CN114989304B - Anti-human Claudin18.2 antibody and application thereof - Google Patents

Anti-human Claudin18.2 antibody and application thereof Download PDF

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CN114989304B
CN114989304B CN202210771141.8A CN202210771141A CN114989304B CN 114989304 B CN114989304 B CN 114989304B CN 202210771141 A CN202210771141 A CN 202210771141A CN 114989304 B CN114989304 B CN 114989304B
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antibody
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CN114989304A (en
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张培发
田媛媛
徐婷婷
李光磊
姜孝明
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Shanghai Letu Life Technology Co ltd
Shenzhen Le Earth Life Science And Technology Investment Co ltd
Shenzhen Letu Biomedical Co ltd
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Shanghai Letu Life Technology Co ltd
Shenzhen Le Earth Life Science And Technology Investment Co ltd
Shenzhen Letu Biomedical Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Abstract

An anti-human claudin18.2 antibody that specifically binds to human CLDN 18.2 (human claudin 18.2) and does not bind to human CLDN 18.1 (human claudin 18.1) and uses thereof. The antibody of the invention can effectively induce apoptosis of tumor cells, and has wide application prospect in the aspects of medicines and/or diagnostic reagents for treating and/or preventing diseases caused by CLDN 18.2 high expression.

Description

Anti-human Claudin18.2 antibody and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to an anti-human Claudin18.2 antibody and application thereof.
Background
In recent years, the incidence and mortality of global cancers are continuously rising, and the global cancers become a serious disease which seriously threatens the life health of human beings and the social development. According to the latest global cancer statistics report, 1810 ten thousand new cases of cancer and 960 ten thousand dead cases of cancer are worldwide in 2018, wherein the incidence rate of gastric cancer is ranked in the fifth place (5.7%), the incidence rate of gastric cancer is ranked in the 103.3 ten thousand, the death rate is ranked in the third place (8.2%), and the death rate is ranked in the 78.2 ten thousand. The incidence of lung cancer is first (11.6%), the number of patients suffering from lung cancer is 209.3 ten thousand, the death rate is also first (18.4%), and the number of deaths is 176.1 ten thousand. Pancreatic cancer incidence rate is 2.5%, incidence rate is 45.8 ten thousand, death rate is 4.5%, and death rate is 43.2 ten thousand. The incidence rate of esophageal cancer is 3.2%, the incidence rate is 57.2 ten thousand, the death rate is 5.3%, and the death rate is 50.8 ten thousand. The national cancer statistics data published by the national cancer center in 1 month of 2019 show that the incidence rate of gastric cancer in 2015 is second in China, the incidence rate of gastric cancer is 40.3 ten thousand, the incidence rate is close to half of the incidence rate of gastric cancer worldwide, the death rate is third, and the death rate is 29.1 ten thousand. The incidence rate and the death rate of lung cancer are the first, the incidence rate and the death rate of 78.7 ten thousand of lung cancer patients and the death rate of 63.1 ten thousand of lung cancer patients. Pancreatic cancer incidence rate is the tenth, mortality rate is the sixth, the number of patients suffering from pancreatic cancer is 9.5 ten thousand, and the number of patients suffering from pancreatic cancer is 8.51 ten thousand. The incidence rate of esophageal cancer is the sixth, the death rate is the fourth, the number of patients suffering from esophageal cancer is 24.6 ten thousand, and the number of dead patients is 18.79 ten thousand.
Tumor attack has been the target of struggle for humans, and tumor treatment approaches include three major traditional techniques: surgical treatment, chemotherapy, radiation therapy. It is an ideal goal to completely remove a tumor while not damaging other normal tissues of the body, which is sometimes achieved by surgical treatment, however the habit of the tumor to infiltrate into its adjacent normal cells or spread to a more distant location makes the surgical treatment effect not very obvious, while chemotherapy and radiation therapy cause certain side effects to normal cells while destroying tumor cells. The antibody treatment has the advantages of strong targeting and low side effect, and is widely applied as a new treatment means.
Antibody treatment is to use a protein specifically expressed on the surface of a tumor cell or an abnormally expressed protein as a target, to block the target by antigen-antibody reaction, to prevent the receptor from acting on its ligand, or to directly kill the tumor cell by ADCC and CDC. Antibody drugs approved by the FDA in the united states, including Alemtuzumab (Alemtuzumab), nivolumab (Nivolumab), rituximab (Rituximab), and Devaluzumab (Durvalumab), etc., are therapeutically effective in this manner.
CLDN 18.2
CLDN 18.2 is a subtype of the CLDNs protein family member CLDN18, playing an important role in cell attachment, and is associated with cell migration, lesions, tumor infiltration and metastasis. The protein consists of 261 amino acids, has four transmembrane domains, two extracellular loops, and the N-and C-termini located in the cell. The regulation of CLDN 18.2 gene expression is not clear at present, and it is mainly believed that after cAMP activating Protein Kinase A (PKA) in tumor cells, activated PKA enters the nucleus, and CREB is activated by amino-terminal kinase-induced domain (KID) phosphorylation, and after being phosphorylated by PKA, the transcriptional activity of CREB increases 10-20 times, causing abnormal expression of CLDN 18.2.
The expression of CLDN 18.2 in humans is tissue specific, in normal tissue cells, only in gastric epithelial cells, whereas CLDN 18.2 is expressed in tight junctions of gastric epithelial cells without exposing epitopes compared to loose cancer cells, so antibodies are not easy to act even if epithelial cells are killed by antibodies, stem cells under epithelial cells can supplement damaged gastric epithelial cells by differentiation, as CLDN 18.2 is not expressed. CLDN 18.2 is highly expressed in certain cancers, such as: high expression in gastric cancer or stomach and esophagus junction cancer, and CLDN 18.2 expression is often observed in pancreatic cancer and lung cancer. CLDN 18.2 is a very potential target because of its differential expression between cancer cells and normal cells.
Disclosure of Invention
According to a first aspect, in an embodiment, an isolated antibody is provided that specifically binds to human CLDN18.2 (human claudin 18.2) and does not bind to human CLDN 18.1 (human claudin 18.1).
According to a second aspect, in an embodiment, there is provided an isolated polynucleotide encoding an antibody of any one of the first aspects.
According to a third aspect, in an embodiment, there is provided a construct comprising the polynucleotide of the second aspect.
According to a fourth aspect, in an embodiment, there is provided an expression system comprising a construct or a polynucleotide of the second aspect having an exogenous gene integrated into the genome of the third aspect.
According to a fifth aspect, in an embodiment, there is provided a pharmaceutical composition comprising an antibody of any one of the first aspects and a pharmaceutically acceptable carrier.
According to a sixth aspect, in an embodiment there is provided the use of an antibody of the first aspect, a polynucleotide of the second aspect, a construct of the third aspect, an expression system of the fourth aspect in the manufacture of a medicament and/or diagnostic agent for the treatment and/or prophylaxis of a disease.
According to the anti-human Claudin18.2 antibody and the application thereof, the antibody disclosed by the invention can effectively induce apoptosis of tumor cells, and has wide application prospects in the aspects of preparing medicines and/or diagnostic reagents for treating and/or preventing diseases caused by Claudin18.2 high expression.
Drawings
FIG. 1 is a diagram of a phage antibody library 3 round enrichment screen Pool ElISA;
FIG. 2 is a SDS-PAGE electrophoresis after purification of antibody expression;
FIG. 3 is a graph of the results of ELISA assays for CLDN18.2 antigen;
fig. 4A: results of flow binding to 293T-CLDN18.1 cells;
Fig. 4B: results of flow binding to 293T-CLDN18.2 cells;
Fig. 4C: results of binding to NUGC4-CLDN18.2 cells;
FIG. 5 is a graph showing results of endocytic efficiency of CLDN18.2 antibody;
FIG. 6A is a graph showing the results of the Scatchard method for affinity detection of the CLDN18.2 antibody IMAB 362;
FIG. 6B is a graph showing the results of the Scatchard method for affinity detection of antibody B1 of CLDN 18.2;
FIG. 6C is a graph showing the results of the Scatchard method for affinity detection of antibody B9 of CLDN 18.2;
FIG. 6D is a graph showing the results of the Scatchard method for affinity detection of antibody B35 of CLDN 18.2;
FIG. 6E is a graph showing the results of the Scatchard method for affinity detection of antibody B41 of CLDN 18.2;
FIG. 6F is a graph showing the results of the Scatchard method for affinity detection of antibody B50 of CLDN 18.2;
FIG. 6G is a graph showing the results of the Scatchard method for affinity detection of antibody B78 of CLDN 18.2;
FIG. 7A is a graph showing the result of CDC action of a CLDN18.2 antibody on 293T-CLDN18.1 cells;
FIG. 7B is a graph showing the CDC effect of a CLDN18.2 antibody on 293T-CLDN18.2 cells;
FIG. 8A is a schematic representation of the ADCC effect of a CLDN18.2 antibody on 293T-CLDN18.1 cells;
FIG. 8B is a schematic representation of the ADCC effect of a CLDN18.2 antibody on 293T-CLDN18.2 cells;
FIG. 9 is a schematic diagram of the structure of the CLDN18.2 antibody.
Detailed Description
The application will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods. In some instances, related operations of the present application have not been shown or described in the specification in order to avoid obscuring the core portions of the present application, and may be unnecessary to persons skilled in the art from a detailed description of the related operations, which may be presented in the description and general knowledge of one skilled in the art.
Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.
The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning. The term "coupled" as used herein includes both direct and indirect coupling (coupling), unless otherwise indicated.
Abbreviations (abbreviations)
ADC-antibody drug conjugates.
ADCC-antibody dependent cellular cytotoxicity.
CDC-complement dependent cytotoxicity.
Complementarity determining regions in the CDR-immunoglobulin variable regions.
FR-antibody framework regions, immunoglobulin variable regions that do not include CDR regions.
VH or V H -immunoglobulin heavy chain variable region.
VL or V L -immunoglobulin light chain variable region.
CH1 or C H1 -first constant region.
CH2 or C H2 -second constant region.
CH3 or C H3 -third constant region.
Herein, unless otherwise specified, nucleotide sequences default from left to right to 5 'to 3' and amino acid sequences default from left to right to N to C.
Depending on the source of the monoclonal antibody, it can be classified into murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies. The murine antibody is a gene 100% derived from mouse, and the fully human antibody is a gene 100% derived from human. The humanized antibody is obtained by grafting CDR regions of a murine antibody into the framework of a human antibody, and the humanized antibody is mostly human in origin and fused with murine components. Chimeric antibodies contain more murine components because the variable region of a murine antibody is directly linked to the constant region of a human antibody. The antibody of any type can be used for treating human diseases, but the degree of human origin also determines the effect of the antibody, and the fully human antibody is the least rejection and the best safety because it is derived from human self. In one embodiment, the invention uses a whole human phage antibody library to screen for human Claudin18.2 antibodies, which are safer in humans than the chimeric antibody IMAB362 (e.g., US20090169547A 1), humanized antibodies (e.g., WO2020200196A 1).
According to a first aspect, in an embodiment, an isolated antibody is provided that specifically binds to human CLDN 18.2 (human claudin 18.2) and does not bind to human CLDN 18.1 (human claudin 18.1), the antibody comprising at least one set of heavy chain variable regions, light chain variable regions:
1) A heavy chain variable region comprising HCDR1, HCDR2, HCDR3, said HCDR1 comprising SEQ ID NO:1, and the HCDR2 comprises the amino acid sequence of SEQ ID NO:2, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:3, an amino acid sequence shown in 3; a light chain variable region comprising LCDR1, LCDR2, LCDR3, said LCDR1 comprising SEQ ID NO:4, and the LCDR2 comprises the amino acid sequence of SEQ ID NO:5, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:6, an amino acid sequence shown in figure 6;
2) A heavy chain variable region comprising HCDR1, HCDR2, HCDR3, said HCDR1 comprising SEQ ID NO:7, said HCDR2 comprises the amino acid sequence of SEQ ID NO:8, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 9; a light chain variable region comprising LCDR1, LCDR2, LCDR3, said LCDR1 comprising SEQ ID NO:10, and the LCDR2 comprises the amino acid sequence of SEQ ID NO:11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:12, an amino acid sequence shown in seq id no;
3) A heavy chain variable region comprising HCDR1, HCDR2, HCDR3, said HCDR1 comprising SEQ ID NO:13, said HCDR2 comprises the amino acid sequence of SEQ ID NO:14, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:15, and a polypeptide comprising the amino acid sequence shown in seq id no; a light chain variable region comprising LCDR1, LCDR2, LCDR3, said LCDR1 comprising SEQ ID NO:16, and the LCDR2 comprises the amino acid sequence of SEQ ID NO:17, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:18, an amino acid sequence shown in seq id no;
4) A heavy chain variable region comprising HCDR1, HCDR2, HCDR3, said HCDR1 comprising SEQ ID NO:19, said HCDR2 comprises the amino acid sequence of SEQ ID NO:20, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:21, an amino acid sequence shown in seq id no; a light chain variable region comprising LCDR1, LCDR2, LCDR3, said LCDR1 comprising SEQ ID NO:22, and the LCDR2 comprises the amino acid sequence of SEQ ID NO:23, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:24, and a polypeptide comprising the amino acid sequence shown in seq id no;
5) A heavy chain variable region comprising HCDR1, HCDR2, HCDR3, said HCDR1 comprising SEQ ID NO:25, said HCDR2 comprises the amino acid sequence of SEQ ID NO:26, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:27, and a polypeptide sequence as set forth in seq id no; a light chain variable region comprising LCDR1, LCDR2, LCDR3, said LCDR1 comprising SEQ ID NO:28, and the LCDR2 comprises the amino acid sequence of SEQ ID NO:29, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:30, an amino acid sequence shown in seq id no;
6) A heavy chain variable region comprising HCDR1, HCDR2, HCDR3, said HCDR1 comprising SEQ ID NO:31, said HCDR2 comprises the amino acid sequence of SEQ ID NO:32, said HCDR3 comprises the amino acid sequence of SEQ ID NO:33, an amino acid sequence shown in seq id no; a light chain variable region comprising LCDR1, LCDR2, LCDR3, said LCDR1 comprising SEQ ID NO:34, and the LCDR2 comprises the amino acid sequence of SEQ ID NO:35, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:36, and a nucleotide sequence shown in seq id no.
In one embodiment, the light chain constant region of the antibody includes, but is not limited to, a human antibody k, a lambda-type light chain constant region, or a variant thereof.
In one embodiment, the heavy chain variable region Framework (FR) sequence of the antibody is selected from human germline heavy chain sequences.
In one embodiment, the heavy chain of the antibody comprises a heavy chain constant region of human IgG1, igG2, igG3, igG4, or a variant thereof.
In one embodiment, the antibody is a full length antibody.
In one embodiment, the antibody is an antibody fragment selected from the group consisting of: fab, fab', F (ab) 2, fd, fv, sc Fv and scFv-Fc fragments, single chain antibodies, minibodies (minibodies) and diabodies. The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab' -SH, F (ab) 2; a diabody; a linear antibody; single chain antibody molecules (e.g., sc Fv). Papain digestion of antibodies produces two identical antigen-binding fragments, called a "Fab" fragment and a residual "Fc" fragment, a name reflecting the ability to crystallize readily. The Fab fragment consists of one complete light (L) chain (VL) and the variable region domain of the heavy (H) chain (VH) and the first constant domain of the heavy chain (CH 1). Pepsin treatment of the antibody produced a single large F (ab) 2 fragment, which corresponds approximately to two disulfide-linked Fab fragments with bivalent antigen-binding activity, and was still able to crosslink the antigen. Fab fragments differ from the F (ab) 2 fragment in that there are additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the name given herein to Fab' in which the cysteine residue of the constant domain carries a free thiol group (thiolgroup). The F (ab ') 2 antibody fragment was originally produced as a pair of cysteine Fab' fragments with a hinge between them. Other chemical couplings of other known antibody fragments are also included.
In one embodiment, the antibody is a monoclonal antibody.
In one embodiment, the antibody is a human antibody.
In one embodiment, the antibody is a chimeric antibody.
In one embodiment, the antibody is a bispecific antibody comprising an unmodified (e.g., naturally occurring) Fc fragment or a modified Fc fragment designed to optimize or alternatively eliminate a particular Fc-mediated function.
In one embodiment, the antibody comprises at least one of the following heavy and light chains:
1) A heavy chain comprising the amino acid sequence of SEQ ID NO:38, and a sequence of amino acids shown in seq id no; a light chain comprising SEQ ID NO:40, an amino acid sequence shown in seq id no;
2) A heavy chain comprising the amino acid sequence of SEQ ID NO: 42; a light chain comprising SEQ ID NO:44, an amino acid sequence shown in seq id no;
3) A heavy chain comprising the amino acid sequence of SEQ ID NO:46, an amino acid sequence shown in seq id no; a light chain comprising SEQ ID NO: 48;
4) A heavy chain comprising the amino acid sequence of SEQ ID NO:50, an amino acid sequence shown in seq id no; a light chain comprising SEQ ID NO:52, an amino acid sequence shown in seq id no;
5) A heavy chain comprising the amino acid sequence of SEQ ID NO:54, an amino acid sequence shown in seq id no; a light chain comprising SEQ ID NO:56, an amino acid sequence shown in seq id no;
6) A heavy chain comprising the amino acid sequence of SEQ ID NO:58, and a sequence of amino acids shown in seq id no; a light chain comprising SEQ ID NO: 60.
According to a second aspect, in an embodiment, there is provided an isolated polynucleotide encoding an antibody of any one of the first aspects.
According to a third aspect, in an embodiment, there is provided a construct comprising the polynucleotide of the second aspect. The construct may generally be obtained by inserting the isolated polynucleotide into a suitable vector, which may be a phage, plasmid, or viral vector, or an artificial chromosome, such as a bacterial or yeast artificial chromosome, which may be selected by those skilled in the art. In other words, the vectors of embodiments of the invention comprise a polynucleotide of interest capable of being expressed in a host cell or an isolated fraction thereof. Vectors are also generally suitable as cloning vectors, i.e.replicable in microbial systems; cloning vectors may be designed for replication in one host, while constructs are designed for expression in a different host. Vectors comprising the polypeptides and proteins of embodiments of the invention may also comprise a selectable marker for propagation or selection in a host cell. The vector may be introduced into a prokaryotic or eukaryotic cell by conventional transformation or transfection techniques.
According to a fourth aspect, in an embodiment, there is provided an expression system comprising a construct or a polynucleotide of the second aspect having an exogenous gene integrated into the genome of the third aspect. The expression system may be a host cell which may express the antibody of any one of the first aspects. In another embodiment of the invention, the host cell may be a eukaryotic cell and/or a prokaryotic cell, more particularly a mouse cell, a human cell, etc.
According to a fifth aspect, in an embodiment, there is provided a pharmaceutical composition comprising an antibody of any one of the first aspects and a pharmaceutically acceptable carrier.
In one embodiment, pharmaceutically acceptable carriers include, but are not limited to, diluents, excipients, solvents, and encapsulating materials.
According to a sixth aspect, in an embodiment there is provided the use of an antibody of the first aspect, a polynucleotide of the second aspect, a construct of the third aspect, an expression system of the fourth aspect in the manufacture of a medicament and/or diagnostic agent for the treatment and/or prophylaxis of a disease.
In one embodiment, the disease includes, but is not limited to, cancer.
In one embodiment, the cancer includes, but is not limited to, a disease associated with cells expressing CLDN 18.2.
In one embodiment, the cancer includes, but is not limited to, a disease associated with cells that highly express CLDN 18.2.
In one embodiment, the cancer includes, but is not limited to, gastric cancer, pancreatic cancer, esophageal cancer, lung cancer.
In one embodiment, the invention provides an antibody that specifically binds to human CLDN 18.2, methods of preparation and uses thereof, particularly in the diagnosis, treatment of diseases associated with CLDN 18.2-expressing cells, including high-expressing CLDN 18.2 tumors such as gastric, pancreatic, esophageal, lung cancers.
Example 1
Fig. 9 is a schematic structural diagram of the CLDN18.2 antibody provided in this example, which is specifically described as follows:
The light chain variable region (V L or VL) and the heavy chain variable region (V H or VH) of an antibody comprise Complementarity Determining Regions (CDRs) and Framework Regions (FR).
The light chain constant region (CL) of the antibody is polymerized with CH1 of the heavy chain constant region through disulfide bonds, the heavy chain constant region further comprises CH2, CH3, and the C-terminal of CH1 is sequentially bonded to CH2, CH3, and a Hinge (HINGE) is bonded between CH1 and CH 2.
The present example provides an anti-human CLDN18.2 antibody comprising a heavy chain variable region (V H) and a light chain variable region (V L), each sequence being selected from a natural human phage antibody library, wherein:
(1) B1 heavy chain variable region nucleic acid sequence
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGTCACGGTGGCACACTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCATCA(SEQ ID NO：37)
(2) B1 heavy chain variable region amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGHGGTLFDYWGQGTLVTVSS(SEQ ID NO：38)
(3) B1 light chain variable region nucleic acid sequence
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGTTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGCAACCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO：39)
(4) B1 light chain variable region amino acid sequence
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISNLEPEDFAVYYCQQRSNWPITFGQGTRLEIK(SEQ ID NO：40)
(5) B9 heavy chain variable region nucleic acid sequence
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAATACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGACGACACGGCTGTGTATTACTGTGCGAGGAGTGGGAGCTACTACTTTCCCTACTACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCATCA(SEQ ID NO：41)
(6) B9 heavy chain variable region amino acid sequence
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGNTIYYADSVKGRFTISRDNAKNSLYLQMNSLRADDTAVYYCARSGSYYFPYYFDYWGQGTLVTVSS(SEQ ID NO：42)
(7) B9 light chain variable region nucleic acid sequence
AACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGAATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACTCCTTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA(SEQ ID NO：43)
(8) B9 light chain variable region amino acid sequence
NIQLTQSPSSLSASVGDRVTITCRASQRISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK(SEQ ID NO：44)
(9) B35 heavy chain variable region nucleic acid sequence
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACGTGGCCAATCTACTTCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCATCA(SEQ ID NO：45)
(10) B35 heavy chain variable region amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDVANLLPWGQGTLVTVSS(SEQ ID NO：46)
(11) B35 light chain variable region nucleic acid sequence
GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACTATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO：47)
(12) B35 light chain variable region amino acid sequence
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPITFGQGTRLEIK(SEQ ID NO：48)
(13) B41 heavy chain variable region nucleic acid sequence
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGATAGGTGTGGGAGCTACGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCATCA(SEQ ID NO：49)
(14) B41 heavy chain variable region amino acid sequence
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGVGATDYWGQGTLVTVSS(SEQ ID NO：50)
(15) B41 light chain variable region nucleic acid sequence
AACATCCAGTTGACCCAGTCTCCATCTTCTGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCATGATCTATGATGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACTATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO：51)
(16) B41 light chain variable region amino acid sequence
NIQLTQSPSSVSASVGDKVTITCRASQGISSWLAWYQQKPGKAPKLMIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK(SEQ ID NO：52)
(17) B50 heavy chain variable region nucleic acid sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGATCCAATGGCTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACAATGGTCACCGTCTCATCA(SEQ ID NO：53)
(18) B50 heavy chain variable region amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSNGYYYYYMDVWGKGTMVTVSS(SEQ ID NO：54)
(19) B50 light chain variable region nucleic acid sequence
GACATCCAGATGACCCAGTCTCCATCTGCCGTATCTGCACGTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGCCTATAAGAACCTGGTTAGCCTGGTATCAGCAGAAGTCAGGGAGAGGCCCGAGACTCTTGATCTCTGCTGCATCCAATTTGCAGAGTGGAGTCCCATCAAGGTTCAGCGGCGGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAACCTGCAACTTGAAGATTCTGCAACTTACTTCTGTCAACAGGCTAGCAGAATCCCGTTCACTTTCGGCGGAGGGACCACGGTTGAGATCAAA(SEQ ID NO：55)
(20) B50 light chain variable region amino acid sequence
DIQMTQSPSAVSARVGDRVTITCRASQPIRTWLAWYQQKSGRGPRLLISAASNLQSGVPSRFSGGGSGTDFTLTISNLQLEDSATYFCQQASRIPFTFGGGTTVEIK(SEQ ID NO：56)
(21) B78 heavy chain variable region nucleic acid sequence
CAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAGCTTCAGTGACTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAGCACCCTTTTTCTGCACGTGAACAACTTGAGACCTGAGGATACGGCTGTCTATTACTGTGCGAAGGCAATAAGGGGTGGTTACTTCGGGTTCCTTGACCACTGGGGCCAGGGAACCCTGGTCACCGTCTCATCA(SEQ ID NO：57)
(22) B78 heavy chain variable region amino acid sequence
QVQLLESGGGLVQPGGSLRLSCAASGFSFSDYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKSTLFLHVNNLRPEDTAVYYCAKAIRGGYFGFLDHWGQGTLVTVSS(SEQ ID NO：58)
(23) B78 light chain variable region nucleic acid sequence
AACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGCTCACTTTCGGCCCTGGGACCAAAGTGGATCTCAAA(SEQ ID NO：59)
(24) B78 light chain variable region amino acid sequence
NIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGPGTKVDLK(SEQ ID NO：60)
(25) Complementarity Determining Region (CDR) sequences in immunoglobulin variable regions of antibodies
TABLE 1
The sequences in Table1 are all of human origin.
Example 1: antibody library packaging
The human antibody library strain was removed from the-80℃refrigerator, thawed on ice, and 1mL of the strain was added to 10mL of Carb+ (50. Mu.g/mL) 2 XYT medium, and shaken at 37℃for 1h at 200 rpm. 100mL of Carb+ (50. Mu.g/mL), tet+ (10. Mu.g/mL) 2 XYT medium was added, and the mixture was shaken at 200rpm for 1 hour, 10 10. Mu.g/mL of M13K07 helper phage was added, and the mixture was allowed to stand at 37℃for 30 minutes and at 200rpm for 30 minutes. Kan+ was added at a final concentration of 50. Mu.g/mL at 37℃overnight at 200 rpm.
The next day, 10000 Xg/min, 10min,4 ℃ and centrifugally collecting supernatant, adding PEG8000/NaCl with 1/4 times of the volume, fully and uniformly mixing, and placing in a refrigerator with the temperature of 4 ℃ for standing for 30 min-1 h. The mixture was centrifuged at 4℃at 12000 Xg/min for 20min, the supernatant was discarded and the pellet was resuspended in 1mL PBS.
The resuspension is the packaged antibody library.
Example 2: antibody library screening
The principle of the immune tube screening is that CLDN 18.2-VLP is coated on the surface of an immune tube with high adsorption capacity, and a phage display antibody library is added into the immune tube and undergoes the panning process of incubation, washing and elution with antigen adsorbed on the surface of the immune tube, and undergoes 2-4 rounds of panning, so that the specific monoclonal antibody aiming at the antigen is finally enriched.
In the first round of screening, 1mL of CLDN18.2-VLP protein with a concentration of 100. Mu.g/mL was added to the immune tube, the coating was removed overnight at 4 ℃, the coating was discarded the next day, 1% of PBSB was added for 1h, 10 12 antibody pools were added to the total amount after two PBST washes, incubated for 1h, washed 6 times with PBST to remove non-specifically bound phage, then 500. Mu.L of 100mM HCl eluent was added to the immune tube for eluting phage specifically binding to the antigen of interest, and 1.0M Tris-HCl (pH 8) was added for neutralization. Adding 10 times volume of eluent into TG1 in logarithmic phase, standing at 37deg.C for 30min, shaking at 200rpm for 30min, adding M13K07 helper phage, standing for 30min, and culturing overnight at 220 rpm. The phage were precipitated the next day for the following 2-3 rounds of screening.
The second round of screening was performed with cells, and the antigen coating concentration for the third round of screening was decreased to 10. Mu.g/mL. Clones from the third round were selected for positive clone screening by ELISA. Among the 44 clones, 6 positive clones capable of specifically binding to CLDN 18.2-VLP protein were screened for subsequent sequencing analysis.
Table 2 phage antibody library 3 rounds of screening enrichment
FIG. 1 shows a Pool ELISA graph of phage antibody library enriched in3 rounds of screening, and the ELISA result of the 3 rounds of enrichment is higher than the enrichment results of the 1 st round and the 2 nd round, which indicates that the sequence enrichment is obvious after the 3 rd round of screening enrichment.
Example 3: full length construction, expression and purification
The clone obtained by screening is sequenced to obtain the DNA sequence of the antibody, the primer is designed, the variable region fragments of the light chain and the heavy chain of the antibody are obtained by PCR amplification, the variable region fragments are respectively constructed on a modified eukaryotic expression vector plasmid pcDNA3.3-TOPO containing the variable region fragments of the light chain and the heavy chain by a homologous recombination method, the structural form of the antibody is IgG1, the constructed vector is used for transforming competent cells TOP10, the coating is carried out overnight, the monoclonal is picked up every other day, the amplification is carried out, and the plasmid is extracted by adopting an endotoxin-free plasmid extraction kit, so that the obtained plasmid is used for expressing eukaryotic cells CHO.
On the day of transfection, cells were adjusted to a final concentration of 6X 10 6 cells/mL with fresh ExpiCHO TM Expression Medium pre-warmed at 37 ℃. Diluting the target plasmid with pre-cooled OptiPROTM-SFM (1 mu g of plasmid is added into 1mL of the culture medium), diluting ExpiFectamineTM-CHO with OptiPROTM-SFM, mixing the two with equal volume, and gently stirring to prepare ExpiFectamineTM-CHO/plasmid DNA mixed solution, incubating for 1-5 min at room temperature, slowly adding into the prepared cell suspension, gently shaking, and finally placing in a cell culture shaker for culture under the condition of 5% CO 2 at 37 ℃. After 18-22 h of transfection, expiCHOTM-enhancement and ExpiCHOTM-Feed are added to the culture, the flask is placed on a shaking table at 37 ℃ and 5% CO 2 for continuous culture, expiCHOTM-Feed of the same volume is added on the 5 th day after transfection, the cell suspension is gently mixed while being slowly added, after 7-15 days of transfection, the cell culture supernatant expressing the protein of interest is centrifuged at 15000 Xg for 10min at high speed, the obtained supernatant is affinity purified, the protein of interest is eluted with 100mM sodium acetate, then neutralized with 1M Tris-HCl, and finally the protein obtained is replaced into PBS buffer by ultrafiltration of concentrated tube.
Purity identification of antibodies
The relative molecular weight and purity of the antibodies were checked by SDS-PAGE.
FIG. 2 shows SDS-PAGE electrophoresis after antibody expression purification, and the purified SDS-PAGE electrophoresis detection analysis shows that the antibody has clear target band, 50KD heavy chain size, 25KD light chain size, no impurity band and purity over 95%.
Example 4: ELISA detection
Hcldn18.2 antigen was coated at a concentration of 2.0 μg/mL,50 μl/well, and 4 ℃ overnight. The following day, PBST was washed 3 times, 1% PBSB was blocked at room temperature for 1h, PBST was washed 3 more times, and the gradient diluted antibody was incubated at room temperature for 1h. PBST was washed 3 times, incubated with Anti-human-IgG-Fc-HRP secondary antibody for 1h, PBST was washed 5 times, developed with TMB, stopped with 1M sulfuric acid, and OD450 was measured.
FIG. 3 is a graph showing ELISA results, in which candidate antibodies B1, B9, and B50 have affinity activity equivalent to that of the control chimeric antibody IMAB362 at the same concentration, and B35, B41, and B78 are lower.
Example 5: cell horizontal flow assay for binding Activity
Cells in the logarithmic growth phase were collected, the cell density was adjusted to 1X 10 6 cells/mL, washed 3 times with PBS, centrifuged at 1000rpm for 5min, and the cells were resuspended in 0.5mL of PBS. Setting an antibody concentration gradient: 0.03, 0.07, 0.1, 0.3, 0.7, 1, 1.5, 3, 7, 10 (. Mu.g/mL), incubation at 4.0℃for 1.0h, PBS wash 3 times, 1000rpm, centrifugation for 5min, cell resuspension in 0.5mL of PBS. Add APC-secondary antibody 5 u L, continue to incubate for 0.5 hours, PBS wash 3 times, 1000rpm, centrifugal 5 minutes, cell heavy suspension in 0.3mL PBS, flow cytometry on machine detection.
FIG. 4A is a schematic representation of flow-through binding to 293T-CLDN18.1 cells, showing that candidate antibodies B1, B9, B35, B41, B50, B78 do not bind to 293T-CLDN18.1 cells at a high dose of 10 μg/mL, indicating that the candidate antibodies do not bind to the CLDN18.1 antigen.
FIG. 4B is a schematic representation of flow-through binding to 293T-CLDN18.2 cells, showing that candidate antibodies B1, B9, B41 bind to 293T-CLDN18.2 cells at equivalent concentrations with comparable binding activity to control chimeric antibody IMAB362, while B35, B41, B78 are lower.
FIG. 4C is a schematic representation of flow-through binding to NUGC4-CLDN18.2 cells, showing that the same candidate antibodies B1, B9, B41 bind to NUGC4-CLDN18.2 cells at equivalent concentrations with comparable binding activity to the control chimeric antibody IMAB362, while B35, B50, B78 are lower.
Example 6: endocytic assay of antibodies
NUGC4-CLDN18.2 cells were collected, washed 3 times with PBS, cell density was adjusted to 2.5×10 6/ml, and the antibody to be assayed was diluted to a concentration of 20 μg/ml. 200 μl of the cell suspension was taken and 200 μl of antibody was added to give a final antibody concentration of 10 μg/mL. Gently stirring, mixing, and incubating on ice for 1 hr. The PBS was centrifuged and washed 3 times to remove unbound antibody. 400. Mu.L of the preheated complete medium was added to each well after removing the supernatant, and the mixture was placed in a 5% CO 2 incubator at 37℃for 4 hours, and the cells were removed. The cells were washed 3 times with PBS and stained with APC anti human IgG Fc. Mu.L of secondary antibody per well at 4℃for 30 minutes. Wash 3 times with PBS. Geometric Mean Fluorescence Intensity (MFI) was measured on a MA900 flow cytometer and the endocytosis efficiency of the CLDN18.2 antibody on NUG C4-CLDN18.2 cells was calculated.
MFI1H is the MFI of the sample after incubation on ice for 1 hour, assuming that the antibody completes binding under this condition and endocytosis has not yet occurred. MFI4H is the MFI of the sample after 4 hours incubation at 37 ℃. The percentage of antibody-mediated endocytosis of cell surface CLDN18.2 was calculated from the following formula: percentage of endocytosis (%) = (MFI 1H-MFI 4H)/MFI 1H x 100%. The percentage of endocytosis is also known as endocytosis efficiency.
FIG. 5 is a graph showing the results of endocytosis efficiency of the CLDN18.2 antibody, showing that the positive control chimeric antibody IMAB362 has the highest endocytosis efficiency, and the candidate antibodies B1, B41 have smaller endocytosis rates B9, B35, B50, and B78.
Example 7: scatchard method for determining antibody affinity
293T-hCDN18.2 cells were collected, 5X 10 5 cells per tube. Each antibody was added at an initial concentration of 20. Mu.g/mL, diluted in a 3-fold gradient, 8 concentration gradients were set, and incubated at 4℃for 1h. Unbound antibody was removed by centrifugation, washed 3 times with PBS, added 5. Mu.L/well APC anti human IgG Fc, incubated at 4deg.C for 30 min in the absence of light, washed 3 times with PBS, and detected on-machine by flow cytometry. The experimental data were analyzed using Flowjo software to obtain MFI, which was converted to the molar concentration of antibody by Scatchard mapping, and the dissociation constant K D value of antibody was calculated.
Scatchard's equation is as follows:
Taking the binding experiment as an example, in the formula, B is the concentration of the ligand binding to the upper receptor, F is the concentration of the free ligand, K d is the equilibrium dissociation constant, and B max is the maximum saturation concentration of ligand binding. The plot of B/F versus B is Scatchard plot, the slope of the resulting curve is 1/K d, and the intercept of the curve on the abscissa is B max. FIGS. 6A-6G are Scatchard plots of CLDN18.2 antibody affinity detection.
TABLE 3 affinity of antibodies
Antibodies to IMAB362 B1 B9 B35 B41 B50 B78
KD(nM) 3.67 3.54 3.14 18.02 3.6 5.16 4.16
The IMAB362 antibody is an existing human-mouse chimeric antibody, and is specifically prepared by referring to the patent specification with publication number US20090169547A 1. The IMAB362 antibody is also described in China patent publication No. CN 107050460A.
It can be seen that the candidate antibody B9 has a higher affinity than the positive control chimeric antibody IMAB362, B1, B41 has an affinity that approximates IMAB362, B50, B78 is slightly lower than IMAB362, B35 has the lowest affinity.
Example 7: evaluation of Complement Dependent Cytotoxicity (CDC) Effect of antibodies
The 293T-hCDN18.1-luci and 293T-hCDN18.2-luci cells were collected by centrifugation, resuspended, the cell concentration adjusted to 2X 10 5/mL, and 50. Mu.L per well, about 1.0X10 4, and 50. Mu.L of antibodies of different concentrations (final concentrations 10, 3.33, 1.11, 0.37, 0.12, 0.041, 0.0137, 0.004, 0.001. Mu.g/mL) were added and incubated in an incubator for 1h. The cell plates were removed, 5. Mu.L of complement was added per well, 250 Xg, centrifuged for 5 minutes and the cell plates incubated in an incubator at 37℃with 5% CO 2 for 6h. After the incubation, 1. Mu.L of 100X luciferin substrate was added to the reaction mixture to perform a reaction for 10min, and the chemiluminescent detection was performed in an ELISA reader.
FIG. 7A is a schematic representation of CDC effects of a CLDN18.2 antibody on 293T-CLDN18.1 cells, showing that candidate antibodies B1, B9, B35, B41, B50, B78 and positive control chimeric antibody IMAB362 were not killer to 293T-CLDN18.1 cells, indicating that candidate antibodies did not bind to the CLDN18.1 antigen. ,
Fig. 7B is a schematic diagram of CDC effect of CLDN18.2 antibody on 293T-CLDN18.2 cells, showing that candidate antibodies at equivalent concentrations, B1, B41 exhibited stronger CDC cell killing effect than positive control chimeric antibody IMAB 362. Candidate antibody B9 was similar to IMAB 362. B35, B50 and B78 have lower killing effect.
Example 8: evaluation of antibody-dependent cellular cytotoxicity (ADCC) Effect of antibodies
293T-hCDN18.1-luci and 293T-hCDN18.2-luci cells were collected, cell concentrations were adjusted to 2X 10 5/mL, and 96-well plates were added at 50. Mu.L per well, about 1.0X10 4 cells. Antibodies (10, 3.33, 1.11, 0.37, 0.12, 0.041, 0.001. Mu.g/mL) were added at various concentrations and incubated in an incubator for 0.5 hours. Effector PBMCs were collected and 50 μl, 1.0x10 5, E were added per well: t=10: 1 (effective target ratio, i.e. the ratio of the number of effector cells to target cells). Incubate in an incubator at 37℃with 5% CO 2 for 4h. After the incubation, 2. Mu.L of 100X luciferin substrate was added for reaction for 10min, and chemiluminescent detection was performed in an ELISA reader.
FIG. 8A is a graph showing the ADCC effect of the CLDN18.2 antibody on 293T-hCLDIN 18.1-luci cells, showing that the candidate antibodies B1, B9, B35, B41, B50, B78 and the positive control chimeric antibody IMAB362 do not kill 293T-CLDN18.1 cells, indicating that the candidate antibodies do not bind to the CLDN18.1 antigen.
FIG. 8B is a graph showing the ADCC effect of the CLDN18.2 antibody on 293T-hCLDIN 18.2-luci cells, showing that the candidate antibody exhibits a stronger ADCC cell killing effect than the positive control chimeric antibody IMAB362 at the same concentration. Candidate antibodies B9, B41 are similar to IMAB 362. B35, B50 and B78 have lower killing effect.
The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.
SEQUENCE LISTING
<110> Shenzhen Dimethom biological medicine Co., ltd
<120> An anti-human Claudin18.2 antibody and uses thereof
<130> 22I33580
<160> 60
<170> PatentIn version 3.3
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1 5 10 15
Gly
<210> 15
<211> 7
<212> PRT
<213> Artificial sequence
<400> 15
Asp Val Ala Asn Leu Leu Pro
1 5
<210> 16
<211> 11
<212> PRT
<213> Artificial sequence
<400> 16
Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala
1 5 10
<210> 17
<211> 7
<212> PRT
<213> Artificial sequence
<400> 17
Ala Ala Ser Ser Leu Gln Ser
1 5
<210> 18
<211> 9
<212> PRT
<213> Artificial sequence
<400> 18
Gln Gln Ala Asn Ser Phe Pro Ile Thr
1 5
<210> 19
<211> 5
<212> PRT
<213> Artificial sequence
<400> 19
Ser Tyr Ala Met Ser
1 5
<210> 20
<211> 17
<212> PRT
<213> Artificial sequence
<400> 20
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 21
<211> 7
<212> PRT
<213> Artificial sequence
<400> 21
Gly Val Gly Ala Thr Asp Tyr
1 5
<210> 22
<211> 11
<212> PRT
<213> Artificial sequence
<400> 22
Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala
1 5 10
<210> 23
<211> 7
<212> PRT
<213> Artificial sequence
<400> 23
Asp Ala Ser Ser Leu Gln Ser
1 5
<210> 24
<211> 9
<212> PRT
<213> Artificial sequence
<400> 24
Gln Gln Ala Asn Ser Phe Pro Leu Thr
1 5
<210> 25
<211> 5
<212> PRT
<213> Artificial sequence
<400> 25
Ser Tyr Ala Met Ser
1 5
<210> 26
<211> 17
<212> PRT
<213> Artificial sequence
<400> 26
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 27
<211> 11
<212> PRT
<213> Artificial sequence
<400> 27
Ser Asn Gly Tyr Tyr Tyr Tyr Tyr Met Asp Val
1 5 10
<210> 28
<211> 11
<212> PRT
<213> Artificial sequence
<400> 28
Arg Ala Ser Gln Pro Ile Arg Thr Trp Leu Ala
1 5 10
<210> 29
<211> 7
<212> PRT
<213> Artificial sequence
<400> 29
Ala Ala Ser Asn Leu Gln Ser
1 5
<210> 30
<211> 9
<212> PRT
<213> Artificial sequence
<400> 30
Gln Gln Ala Ser Arg Ile Pro Phe Thr
1 5
<210> 31
<211> 5
<212> PRT
<213> Artificial sequence
<400> 31
Asp Tyr Ala Met Ser
1 5
<210> 32
<211> 17
<212> PRT
<213> Artificial sequence
<400> 32
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 33
<211> 12
<212> PRT
<213> Artificial sequence
<400> 33
Ala Ile Arg Gly Gly Tyr Phe Gly Phe Leu Asp His
1 5 10
<210> 34
<211> 11
<212> PRT
<213> Artificial sequence
<400> 34
Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn
1 5 10
<210> 35
<211> 7
<212> PRT
<213> Artificial sequence
<400> 35
Ala Ala Ser Ser Leu Gln Ser
1 5
<210> 36
<211> 9
<212> PRT
<213> Artificial sequence
<400> 36
Gln Gln Ser Tyr Ser Thr Pro Leu Thr
1 5
<210> 37
<211> 354
<212> DNA
<213> Artificial sequence
<400> 37
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaggtcac 300
ggtggcacac tctttgacta ctggggccag ggaaccctgg tcaccgtctc atca 354
<210> 38
<211> 118
<212> PRT
<213> Artificial sequence
<400> 38
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Gly His Gly Gly Thr Leu Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 39
<211> 321
<212> DNA
<213> Artificial sequence
<400> 39
gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttagc agttacttag cctggtacca acagaaacct 120
ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180
aggttcagtg gcagtgggtc tgggacagat ttcactctca ccatcagcaa cctagagcct 240
gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctatcac cttcggccaa 300
gggacacgac tggagattaa a 321
<210> 40
<211> 107
<212> PRT
<213> Artificial sequence
<400> 40
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile
85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105
<210> 41
<211> 363
<212> DNA
<213> Artificial sequence
<400> 41
gaagtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120
ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtaatac catatactac 180
gcagactctg tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat 240
ctgcaaatga acagcctgag agccgacgac acggctgtgt attactgtgc gaggagtggg 300
agctactact ttccctacta ctttgactac tggggccagg gcaccctggt caccgtctca 360
tca 363
<210> 42
<211> 121
<212> PRT
<213> Artificial sequence
<400> 42
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser Ser Ser Gly Asn Thr Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Ser Tyr Tyr Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 43
<211> 321
<212> DNA
<213> Artificial sequence
<400> 43
aacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagaattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaacag agttacagta ctccttacac ttttggccag 300
gggaccaagc tggagatcaa a 321
<210> 44
<211> 107
<212> PRT
<213> Artificial sequence
<400> 44
Asn Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 45
<211> 348
<212> DNA
<213> Artificial sequence
<400> 45
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagacgtg 300
gccaatctac ttccctgggg ccagggaacc ctggtcaccg tctcatca 348
<210> 46
<211> 116
<212> PRT
<213> Artificial sequence
<400> 46
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Val Ala Asn Leu Leu Pro Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 47
<211> 321
<212> DNA
<213> Artificial sequence
<400> 47
gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ctatcagcag cctgcagcct 240
gaagattttg caacttacta ttgtcaacag gctaacagtt tccctatcac cttcggccaa 300
gggacacgac tggagattaa a 321
<210> 48
<211> 107
<212> PRT
<213> Artificial sequence
<400> 48
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile
85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105
<210> 49
<211> 348
<212> DNA
<213> Artificial sequence
<400> 49
caggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gataggtgtg 300
ggagctacgg actactgggg ccagggaacc ctggtcaccg tctcatca 348
<210> 50
<211> 116
<212> PRT
<213> Artificial sequence
<400> 50
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Gly Val Gly Ala Thr Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 51
<211> 321
<212> DNA
<213> Artificial sequence
<400> 51
aacatccagt tgacccagtc tccatcttct gtgtctgcat ctgtaggaga caaagtcacc 60
atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120
gggaaagccc ctaagctcat gatctatgat gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ctatcagcag cctgcagcct 240
gaagattttg caacttacta ttgtcaacag gctaacagtt tccctctcac tttcggcgga 300
gggaccaagg tggagatcaa a 321
<210> 52
<211> 107
<212> PRT
<213> Artificial sequence
<400> 52
Asn Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Met Ile
35 40 45
Tyr Asp Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 53
<211> 360
<212> DNA
<213> Artificial sequence
<400> 53
gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggctgttt attactgtgc gagatccaat 300
ggctactact actactacat ggacgtctgg ggcaaaggga caatggtcac cgtctcatca 360
<210> 54
<211> 120
<212> PRT
<213> Artificial sequence
<400> 54
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Asn Gly Tyr Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 55
<211> 321
<212> DNA
<213> Artificial sequence
<400> 55
gacatccaga tgacccagtc tccatctgcc gtatctgcac gtgtgggaga cagagtcacc 60
atcacttgtc gggcgagtca gcctataaga acctggttag cctggtatca gcagaagtca 120
gggagaggcc cgagactctt gatctctgct gcatccaatt tgcagagtgg agtcccatca 180
aggttcagcg gcggtggatc tgggacagat ttcactctca ccatcagtaa cctgcaactt 240
gaagattctg caacttactt ctgtcaacag gctagcagaa tcccgttcac tttcggcgga 300
gggaccacgg ttgagatcaa a 321
<210> 56
<211> 107
<212> PRT
<213> Artificial sequence
<400> 56
Asp Ile Gln Met Thr Gln Ser Pro Ser Ala Val Ser Ala Arg Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Pro Ile Arg Thr Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Ser Gly Arg Gly Pro Arg Leu Leu Ile
35 40 45
Ser Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Gln Leu
65 70 75 80
Glu Asp Ser Ala Thr Tyr Phe Cys Gln Gln Ala Ser Arg Ile Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Thr Val Glu Ile Lys
100 105
<210> 57
<211> 363
<212> DNA
<213> Artificial sequence
<400> 57
caggtgcagc tgttggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cagcttcagt gactatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagag cacccttttt 240
ctgcacgtga acaacttgag acctgaggat acggctgtct attactgtgc gaaggcaata 300
aggggtggtt acttcgggtt ccttgaccac tggggccagg gaaccctggt caccgtctca 360
tca 363
<210> 58
<211> 121
<212> PRT
<213> Artificial sequence
<400> 58
Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asp Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu Phe
65 70 75 80
Leu His Val Asn Asn Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ala Ile Arg Gly Gly Tyr Phe Gly Phe Leu Asp His Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 59
<211> 321
<212> DNA
<213> Artificial sequence
<400> 59
aacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaacag agttacagta ccccgctcac tttcggccct 300
gggaccaaag tggatctcaa a 321
<210> 60
<211> 107
<212> PRT
<213> Artificial sequence
<400> 60
Asn Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Leu Lys
100 105

Claims (8)

1. An isolated antibody that specifically binds to human CLDN 18.2 and does not bind to human CLDN 18.1, comprising a heavy chain variable region and a light chain variable region:
The 3 CDR regions of the heavy chain variable region are HCDR1, HCDR2, HCDR3, respectively;
the HCDR1 is SEQ ID NO:1, and a polypeptide sequence shown in the specification;
the HCDR2 is SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2);
The HCDR3 is SEQ ID NO:3, an amino acid sequence shown in 3;
the 3 CDR regions of the light chain variable region are LCDR1, LCDR2 and LCDR3 respectively;
the LCDR1 is SEQ ID NO:4, and a polypeptide sequence shown in the figure;
The LCDR2 is SEQ ID NO:5, and a polypeptide sequence shown in the figure;
The LCDR3 is SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.
2. The antibody of claim 1, wherein the light chain constant region of the antibody comprises a human antibody k, λ light chain constant region;
or, the heavy chain variable region Framework (FR) sequence of the antibody is selected from the group consisting of human germline heavy chain sequences;
Or, the antibody is a full length antibody;
Or, the antibody is an antibody fragment selected from the group consisting of: fab, fab', F (ab) 2, fd, fv, scFv, and scfv-Fc fragments;
Or, the antibody is a monoclonal antibody;
Or, the antibody is a human antibody;
Alternatively, the antibody is a chimeric antibody.
3. The antibody of claim 1, wherein the heavy chain of the antibody is SEQ ID NO:38, and a sequence of amino acids shown in seq id no; the light chain is SEQ ID NO:40, and a polypeptide having the amino acid sequence shown in seq id no.
4. An isolated polynucleotide encoding the antibody of any one of claims 1-3.
5. A construct comprising the polynucleotide of claim 4.
6. An expression system comprising the construct or genome of claim 5 integrated with an exogenous polynucleotide of claim 4.
7. A pharmaceutical composition comprising the antibody of any one of claims 1-3 and a pharmaceutically acceptable carrier.
8. Use of an antibody according to any one of claims 1 to 3, a polynucleotide according to claim 4, a construct according to claim 5 or an expression system according to claim 6 for the preparation of a medicament for the treatment and/or prevention of a disease caused by abnormal expression of CLDN 18.2 and/or a disease caused by abnormal expression of CLDN 18.2, which is cancer, which is a disease associated with cells that highly express CLDN 18.2, which is gastric cancer, pancreatic cancer, esophageal cancer or lung cancer.
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CN113735974A (en) * 2020-05-29 2021-12-03 杭州邦顺制药有限公司 Antibodies against Claudin18.2 and uses thereof
WO2022093310A1 (en) * 2020-10-26 2022-05-05 Shattuck Labs, Inc. Homodimeric and heterodimeric proteins comprising butyrophilin
CN114555640A (en) * 2020-06-23 2022-05-27 德琪(杭州)生物有限公司 Antibodies and methods for treating CLAUDIN-related diseases

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