CN114685670A - CLDN18.2 antibodies and uses thereof - Google Patents

Abstract

The invention provides a CLDN18.2 antibody and application thereof. In particular, the invention also describes 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.

Description

CLDN18.2 antibodies and uses thereof

Technical Field

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

Background

Gastrointestinal tract and pancreatic tumors are a great threat to human life, and although treatment means such as surgical treatment, radiotherapy and chemotherapy, interventional therapy and the like have certain curative effect on the tumors, the survival rate of patients is not improved obviously.

In the cell immunotherapy, an expression vector of a Chimeric Antigen Receptor (CAR) is constructed by a molecular biology technique, and the expression vector is introduced into an immune cell isolated from a human body, so that the CAR is expressed on the cell surface, and then the cell surface is subjected to amplification culture, and the cell surface is returned to the human body. The CAR is formed by connecting an antigen recognition domain, a hinge region, a transmembrane region and an intracellular signal domain in sequence, and immune cells expressing the CAR can specifically recognize and combine 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 malignant tumor and is considered to be one of the most difficult cancers to cure worldwide. Despite recent advances in treatment options, gastric cancer recurrence is elusive, with five-year survival rates of approximately 5-20% for advanced gastric cancer patients and an overall median survival time of approximately 10 months. With the deep research on the generation and development molecular mechanisms of gastric cancer, targeted therapy becomes an effective treatment scheme for advanced cancers, and targets mainly comprise EGFR, HER-2, VEGF, VEGFR and the like. CLDN18.2 is expressed only on differentiated gastric mucosal epithelial cells in normal tissues as a highly specifically expressed cell surface molecule, and thus there is a need to develop therapeutic antibodies against CLDN18.2 with greater potential against gastric cancer, lower toxicity, lower dosage.

In view of the above, there is an urgent need in the art to develop a novel CLDN18.2 antibody and its use.

Disclosure of Invention

The present invention aims to provide a novel CLDN18.2 antibody and uses thereof.

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

CDR1 shown in SEQ ID NO. 2,

CDR2 shown in SEQ ID NO. 3, and

CDR3 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 a heavy chain of an antibody, said heavy chain having a heavy chain variable region and a heavy chain constant region as described in 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, said light chain variable region having complementarity determining regions CDRs selected from the group consisting of:

CDR 1' shown in SEQ ID NO. 6,

CDR 2' shown in SEQ ID NO. 7, and

CDR 3' shown in SEQ ID NO. 8.

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

In a fourth aspect of the invention, there is provided a light chain of an antibody, said light chain having a light chain variable region and a light chain constant region as described in 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) a heavy chain variable region according to the first aspect of the invention; and/or

(2) A 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; in another preferred embodiment of the heavy chain of the second aspect of the invention, the antibody of the invention is an antibody specific for a CLDN18.2 protein.

In another preferred embodiment, the antibody comprises: a single chain antibody, a diabody, a monoclonal antibody, a chimeric antibody (e.g., a human-murine 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) the sequence of a heavy chain variable region according to the first aspect of the invention, the sequence of a heavy chain according to the second aspect of the invention, the sequence of a light chain variable region according to the third aspect of the invention, the sequence of a light chain according to the fourth aspect of the invention, or the sequence of an antibody according to the fifth aspect of the invention; and

(ii) optionally a tag sequence to facilitate 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) A 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 carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

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 into which has been integrated 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 coupling 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 injection.

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

In a twelfth aspect of the invention, there is provided a 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 manufacture of a medicament, a reagent, a detection plate or a kit;

the reagent, assay plate or kit is for: detecting CLDN18.2 protein in a sample;

the agent is for use in the treatment or prevention of a tumor expressing a CLDN18.2 protein.

In another preferred embodiment, the tumor comprises: gastric cancer, esophageal cancer, bile duct cancer, pancreatic cancer, lung cancer, ovarian cancer, and 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 and immune microparticles coated with antibodies.

In a thirteenth aspect of the present invention, there is provided a method for detecting a 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 the formation of an antigen-antibody complex, wherein the formation of a complex is indicative of the presence of a 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 a host cell according to the ninth aspect of the invention under conditions suitable for expression;

(b) isolating a 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 to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a hybridoma methodology of the present 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, and the binding intensity of the detection antibody and CLDN18.2 is measured; the ordinate SSC is the biased dispersion of the cells and measures the complexity of the cells.

FIG. 4 shows the titer test of subclones.

FIG. 5 shows a flow chart of the results of the identification of the expression of the C18-16-1H1-1A8 recombinant antibody. Wherein FL1-H is the fluorescence intensity, the binding intensity of the detection antibody to CLDN 18.2; SSC-H subset is the biased dispersion of cells and detects the complexity of the cells.

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

FIG. 7 shows a light chain expression vector construction map.

FIG. 8 is a graph showing the results of the identification of FACS binding of the antibody to 18.2-K562. Among them, IgG1 was a negative control.

Detailed Description

The present inventors have extensively and intensively studied and, as a result of extensive screening, have unexpectedly found an anti-CLDN 18.2 monoclonal antibody. The experimental result shows that the monoclonal antibody has high specificity and strong affinity, and can be specifically combined with CLDN 18.2. The present invention has been completed based on 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 cell bypass permeability and conductance. Its 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 is transcribed to produce two protein subtypes by alternative splicing: CLDN18.1 and CLDN18.2, both subtypes consisting of 261 amino acids, differing by only 8 amino acids in the N-terminal extracellular region.

The CLDN18.2 subtype is a stomach-specific subtype, under normal physiological conditions, Claudin 18.2(CLDN18.2) is expressed only on the surface of human gastric mucosal epithelial short-lived cells; however, the target is highly expressed in a plurality of tumors such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer and the like, for example, the expression of the target exists in 50% -80% of gastric cancer patients. CLDN18.2 is usually buried in the gastric mucosa, monoclonal antibodies in normal tissues are substantially inaccessible, and the development of malignant tumors can result in the disruption of tight junctions, exposing the CLDN18.2 epitope on the surface of tumor cells to become a specific target. Thus, CLDN18.2 confers specificity to targeted therapies. The recent discovery of expression of CLDN18.2 in pancreatic (50%), esophageal and lung cancers has also shown potential for diagnosis and treatment of other tumors.

Term(s) for

As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one 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 at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the 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 segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly 5 classes of 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 IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as 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.

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 a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. 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 also 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 production of the antibody by any particular method.

The invention also includes monoclonal antibodies having the corresponding amino acid sequences of the monoclonal antibodies against CLDN18.2 protein, monoclonal antibodies having the variable region chains of the monoclonal antibodies against CLDN18.2 protein, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having light and heavy chains with hypervariable regions (complementarity determining regions, CDRs) so long as the hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous to the hypervariable regions of the light and heavy chains of the invention.

As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the anti-CLDN 18.2 protein monoclonal antibody or fragment thereof. The present invention also includes a cell surface marker or antigen bound to the anti-CLDN 18.2 protein monoclonal antibody or fragment thereof.

The invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab')₂A fragment; an antibody heavy chain; the light chain of the antibody.

As used herein, the terms "heavy chain variable region" and "V_H"may be used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary 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), the coding nucleotide sequence of which is ggctacactttcaccagctactggatgcac (SEQ ID NO: 15);

CDR2 having an amino acid sequence of MIHPNSGSTN (SEQ ID NO:3) and a coding nucleotide sequence of atgattcatcctaatagtggtagtactaac (SEQ ID NO: 16);

CDR3 having an amino acid sequence of GGYYGNSLDF (SEQ ID NO:4) and a coding nucleotide sequence of 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 invention, the heavy chain of the antibody comprises the above-described heavy chain variable region 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 (mIgG2c) is:

AKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEVLHNHLTTKTISRSLGK(SEQ ID No:13)。

in another preferred embodiment, the nucleotide sequence encoding the heavy chain constant region (mIgG2c) is:

GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGGTACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGCCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAACACCTGGCCCAGCCAGACCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAAGTGGACAAGAAAATTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGTGCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAATAG(SEQ ID No:11)。

as used herein, the terms "light chain variable region" and "V_L"may be used interchangeably.

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:

CDR 1', the amino acid sequence of which is KSSQSLLNSGNQKNYLT (SEQ ID NO:6), the coding nucleotide sequence of which is aagtccagtcagagtctgttaaacagtggaaatcaaaagaactacttgacc (SEQ ID NO: 18);

CDR 2', the amino acid sequence of which is WASTRES (SEQ ID NO:7), the coding nucleotide sequence of which is tgggcatccactagggaatct (SEQ ID NO: 19);

CDR 3' has an amino acid sequence of QNAYSYPFT (SEQ ID NO:8) and a coding nucleotide sequence of 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 invention, the light chain of the antibody comprises the light chain variable region and a light chain constant region, which may be murine or human.

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 and refer to an antibody having the ability of specifically binding 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 the initial methionine.

In another preferred embodiment, the antibody is a murine or human murine chimeric monoclonal antibody against CLDN18.2 protein, whose heavy chain constant region and/or light chain constant region may be humanized. More preferably, the humanized heavy or light chain constant region is that of human IgG1, IgG2, or the like.

The invention also provides other proteins or fusion expression products having an antibody 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 heavy and light chains with variable regions, provided that the variable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy and light chains of the antibody of the invention.

In general, the antigen binding properties of an antibody can be described by 3 specific regions 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, and the β -sheets formed by the FRs between them 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 antibodies of the same type.

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

The invention includes not only intact monoclonal antibodies, but also fragments of immunologically active antibodies or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. 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 those skilled in the art in view of the teachings herein.

The antibody of the present invention refers to a polypeptide having CLDN18.2 protein binding activity comprising the above-described CDR regions. The term also includes variants of the polypeptides comprising the CDR regions described above that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally 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 that hybridizes under high or low stringency conditions with DNA encoding 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 human antibodies or fragments thereof. In addition to substantially full-length polypeptides, the invention also encompasses fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, 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.

In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

TABLE 1

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

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) for the mature polypeptide as well as non-coding sequences.

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

The present invention also relates to polynucleotides which hybridize to the above-described sequences and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. And the polypeptide coded by the hybridizable polynucleotide has the same biological functions and activities 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 obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

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

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coliCompetent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The obtained transformant 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 culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated 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 such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.

Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.

Therapeutic agents that may be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (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. viral particles (Peng et al, 2004, Gene therapy 11, 1234); 6. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 7. nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-described antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, typically having a pH of from about 5 to about 8, preferably a pH of from about 6 to about 8, although the pH will 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 present invention can be directly used for binding to CLDN18.2 protein molecules, and thus can be used for the prevention and treatment of tumors. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. 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.

In the case of pharmaceutical compositions, 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/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Hybridoma cell strain

The invention also provides a hybridoma cell strain capable of producing the monoclonal antibody for the CLDN18.2 protein; preferably, the invention provides a hybridoma cell strain with high titer aiming at the CLDN18.2 protein monoclonal antibody.

After obtaining the hybridoma producing the CLDN18.2 protein monoclonal antibody of the present invention, one skilled in the art can conveniently prepare an antibody using the hybridoma cell strain. In addition, the structure of the antibody of the present invention (e.g., the heavy chain variable region and the light chain variable region of the antibody) can be easily known by those skilled in the art, and then the monoclonal antibody of the present invention can be prepared by recombinant methods.

Preparation of monoclonal antibodies

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, the antigens of the invention can be administered to an animal to induce the production of monoclonal antibodies. For Monoclonal Antibodies, they can be prepared using hybridoma technology (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 hybrids, 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-level production of antibody by selected antibody-producing cells, and are sensitive to medium (HAT medium matrix), including myeloma Cell lines, such as 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, rockwell, 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, Techniques for the Production and use of Monoclonal Antibodies (Monoclonal Antibodies Production Techniques and Applications), pp 51-63 (Marcel Dekker, Inc., New York, 1987).

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

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

The present invention provides a monoclonal antibody against CLDN18.2 protein, in particular a monoclonal antibody against CLDN18.2 protein. In a preferred embodiment of the present invention, the monoclonal antibody is prepared by culturing hybridoma cells. Taking supernatant fluid of hybridoma cell culture, carrying out saturated ammonium sulfate precipitation to obtain IgG, and purifying the antibody obtained by crude extraction through an affinity chromatography column (Protein G-Sepharose).

In a preferred embodiment of the invention, the monoclonal antibody is prepared by a method for producing the monoclonal antibody by Balb/C mouse ascites. The hybridoma cells are inoculated into the abdominal cavity of the sensitized mouse, and the abdomen is obviously swelled after about 10 days. Ascites is extracted, and after the crude extraction by saturated ammonium sulfate precipitation, the antibody of the crude extraction is purified by an affinity chromatography column (Protein G-Sepharose).

Labeled immunoglobulins

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

The colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the present invention, the monoclonal antibody having CLDN18.2 protein is labeled with colloidal gold to obtain a colloidal gold-labeled monoclonal antibody.

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

Method and sample

The present invention relates to a method for detecting tumors in cytolytic samples of cells and/or tissues. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving 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 may be any sample comprising cells present in a cell preservation solution, as used in liquid-based cytology methods.

Reagent kit

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

The present invention further designs a detection kit for detecting the level of CLDN18.2, which comprises an antibody 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, etc. The test kit may be an in vitro diagnostic device.

The main advantages of the present invention include:

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

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

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

The invention is further illustrated by the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Experimental 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 methodology is shown in FIG. 1.

A8 antibody related sequence

TABLE 2A 8 antibody related sequences

Note: the Leader sequence is underlined. 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 1 preparation of CLDN18.2 antibody

1. Immunization of mice

2. Serum detection

3. Fusion of

4. Post-fusion screening

5. Subcloning and establishing strain

6. Antibody sequence analysis

7. Expression validation of antibody sequences

8. Affinity detection of antibodies

Example 2 hLDN18.2 Positive hybridomas

As shown in fig. 2, the hcldn18.2 positive hybridomas in the present invention are summarized.

Example 3 hybridoma supernatant Cross-reaction assay

CHO-hCLDNN 18.1+ hybridoma supernatant + goat anti-mouse IgGFc-FITC

Appropriate amount of CHO-hLDN18.1 cells were dispensed into 1.5ml EP tubes, centrifuged and resuspended in 50. mu.l PBS, 50. mu.l of goat anti-mouse IgG Fc-FITC diluted in PBS 1:500 was added, left to stand at 4 ℃ for 15 minutes, centrifuged and supernatant replaced and resuspended in 200. mu.l PBS for FACS flow analysis.

As shown in fig. 3, the cross-reaction with 18.1 was detected by flow method using CHO stable cells (Cell Pool), and the antibody (C18-16-1H1-1a8) was a CLDN 18.2-specific antibody, which did not react with CLDN 18.1.

Example 4 hybridoma supernatant titer assay

10-fold dilution:

CHO-hLDN18.2 + hybridoma supernatant (10-fold dilution) + goat anti-mouse IgGFc-FITC

Appropriate amount of CHO-hLDN18.2 cells were dispensed into 1.5ml EP tubes, centrifuged and resuspended in 50. mu.l PBS, 50. mu.l of goat anti-mouse IgG Fc-FITC diluted in PBS 1:500 was added, left to stand at 4 ℃ for 15 minutes, centrifuged and supernatant replaced and resuspended in 200. mu.l PBS for FACS flow analysis.

The titer of the hybridoma supernatant from the positive clone of mouse 16 was determined as shown in FIG. 4. C18-16-1H1-1A8(A8) was selected for expression verification of the antibody sequence.

The information relating to the A8 antibody is as follows:

cell line C18-16-1H1-1A8

Fusion of SP2/0 origin cell and C57 mouse B cell

Subtype IgG2c, kappa

Heavy chain V gene mIgHV1-64

Light chain V gene MIgKV 8-19.

Example 5 expression verification of antibody sequences

Mu.g each of the light and heavy chain expression vectors (as shown in FIGS. 6 and 7) were co-transfected into 293T cells in 10 cm dishes using the calcium phosphate method, and after three days, supernatants were collected for FACS validation in CHO-hLDN 18.2 cells.

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

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

Example 6A8 Activity assay

6.1. Cell plating: preparing 18.2-K562, 18.1-K562 and K562 into cell suspension, and adjusting the density to 1 × 10⁶and/mL. Taking 3 round bottom plates with 96 holes, and markingplate1, plate2, plate 3. mu.L of suspensions of 18.2-K562, 18.1-K562 and K562 were added to the plates 1, 2 and 3, respectively, using a 100. mu.L pipetting gun. The mixture was centrifuged in a centrifuge at 300g for 5 min. The supernatant was discarded.

6.2. Adding an antibody diluent: the antibody was diluted with FACS Buffer to 8 dilutions with concentration gradients 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. The antibody dilutions were added to 18.2-K562, 18.1-K562, 100. mu.L per well, using a 100. mu.L pipet gun, and incubated for 60min at 4 ℃. Washing the plate: the FACS Buffer plates were washed 2 times.

6.3. Adding a secondary antibody: FACS Buffer 1 was used: a secondary Anti-Human IgG FITC dilution of 150, 100. mu.L/well, was added to each well and incubated at 4 ℃ for 30 min. Washing the plate: FACS buffer washes plates 3 times.

6.4. And (3) loading: sample detection was performed on a flow cytometer.

The results of the experiment are shown in FIG. 8, and in the graph of the results of the identification of FACS binding of the antibody to 18.2-K562, the results show that the affinity of the antibody is: 0.6753 μ g/ml. Of these, hIgG1 was a negative control. The results of the characterization of FACS binding of the antibody to 18.1-K562 and of the antibody to K562 show that the antibody does not bind to 18.1-K562.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Sequence listing

<110> Shanghai Lai Fisher medical science and technology Co Ltd

<120> CLDN18.2 antibodies and uses thereof

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<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 (10)

1. An antibody heavy chain variable region comprising the following three Complementarity Determining Regions (CDRs):

CDR1 shown in SEQ ID NO. 2,

CDR2 shown in SEQ ID NO. 3, and

CDR3 shown in SEQ ID NO. 4.

2. An antibody heavy chain having the heavy chain variable region and the heavy chain constant region of claim 1.

3. An antibody light chain variable region having Complementarity Determining Regions (CDRs) selected from the group consisting of:

CDR 1' shown in SEQ ID NO. 6,

CDR 2' of SEQ ID NO. 7, and

CDR 3' shown in SEQ ID NO. 8.

4. An antibody light chain comprising the light chain variable region and the light chain constant region of claim 3.

5. An antibody, wherein said antibody has:

(1) the heavy chain variable region of claim 1; and/or

(2) The light chain variable region of claim 3.

6. A recombinant protein, said recombinant protein having:

(i) the sequence of the heavy chain variable region of claim 1, the sequence of the heavy chain of claim 2, the sequence of the light chain variable region of claim 3, the sequence of the light chain of claim 4, or the sequence of the antibody of claim 5; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

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

(1) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5; or

(2) The recombinant protein of 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 having the polynucleotide of claim 7 integrated therein.

10. An immunoconjugate, comprising:

(a) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, the antibody of claim 5, or the recombinant protein of claim 6; and

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

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