results are as Figure 8As shown in the identification result graph of the FACS binding of the antibody to 18.2-K562, the result shows that the antibody affinity is: 0.6753 μg / ml. Among them, hIgG1 is the negative control. The identification results of the FACS binding of the antibody to 18.1-K562 and the antibody to K562 both show that the antibody has no binding to 18.1-K562.

[0214] All the documents mentioned in the present invention are cited herein as references, as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application. Sequence Listing <110> Shanghai Laifu Medical Technology Co., Ltd. <120> CLDN18.2 Antibody and Its Application <130> P2020-1721 <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 138 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn 65 70 75 80 Gly Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp 115 120 125 Gly Gln Gly Thr Ser Leu Thr Val Ser Ser 130 135 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <400> 2 Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <400> 3 Met Ile His Pro Asn Ser Gly Ser Thr Asn 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <400> 4 Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe 1 5 10 <210> 5 <211> 133 <212> PRT <213> Artificial Sequence <400> 5 Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr 20 25 30 Val Thr Ala Arg Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr 115 120 125 Lys Leu Glu Ile Lys 130 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <400> 6 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <400> 7 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <400> 8 Gln Asn Ala Tyr Ser Tyr Pro Phe Thr 1 5 <210> 9 <211> 414 <212> DNA <213> Artificial Sequence <400> 9 atgggatgga gctatatcat cctctttttg gtagcaacag ctacaggtgt ccactcccag 60 gtccaactgc agcagcctgg ggctgagctg gtaaagcctg gggcttcagt gaagttgtcc 120 tgcaaggctt ctggctacac tttcaccagc tactggatgc actgggtgag gcagaggcct 180 ggacaaggcc ttgagtggat tggaatgatt catcctaata gtggtagtac taactacaat 240 gggaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg 300 caactcagca gcctgacatc tgaggactct gcggtctatt tctgtgcaag agggggctac 360 tatggtaact cccttgactt ctggggccaa ggcacctctc tcacagtctc ctca 414 <210> 10 <211> 399 <212> DNA <213> Artificial Sequence <400> 10 atggaatcac agactcaggt cctcatgtcc ctgctgttct gggtatctgg tacctgtggg 60 gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaagaga gaaggtcact 120 atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 180 tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg 240 gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 300 atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatgc ttatagttat 360 ccattcacgt tcggctcggg gacaaagttg gaaataaaa 399 <210> 11 <211> 1008 <212> DNA <213> Artificial Sequence <400> 11 gccaaaacaa cagccccatc ggtctatcca ctggcccctg tgtgtggagg tacaactggc 60 tcctcggtga ctctaggatg cctggtcaag ggttatttcc ctgagccagt gaccttgacc 120 tggaactctg gatccctgtc cagtggtgtg cacaccttcc cagctctcct gcagtctggc 180 ctctacaccc tcagcagctc agtgactgta acctcgaaca cctggcccag ccagaccatc 240 acctgcaatg tggcccaccc ggcaagcagc accaaagtgg acaagaaaat tgagcccaga 300 gtgcccataa cacagaaccc ctgtcctcca ctcaaagagt gtcccccatg cgcagctcca 360 gacctcttgg gtggaccatc cgtcttcatc ttccctccaa agatcaagga tgtactcatg 420 atctccctga gccccatggt cacatgtgtg gtggtggatg tgagcgagga tgacccagac 480 gtccagatca gctggtttgt gaacaacgtg gaagtacaca cagctcagac acaaacccat 540 agagaggatt acaacagtac tctccgggtg gtcagtgccc tccccatcca gcaccaggac 600 agagaggatt acaacagtac tctccgggtg gtcagtgccc tccccatcca gcaccaggac 600 tggatgagtg gcaaggagtt caaatgcaag gtcaacaaca gagccctccc atcccccatc 660 tggatgagtg gcaaggagtt caaatgcaag gtcaacaaca gagccctccc atcccccatc 660 gagaaaacca tctcaaaacc cagagggcca gtaagagctc cacaggtata tgtcttgcct 720 gagaaaacca tctcaaaacc cagagggcca gtaagagctc cacaggtata tgtcttgcct 720 ccaccagcag aagagatgac taagaaagag ttcagtctga cctgcatgat cacaggcttc 780 ccaccagcag aagagatgac taagaaagag ttcagtctga cctgcatgat cacaggcttc 780 ttacctgccg aaattgctgt ggactggacc agcaatgggc gtacagagca aaactacaag 840 ttacctgccg aaattgctgt ggactggacc agcaatgggc gtacagagca aaactacaag 840 aacaccgcaa cagtcctgga ctctgatggt tcttacttca tgtacagcaa gctcagagta 900 aacaccgcaa cagtcctgga ctctgatggt tcttacttca tgtacagcaa gctcagagta 900 caaaagagca cttgggaaag aggaagtctt ttcgcctgct cagtggtcca cgaggtgctg 960 caaaagagca cttgggaaag aggaagtctt ttcgcctgct cagtggtcca cgaggtgctg 960 cacaatcacc ttacgactaa gaccatctcc cggtctctgg gtaaatag 1008 cacaatcacc ttacgactaa gaccatctcc cggtctctgg gtaaatag 1008 <210> 12 <211> 324 <212> DNA <213> Artificial Sequence <400> 12 cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60 cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60 ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120 ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120 tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180 agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa 240 cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300 agcttcaaca ggaatgagtg ttag 324 <210> 13 <211> 335 <212> PRT <213> Artificial Sequence <400> 13 Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly 1 5 10 15 Gly Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val Thr Ser Asn Thr Trp Pro Ser Gln Thr Ile 65 70 75 80 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Glu Pro Arg Val Pro Ile Thr Gln Asn Pro Cys Pro Pro Leu Lys 100 105 110 Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu Gly Gly Pro Ser Val 115 120 125 Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser 130 135 140 Pro Met Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp 145 150 155 160 Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln 165 170 175 Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser 180 185 190 Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys 195 200 205 Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile 210 215 220 Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro 225 230 235 240 Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Cys Met 245 250 255 Ile Thr Gly Phe Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn 260 265 270 Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser 275 280 285 Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr 290 295 300 Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val Val His Glu Val Leu 305 310 315 320 His Asn His Leu Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly Lys 325 330 335 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <400> 14 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <400> 15 ggctacactt tcaccagcta ctggatgcac 30 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <400> 16 atgattcatc ctaatagtgg tagtactaac 30 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <400> 17 gggggctact atggtaactc ccttgacttc 30 <210> 18 <211> 51 <212> DNA <213> Artificial Sequence <400> 18 aagtccagtc agagtctgtt aaacagtgga aatcaaaaga actacttgac c 51 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <400> 19 tgggcatcca ctagggaatc t 21 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <400> 20 cagaatgctt atagttatcc attcacg 27

Claims

1. An anti-CLDN18.2 antibody, characterized in that, The antibody has: (1) A heavy chain, and the heavy chain variable region of the heavy chain has the following three complementarity-determining regions CDR: CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:4; and (2) A light chain, and the light chain variable region of the light chain has the following three complementarity-determining regions CDR: CDR1’ shown in SEQ ID NO:6, CDR2’ shown in SEQ ID NO:7, and CDR3’ shown in SEQ ID NO:

8.

2. The antibody according to claim 1, wherein The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:

1.

3. The antibody according to claim 1, wherein The light chain variable region has the amino acid sequence shown in SEQ ID NO:

5.

4. The antibody according to claim 1, wherein, The heavy chain constant region of the heavy chain has the amino acid sequence shown in SEQ ID NO:

13.

5. The antibody according to claim 1, wherein The light chain constant region of the light chain has the amino acid sequence shown in SEQ ID NO:

14.

6. A recombinant protein, characterized in that, The recombinant protein consists of: (i) The sequence of the antibody as claimed in claim 1; and (ii) Optionally, a tag sequence for assisting expression and / or purification.

7. A polynucleotide, characterized in that, It encodes a polypeptide selected from the group consisting of: (1) The antibody as claimed in claim 1; or (2) The recombinant protein as claimed in claim 6.

8. A carrier, characterized in that, It contains the polynucleotide as claimed in claim 7.

9. A genetically engineered host cell, characterized in that, It contains the vector as claimed in claim 8 or the polynucleotide as claimed in claim 7 is integrated into the genome.

10. An immunoconjugate, characterized in that, The immunoconjugate consists of: (a) The antibody as claimed in claim 1, or the recombinant protein as claimed in claim 6; and (b) A conjugate part: a detectable marker.

11. Use of an antibody as claimed in claim 1, or a recombinant protein as claimed in claim 6, or an immunoconjugate as claimed in claim 10 for preparing a reagent, a test plate or a kit; The reagent, test plate or kit is used for: detecting CLDN18.2 protein in a sample.

12. A method for preparing a recombinant polypeptide, characterized in that, The method comprises: (a) Culturing the host cell as claimed in claim 9 under conditions suitable for expression; (b) Isolating the recombinant polypeptide from the culture, and the recombinant polypeptide is the antibody as claimed in claim 1 or the recombinant protein as claimed in claim 6.

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