CN114685670B

CN114685670B - CLDN18.2 antibodies and their applications

Info

Publication number: CN114685670B
Application number: CN202011635146.5A
Authority: CN
Inventors: 董坚; 孙亚如; 杨圣; 聂紫; 李文荣
Original assignee: Shanghai Life Medical Science & Technology Co ltd
Current assignee: Shanghai Life Medical Science & Technology Co ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2025-06-27
Anticipated expiration: 2040-12-31
Also published as: CN114685670A

Abstract

The invention provides a CLDN18.2 antibody and application thereof. In particular, nucleic acids encoding the antibodies, compositions comprising the antibodies, methods of making the antibodies, and methods of using the antibodies to treat or prevent diseases, such as cancer and/or inflammatory diseases, are also described.

Description

CLDN18.2 antibodies and uses thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a CLDN18.2 antibody and application thereof.

Background

Gastrointestinal tract and pancreas tumor are very threatening to human life, and although the treatment means such as operation treatment, radiotherapy and chemotherapy, interventional treatment and the like have certain curative effects on the tumor, the survival rate of patients is not improved obviously.

Cellular immunotherapy is an emerging tumor treatment method, which constructs an expression vector of a chimeric antigen receptor (CHIMERIC ANTIGEN receptor, CAR) by molecular biology technology, introduces the expression vector into immune cells isolated from a human body, makes the cell surface express the CAR, and then performs amplification culture, and returns the CAR to the human body. The CAR is formed by sequentially connecting an antigen recognition domain, a hinge region, a transmembrane region and an intracellular signal domain, and immune cells expressing the CAR can specifically recognize and bind to target cells and kill the target cells by releasing specific immune factors.

Gastric cancer is one of the most common cancers in the world, and in china, gastric cancer is the second most common malignancy, and is considered one of the most refractory cancers worldwide. Despite recent advances in treatment options, recurrence of gastric cancer is inevitable, with five-year survival rates of about 5-20% for advanced gastric cancer patients, and a median overall survival period of about 10 months. Along with the deep research of the molecular mechanism of gastric cancer occurrence and development, targeted therapy becomes an effective treatment scheme of advanced cancers, and targets mainly comprise EGFR, HER-2, VEGF, VEGFR and the like. CLDN18.2 is expressed as a highly specific expressed cell surface molecule only on differentiated gastric mucosal epithelial cells in normal tissues, and thus there is a need to develop therapeutic antibodies against CLDN18.2 with greater potential against gastric cancer, lower toxicity, lower drug dose.

In view of the foregoing, there is a need in the art to develop a novel CLDN18.2 antibody and its uses.

Disclosure of Invention

The invention aims at providing a novel CLDN18.2 antibody and application thereof.

In a first aspect of the invention there is provided a heavy chain variable region of an antibody, said heavy chain variable region comprising the following three complementarity determining region CDRs:

CDR1 shown in SEQ ID NO. 2,

CDR2 as shown in SEQ ID NO 3, and

CDR3 as shown in SEQ ID No. 4.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 1.

In a second aspect of the invention there is provided an antibody heavy chain having a heavy chain variable region and a heavy chain constant region according to the first aspect of the invention.

In another preferred embodiment, the heavy chain constant region is of human or murine origin.

In another preferred embodiment, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO. 13.

In a third aspect of the invention, there is provided a light chain variable region of an antibody, the light chain variable region having complementarity determining region CDRs selected from the group consisting of:

CDR1' shown in SEQ ID NO. 6,

CDR2' shown in SEQ ID NO 7, and

CDR3' shown in SEQ ID NO. 8.

In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 5.

In a fourth aspect of the invention there is provided an antibody light chain having a light chain variable region and a light chain constant region according to the third aspect of the invention.

In another preferred embodiment, the constant region of the light chain is of human or murine origin.

In another preferred embodiment, the light chain constant region has the amino acid sequence shown in SEQ ID NO. 14.

In a fifth aspect of the invention, there is provided an antibody having:

(1) The heavy chain variable region according to the first aspect of the invention, and/or

(2) The light chain variable region according to the third aspect of the invention.

In another preferred embodiment the antibody has a heavy chain according to the second aspect of the invention, and in another preferred embodiment the heavy chain according to the second aspect of the invention is an antibody specific for the CLDN18.2 protein.

In another preferred embodiment, the antibody comprises a single chain antibody, a double chain antibody, a monoclonal antibody, a chimeric antibody (e.g., a human mouse chimeric antibody), a murine antibody, or a humanized antibody.

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

(i) A sequence of a heavy chain variable region according to the first aspect of the invention, a sequence of a heavy chain according to the second aspect of the invention, a sequence of a light chain variable region according to the third aspect of the invention, a sequence of a light chain according to the fourth aspect of the invention, or a sequence of an antibody according to the fifth aspect of the invention, and

(Ii) Optionally a tag sequence to assist expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag.

In another preferred embodiment, the recombinant protein is specific for an anti-CLDN 18.2 protein.

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

(1) A heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, or

(2) The recombinant protein according to the sixth aspect of the invention.

In another preferred embodiment, the polynucleotide has the sequence shown in SEQ ID NO. 9, 10, 11, 12.

In an eighth aspect of the invention there is provided a vector comprising a polynucleotide according to the seventh aspect of the invention.

In another preferred embodiment, the vector comprises a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vector.

In a ninth aspect of the invention there is provided a genetically engineered host cell comprising a vector or genome according to the eighth aspect of the invention incorporating a polynucleotide according to the seventh aspect of the invention.

In a tenth aspect of the invention, there is provided an immunoconjugate comprising:

(a) A heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, an antibody according to the fifth aspect of the invention, or a recombinant protein according to the sixth aspect of the invention, and

(B) A conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) A heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, or an immunoconjugate according to the tenth aspect of the invention, and

(Ii) A pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

In another preferred embodiment, the pharmaceutical composition is used for preparing medicines for treating tumors selected from the group consisting of gastric cancer, esophageal cancer, cholangiocarcinoma, pancreatic cancer, lung cancer, ovarian cancer and colon cancer.

In a twelfth aspect of the invention there is provided the use of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, or an immunoconjugate according to the tenth aspect of the invention, for the preparation of a medicament, reagent, assay plate or kit;

The reagent, the detection plate or the kit is used for detecting CLDN18.2 protein in a sample;

the agent is used for treating or preventing tumors expressing CLDN18.2 protein.

In another preferred example, the tumor comprises gastric cancer, esophageal cancer, cholangiocarcinoma, pancreatic cancer, lung cancer, ovarian cancer, colon cancer.

In another preferred embodiment, the tumor is selected from the group consisting of gastric cancer and pancreatic cancer.

In another preferred embodiment, the reagent comprises a chip, an immune microparticle coated with an antibody.

In a thirteenth aspect of the invention, there is provided a method of detecting CLDN18.2 protein in a sample, the method comprising the steps of:

(1) Contacting the sample with an antibody according to the fifth aspect of the invention;

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

In a fourteenth aspect of the present invention, there is provided a method of producing a recombinant polypeptide, the method comprising:

(a) Culturing the host cell of the ninth aspect of the invention under conditions suitable for expression;

(b) Isolating the recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the hybridoma method system of the invention.

Figure 2 shows a summary of hcldn18.2 positive hybridomas.

FIG. 3 shows cross-reactive FACS detection of subcloned hybridoma supernatants. Wherein, the abscissa is fluorescence intensity, the binding intensity of the detection antibody and the CLDN18.2, and the ordinate SSC is deflection dispersion of the cells, and the complexity of the cells is detected.

Figure 4 shows the potency detection of subclones.

FIG. 5 shows a flow chart of the results of the identification of the expression of the recombinant C18-16-1H1-1A8 antibody. Wherein FL1-H is fluorescence intensity, the binding intensity of the detection antibody and CLDN18.2, SSC-H subset is deflection dispersion of cells, and the complexity of the cells is detected.

FIG. 6 shows a heavy chain expression vector construction map.

FIG. 7 shows an expression vector construction map of the light chain.

FIG. 8 shows a graph of the identification of FACS binding of antibodies to 18.2-K562. Wherein, igG1 is a negative control.

Detailed Description

The inventors have studied extensively and intensively and have unexpectedly found an anti-CLDN 18.2 monoclonal antibody through extensive screening. Experimental results show that the monoclonal antibody has high specificity and strong affinity, and can be specifically combined with CLDN18.2. The present invention has been completed on the basis of this finding.

CLDN18.2

Claudins is a family of proteins that function to maintain tight junctions that control the exchange of molecules between cells. Widely distributed in stomach, pancreas and lung tissues, and can be used for diagnosis and treatment. Claudin (CLDN) has 4 transmembrane domains and is involved in the regulation of physiological processes in the body such as paracellular permeability and electrical conductance. The family comprises at least 24 members, CLDN18 is a member of the Claudin protein family, encoded in humans by the CLDN18 gene. The human CLDN18 gene has two different exons 1, and two protein subtypes, CLDN18.1 and CLDN18.2, are generated by alternative splicing after transcription, and are composed of 261 amino acids, and only 8 amino acid differences exist in the N-terminal extracellular region.

The CLDN18.2 subtype is a stomach specific subtype, and Claudin 18.2 (CLDN 18.2) is only expressed on the surface of short-lived cells of human gastric mucosa epithelium under normal physiological conditions, but is highly expressed in various tumors such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer and the like, for example, 50-80% of gastric cancer patients have the expression of the target. CLDN18.2 is usually buried in gastric mucosa, and monoclonal antibodies in normal tissues are basically not contacted, so that malignant tumors can cause the damage of tight junctions, and CLDN18.2 epitopes on the surfaces of tumor cells are exposed and become specific targets. CLDN18.2 therefore confers specificity to targeted therapies. Recently, expression of CLDN18.2 in pancreatic cancer (50%), esophageal cancer and lung cancer has also been found to show potential for diagnosis and treatment of other tumors.

Terminology

As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, the constant region of the light chain being opposite the first constant region of the heavy chain and the variable region of the light chain being opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together 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 publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant region. There are mainly class 5 immunoglobulins IgA, igD, igE, igG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, igG2, igG3, igG4, igA and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.

The invention also includes monoclonal antibodies having the corresponding amino acid sequences of the anti-CLDN 18.2 protein monoclonal antibodies, monoclonal antibodies having the variable region chains of the anti-CLDN 18.2 protein monoclonal antibodies, and other proteins or protein conjugates and fusion expression products having these chains. In particular, the 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 comprising a hypervariable region (complementarity determining region, CDR), provided that the hypervariable region is identical or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light chain and heavy chain of the invention.

Immunoconjugates and fusion expression products include conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules conjugated to the anti-CLDN 18.2 protein monoclonal antibody or fragment thereof, as known to those skilled in the art. The invention also includes cell surface markers or antigens conjugated to the anti-CLDN 18.2 protein monoclonal antibodies or fragments thereof.

The 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.

As used herein, the term "heavy chain variable region" is used interchangeably with "V _H".

As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determining region, CDR)".

In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises the following three complementarity determining regions CDRs:

CDR1, the amino acid sequence of which is GYTFTSYWMH (SEQ ID NO: 2), and the encoding nucleotide sequence of which is GGCTACACTTTCACCAGCTACTGGATGCAC (SEQ ID NO: 15);

CDR2 having the amino acid sequence MIHPNSGSTN (SEQ ID NO: 3) and the coding nucleotide sequence ATGATTCATCCTAATAGTGGTAGTACTAAC (SEQ ID NO: 16);

CDR3, which has the amino acid sequence GGYYGNSLDF (SEQ ID NO: 4), and whose coding nucleotide sequence is GGGGGCTACTATGGTAACTCCCTTGACTTC (SEQ ID NO: 17).

In another preferred embodiment, the amino acid sequence of the heavy chain variable region is:

MGWSYIILFLVATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVRQRPGQGLEWIGMIHPNSGSTNYNGKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARGGYYGNSLDFWGQGTSLTVSS(SEQ ID NO.:1);

the coding nucleotide sequence is as follows:

atgggatggagctatatcatcctctttttggtagcaacagctacaggtgtccactcccaggtccaactgcagcagcctggggctgagctggtaaagcctggggcttcagtgaagttgtcctgcaaggcttctggctacactttcaccagctactggatgcactgggtgaggcagaggcctggacaaggccttgagtggattggaatgattcatcctaatagtggtagtactaactacaatgggaagttcaagagcaaggccacactgactgtagacaaatcctccagcacagcctacatgcaactcagcagcctgacatctgaggactctgcggtctatttctgtgcaagagggggctactatggtaactcccttgacttctggggccaaggcacctctctcacagtctcctca(SEQ ID NO.:9).

In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the heavy chain variable region described above and a heavy chain constant region, which may be of murine or human origin.

In another preferred embodiment, the amino acid sequence of the heavy chain constant region (mIgG 2 c) is:

AKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEVLHNHLTTKTISRSLGK(SEQ ID No:13).

in another preferred embodiment, the nucleotide sequence encoding the heavy chain constant region (mIgG 2 c) is:

GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGGTACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGCCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAACACCTGGCCCAGCCAGACCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAAGTGGACAAGAAAATTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGTGCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAATAG(SEQ ID No:11).

As used herein, the term "light chain variable region" is used interchangeably with "V _L".

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

CDR1', which has the amino acid sequence KSSQSLLNSGNQKNYLT (SEQ ID NO: 6), which encodes the nucleotide sequence AAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGACC (SEQ ID NO: 18);

CDR2', having the amino acid sequence WASTRES (SEQ ID NO: 7), having the coding nucleotide sequence TGGGCATCCACTAGGGAATCT (SEQ ID NO: 19);

CDR3', which has the amino acid sequence QNAYSYPFT (SEQ ID NO: 8), and which encodes the nucleotide sequence CAGAATGCTTATAGTTATCCATTCACG (SEQ ID NO: 20).

In another preferred embodiment, the amino acid sequence of the light chain variable region is:

MESQTQVLMSLLFWVSGTCGDIVMTQSPSSLTVTAREKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNAYSYPFTFGSGTKLEIK(SEQ ID NO.:5),

the coding nucleotide sequence is as follows:

atggaatcacagactcaggtcctcatgtccctgctgttctgggtatctggtacctgtggggacattgtgatgacacagtctccatcctccctgactgtgacagcaagagagaaggtcactatgagctgcaagtccagtcagagtctgttaaacagtggaaatcaaaagaactacttgacctggtaccagcagaaaccagggcagcctcctaaactgttgatctactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctggaacagatttcactctcaccatcagcagtgtgcaggctgaagacctggcagtttattactgtcagaatgcttatagttatccattcacgttcggctcggggacaaagttggaaataaaa(SEQ ID NO.:10).

In a preferred embodiment of the present invention, the light chain of the antibody comprises the light chain variable region described above and a light chain constant region, which may be murine or human in origin.

In another preferred embodiment, the amino acid sequence of the light chain constant region (IgK) is:

RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQN GVLNSWTDQDSKDSTYSMSS TLTLTKDEYE RHNSYTCEAT HKTSTSPIVK SFNRNEC(SEQ ID No:14).

in another preferred embodiment, the nucleotide sequence encoding the light chain constant region (IgK) is:

CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG(SEQ ID No:12).

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

In another preferred embodiment, the antibody is a murine or human murine chimeric monoclonal antibody against CLDN18.2 protein, the heavy chain constant region and/or the light chain constant region of which may be a humanized heavy chain constant region or a light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or light chain constant region of human IgG1, igG2, or the like.

The invention also provides other proteins or fusion expression products having the antibodies of the invention. In particular, the 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 comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy chain and light chain of the antibodies of the invention.

In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.

The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest because they are involved, at least in part, in binding to an antigen. Thus, the invention includes those molecules having monoclonal antibody light and heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.

The invention includes not only intact monoclonal antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

The antibody of the invention refers to a polypeptide having CLDN18.2 protein binding activity and comprising the CDR regions described above. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. Such variants include, but are not limited to, deletions, insertions and/or substitutions of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and the addition of one or more (typically less than 20, preferably less than 10, more preferably less than 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variants of the polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

The invention also provides other polypeptides, such as fusion proteins comprising a human antibody or fragment thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.

In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively mutated polypeptides are preferably produced by amino acid substitution according to Table 1.

TABLE 1

The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to or degenerate as shown in SEQ ID No. 9, 10, 11, 12, 15, 16, 17, 18, 19, 20. As used herein, a "degenerate variant" refers in the present invention to a nucleic acid sequence encoding a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but differing from the coding region sequences set forth in SEQ ID NOS.9, 10, 11, 12, 15, 16, 17, 18, 19, 20.

Polynucleotides encoding the mature polypeptides of the present invention include coding sequences encoding only the mature polypeptide, coding sequences and various additional coding sequences for the mature polypeptide, coding sequences (and optionally additional coding sequences) for the mature polypeptide, and non-coding sequences.

The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means (1) hybridization and elution at a relatively low ionic strength and a relatively high temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃, or (2) hybridization with a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42 ℃, etc., or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95%. Also, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO. 1 and/or SEQ ID NO. 5.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.

The host cell may 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 E.coli, streptomyces, salmonella typhimurium, fungal cells such as yeast, drosophila S2 or Sf9 insect cells, CHO, COS7, 293 cell animal cells, and the like.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl ₂ method using procedures well known in the art. Another approach is to use MgCl ₂. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. may be used.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

The antibodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.

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

Therapeutic agents that may be conjugated or coupled to the antibodies of the invention include, but are not limited to, 1. Radionuclides (Koppe et al, 2005, cancer metastasis comment (CANCER METASTASIS REVIEWS) 24,539); biological toxicity (Chaudhary et al, 1989, nature et al 339,394; epel et al, 2002, J.Chemie of THE AMERICAN CHEMICAL Society) 51,565; 3 cytokines such as IL-2 et al (Gillies et al, 1992; proc. Natl. Acad. Sci. U.S. (PNAS) 89,1428; card et al, 2004, cancer immunology and immunotherapy (Cancer Immunology and Immunotherapy) 53,345; halin et al, 2003, cancer research (CANCER RESEARCH) 63,3202; 4. Gold nanoparticles/nanorods (Lapotko et al, 2005, cancer communication (CANCER LETTERS) 239,36; huang et al, 2006, journal of 35 of THE AMERICAN CHEMICAL Society), 5. Viral particles (Peng et al, 2004, gene therapy (GENE THERAPY) 11,1234; liposome (Mamot et al, 2005, cancer research (CANCER RESEARCH), 7. Nanomagnets; 8. Prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase (28), or any other chemotherapeutic agent, e.g., in the form of 35; 6710, etc.).

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the invention can be directly used for binding to CLDN18.2 protein molecules, and thus can be used for preventing and treating tumors. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the monoclonal antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

Hybridoma cell strain

The invention also provides a hybridoma cell strain capable of producing the CLDN18.2 protein monoclonal antibody, and preferably provides a hybridoma cell strain with high titer against the CLDN18.2 protein monoclonal antibody.

After obtaining the hybridoma producing the CLDN18.2 protein monoclonal antibody of the invention, one skilled in the art can conveniently use the hybridoma cell line to make antibodies. Furthermore, the structure of the antibodies of the invention (e.g., the heavy and light chain variable regions of the antibodies) can be readily known to those skilled in the art, and then the monoclonal antibodies of the invention can be prepared by recombinant methods.

Preparation of monoclonal antibodies

Antibodies of the invention may be prepared by various techniques known to those skilled in the art. For example, the antigens of the invention may be administered to animals to induce monoclonal antibody production. For monoclonal antibodies, hybridoma technology can be used to prepare (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 using recombinant DNA methods (U.S. Pat. No. 4,816,567).

Representative myeloma cells are those that fuse efficiently, support stable high levels of antibody production by the antibody-producing cell of choice, and are sensitive to the medium (HAT medium matrix), including myeloma cell lines, e.g., murine myeloma cell lines, including those 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, rocyvale, 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 antibody production techniques and applications (Monoclonal Antibodies Production Techniques and Applications), pages 51-63 (MARCEL DEKKER, inc., new York, 1987) ].

The culture medium in which the hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies having the desired specificity, such as by an in vitro binding assay, e.g., an enzyme-linked immunosorbent assay (ELISA) or a Radioimmunoassay (RIA). The location of cells expressing the antibody can be detected by FACS. The hybridoma clones can then be subcloned by limiting dilution steps (subcloned) and grown by standard methods (Goding, monoclonal antibody (Monoclonal Antibodies): principles and practices (PRINCIPLES AND PRACTICE), ACADEMIC PRESS (1986) pages 59-103). Suitable media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, hybridoma cells can grow as ascites tumors in animals.

Monoclonal antibodies secreted by the subclones are suitably isolated from culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

The invention provides a monoclonal antibody aiming at CLDN18.2 protein, in particular to a monoclonal antibody aiming at CLDN18.2 protein. In a preferred embodiment of the invention, the monoclonal antibodies are prepared by culturing hybridoma cells. Taking supernatant of hybridoma cell culture, roughly extracting IgG by a saturated ammonium sulfate precipitation method, and purifying the roughly extracted antibody by an affinity chromatography column (Protein G-Sephrose).

In a preferred embodiment of the present invention, the monoclonal antibody is prepared by the method of producing a monoclonal antibody by using Balb/C mouse ascites. Approximately hybridoma cells were inoculated into the abdominal cavity of sensitized mice, and obvious abdominal distension was seen around 10 days. Extracting ascites, coarse extracting with saturated ammonium sulfate precipitation, and purifying the coarse extracted antibody with affinity chromatographic column (Protein G-Sephrose).

Labeled immunoglobulins

In a preferred embodiment of the invention, the immunoglobulin is provided with a detectable label. More preferably, the label is selected from the group consisting of a colloidal gold label, a colored label, and a fluorescent label.

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

The CLDN18.2 protein monoclonal antibody has good specificity and high potency.

Methods and samples

The present invention relates to a method for detecting tumors in a sample lysed in cells and/or tissues. The method steps are generally as follows, obtaining a cell and/or tissue sample, lysing the sample in a medium, and detecting the level of CLDN18.2 protein in the lysed sample. The sample used in the method of the invention may be any sample comprising cells present in a cell preservation fluid, as used in liquid-based cell assays.

Kit for detecting a substance in a sample

The invention also provides a kit comprising the antibody (or fragment thereof) of the invention or the detection plate of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.

The invention further relates to a detection kit for detecting the level of CLDN18.2, which comprises an antibody for recognizing CLDN18.2 protein, a lysis medium for dissolving a sample, general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, and the like. The detection kit may be an in vitro diagnostic device.

The main advantages of the invention include:

(1) The antibody of the invention has high specificity and strong affinity, can be prepared in large quantity, and has easy control of monoclonal antibody quality.

(2) The antibody of the invention can be used for targeting drugs specifically targeting CLDN18.2 positive tumor cells, antibody drug conjugates or multifunctional antibodies.

(3) The antibodies of the invention can be used for preparing reagents for diagnosing tumors or for preparing chimeric antigen receptor immune cells.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the detailed conditions in the examples below, are generally followed by conventional conditions such as those described in Sambrook et al, molecular cloning, a laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. Percentages and parts are by weight unless otherwise indicated.

The experiment system of the invention

The invention adopts a hybridoma method system, obtains positive hybridoma cells through DNA and cell immunity and flow screening, and has stable screening system.

The hybridoma method system is shown in FIG. 1.

A8 antibody-related sequences

TABLE 2A 8 antibody related sequences

Note that the underlined part is the Leader sequence. Wherein VL is a light chain variable region, CL is a light chain constant region, CH is a heavy chain constant region, and VH is a heavy chain variable region.

Example 1CLDN18.2 preparation of antibodies

1. Immunized mice

2. Serum detection

3. Fusion of

4. Post-fusion screening

5. Subcloning strain

6. Antibody sequence analysis

7. Expression validation of antibody sequences

8. Affinity detection of antibodies

Example 2 hCDN18.2 Positive hybridoma

As shown in FIG. 2, hCDN18.2 positive hybridomas in the present invention were pooled.

Example 3 hybridoma supernatant Cross-reaction assay

CHO-hCDN18.1+ hybridoma supernatant+sheep anti-mouse IgGFc-FITC

Appropriate amounts of CHO-hCDN18.1 cells were dispensed into 1.5ml EP tubes, centrifuged and resuspended in 50. Mu.l PBS, 50. Mu.l goat anti-mouse IgG Fc-FITC diluted 1:500 in PBS were added, allowed to stand at 4℃for 15 min, the supernatant was centrifuged and replaced, and resuspended in 200. Mu.l PBS for FACS flow analysis.

As shown in fig. 3, cross-reactivity with 18.1 was detected by flow-through method using CHO stable cells (Cell Pool), and antibody (C18-16-1H 1-1 A8) was a CLDN 18.2-specific antibody, not reacting with CLDN 18.1.

Example 4 hybridoma supernatant titer detection

Dilution by 10-fold ratio:

CHO-hcldn18.2+ hybridoma supernatant (10 fold dilution) +goat anti-mouse IgGFc-FITC

Appropriate amounts of CHO-hCDN18.2 cells were dispensed into 1.5ml EP tubes, centrifuged and resuspended in 50. Mu.l PBS, 50. Mu.l goat anti-mouse IgG Fc-FITC diluted 1:500 in PBS were added, allowed to stand at 4℃for 15 min, the supernatant was centrifuged and replaced, and resuspended in 200. Mu.l PBS for FACS flow analysis.

Hybridoma supernatant titers of the positive clones of the 16-size mice are shown in FIG. 4. C18-16-1H1-1A8 (A8) was selected for expression verification of the antibody sequence.

The information related to the A8 antibody is as follows:

Cell line C18-16-1H1-1A8

Fusion of source SP2/0 cells with C57 mouse B cells

Subtype IgG2c, kappa

Heavy chain V genes mIgHV-64

Light chain V gene MIgKV-19.

Example 5 expression validation of antibody sequences

10 Μg of each light and heavy chain expression vector (as shown in FIGS. 6 and 7) was co-transferred into 293T cells in 10 cm dishes using the calcium phosphate method, and three days later supernatants were collected for FACS validation in CHO-hCDN 18.2 cells.

The results of the flow test are shown in FIG. 5.

The recombinant antibody of C18-16-1H1-1A8 in the supernatant of 293T has better activity and correct sequence.

Example 6A8 Activity detection of antibodies

6.1. Cell plating, namely preparing cell suspensions from 18.2-K562, 18.1-K562 and K562, and adjusting the density to 1X 10 ⁶/mL. Taking 3 blocks of 96-hole round bottom plates, and marking the round bottom plates as plate1, plate2 and plate3. 100. Mu.L of 18.2-K562, 18.1-K562, K562 suspensions were added to plate1, plate2, plate3, respectively, using a 100. Mu.L pipette. Centrifugation was performed in a centrifuge at 300g for 5min. The supernatant was removed by shaking.

6.2. Antibody dilution by FACS Buffer was performed to give 8 concentration gradient dilutions of 20. Mu.g/mL, 6.667. Mu.g/mL, 2.222. Mu.g/mL, 0.741. Mu.g/mL, 0.247. Mu.g/mL, 0.082. Mu.g/mL, 0.027. Mu.g/mL, 0.002. Mu.g/mL, etc. Antibody dilutions were added to 18.2-K562, 18.1-K562, respectively, using a 100. Mu.L pipette, and incubated at 4℃for 60min per well. Wash plate, FACS Buffer wash plate 2 times.

6.3. Secondary antibody was added by diluting the secondary antibody Anti-Human IgG FITC with FACS Buffer 1:150, 100. Mu.L/well was added to each well and incubated at 4℃for 30min. Wash plate, FACS buffer wash plate 3 times.

6.4. And (5) loading the sample on a flow cytometer for sample detection.

The results of the experiment are shown in FIG. 8, which shows that the antibody affinity is 0.6753. Mu.g/ml in the graph of the identification of FACS binding of the antibody to 18.2-K562. Wherein hIgG1 was the negative control. The identification of antibody binding to 18.1-K562 and FACS of antibody binding to K562 showed that antibody did not bind to 18.1-K562.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

<110> Shanghai Miao medical science and technology Co., ltd

<120> CLDN18.2 antibodies and uses thereof

<130> P2020-1721

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 138

<212> PRT

<213> Artificial sequence (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 (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 (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 (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 (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 (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 (ARTIFICIAL SEQUENCE)

<400> 7

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 8

Gln Asn Ala Tyr Ser Tyr Pro Phe Thr

1 5

<210> 9

<211> 414

<212> DNA

<213> Artificial sequence (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 (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 (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

tggatgagtg gcaaggagtt caaatgcaag gtcaacaaca gagccctccc atcccccatc 660

gagaaaacca tctcaaaacc cagagggcca gtaagagctc cacaggtata tgtcttgcct 720

ccaccagcag aagagatgac taagaaagag ttcagtctga cctgcatgat cacaggcttc 780

ttacctgccg aaattgctgt ggactggacc agcaatgggc gtacagagca aaactacaag 840

aacaccgcaa cagtcctgga ctctgatggt tcttacttca tgtacagcaa gctcagagta 900

caaaagagca cttgggaaag aggaagtctt ttcgcctgct cagtggtcca cgaggtgctg 960

cacaatcacc ttacgactaa gaccatctcc cggtctctgg gtaaatag 1008

<210> 12

<211> 324

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60

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 (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 (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 (ARTIFICIAL SEQUENCE)

<400> 15

ggctacactt tcaccagcta ctggatgcac 30

<210> 16

<211> 30

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 16

atgattcatc ctaatagtgg tagtactaac 30

<210> 17

<211> 30

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 17

gggggctact atggtaactc ccttgacttc 30

<210> 18

<211> 51

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 18

aagtccagtc agagtctgtt aaacagtgga aatcaaaaga actacttgac c 51

<210> 19

<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 19

tgggcatcca ctagggaatc t 21

<210> 20

<211> 27

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 20

cagaatgctt atagttatcc attcacg 27

Claims

1. An anti-CLDN 18.2 antibody, comprising:

(1) A heavy chain having the following three complementarity determining region CDRs:

CDR1 shown in SEQ ID NO. 2,

CDR2 as shown in SEQ ID NO 3, and

CDR3 as shown in SEQ ID NO. 4, and

(2) A light chain having the following three complementarity determining region CDRs:

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 of claim 1, wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID No. 1.

3. The antibody of claim 1, wherein the light chain variable region has the amino acid sequence set forth in SEQ ID No. 5.

4. The antibody of claim 1, wherein the heavy chain constant region of the heavy chain has the amino acid sequence set forth in SEQ ID No. 13.

5. The antibody of claim 1, wherein the light chain constant region of the light chain has the amino acid sequence set forth in SEQ ID No. 14.

6. A recombinant protein, characterized in that said recombinant protein consists of:

(i) The sequence of the antibody according to claim 1, and

(Ii) Optionally a tag sequence to assist expression and/or purification.

7. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) The antibody of claim 1, or

(2) The recombinant protein according to claim 6.

8. A vector comprising the polynucleotide of claim 7.

9. A genetically engineered host cell comprising the vector or genome of claim 8 integrated with the polynucleotide of claim 7.

10. An immunoconjugate, characterized in that the immunoconjugate consists of:

(a) The antibody according to claim 1, or the recombinant protein according to claim 6, and

(B) Coupling moiety, detectable label.

11. Use of the antibody of claim 1, or the recombinant protein of claim 6, or the immunoconjugate of claim 10, for the preparation of a reagent, assay plate, or kit;

The reagent, the detection plate or the kit is used for detecting CLDN18.2 protein in a sample.

12. A method of producing a recombinant polypeptide, the method comprising:

(a) Culturing the host cell of claim 9 under conditions suitable for expression;

(b) Isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody of claim 1 or the recombinant protein of claim 6.