CN116102649A - anti-CLDN 18.2 monoclonal antibody and application thereof - Google Patents

anti-CLDN 18.2 monoclonal antibody and application thereof Download PDF

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CN116102649A
CN116102649A CN202111333547.XA CN202111333547A CN116102649A CN 116102649 A CN116102649 A CN 116102649A CN 202111333547 A CN202111333547 A CN 202111333547A CN 116102649 A CN116102649 A CN 116102649A
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antibody
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瞿爱东
梁红远
张坤明
吴丽娜
祝婧烨
邱建华
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SHANGHAI INSTITUTE OF BIOLOGICAL PRODUCTS CO LTD
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SHANGHAI INSTITUTE OF BIOLOGICAL PRODUCTS CO LTD
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Priority to PCT/CN2022/131502 priority patent/WO2023083327A1/en
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Abstract

The invention provides anti-CLDN 18.2 monoclonal antibodies and formulations thereof. In particular, the invention provides a novel anti-CLDN 18.2 humanized antibody. The antibody of the invention can be combined with antigen with high specificity, has high affinity and high biological activity, can be combined with human CLDN18.2 antigen molecule with specificity, and can inhibit tumor with high efficiency in vitro and in vivo.

Description

anti-CLDN 18.2 monoclonal antibody and application thereof
Technical Field
The invention relates to the field of medicines, in particular to an anti-CLDN 18.2 humanized monoclonal antibody and a preparation thereof.
Background
Tight Junctions (TJs) are multi-molecular complexes that regulate the permeability between epithelial tissue cells. In tight junction complexes, tight junction proteins (claudins) form tight channels between adjacent cells, regulating the flow of ions, solutes, etc.
CLDN18 is a major component of tight junctions in lung and stomach epithelial cells, CLDN18 has two subtypes, CLDN18.1 and CLDN18.2, CLDN18.1 is predominantly expressed in the lung and CLDN18.2 is predominantly expressed in the stomach.
The CLDN18.2 was found to be highly expressed in a variety of tumors such as primary and metastatic gastric Cancer, pancreatic Cancer, esophageal Cancer, lung Cancer, cholangiocarcinoma, ovarian Cancer, etc. (Sahin U, et al Clin Cancer Res,2008;14 (23): 7624-34). Because tumor cells have the characteristics of rapid proliferation, susceptibility to invasion and metastasis and the like, and lose a tight connection structure, the CLDN18.2 molecules on the surface of the tumor cells are easier to expose, and the CLDN18.2 becomes an ideal tumor treatment target (Hewitt KJ, et al BMC Cancer,2006; 6:186).
The chimeric antibody IMAB362 against CLDN18.2 developed by Ganymed Pharmaceuticals AG has entered phase III clinical studies. In phase II clinical studies on advanced or recurrent gastroesophageal Cancer, IMAB362 significantly prolonged the patient's PFS and OS in combination with first-line chemotherapeutic EOX (epirubicin, oxaliplatin, capecitabine) compared to chemotherapy alone, as a potential drug for the treatment of gastric Cancer, esophageal Cancer, and the like (Lordick F, et al, cancer,2021;24 (3): 721-730.).
Because of the potential of CLDN18.2 in tumor therapy, there is a great clinical need to develop highly active antibody drugs.
The high similarity of human CLDN18.1 to CLDN18.2 presents challenges for the development of CLDN18.2 specific antibodies. In addition, mouse CLDN18.2 is identical to human CLDN18.2 extracellular loop 1 sequence, and screening of antibodies specific for human CLDN18.2 in mice requires breaking immune tolerance in mice. Therefore, numerous technical difficulties need to be overcome in developing highly active drugs specifically recognizing human CLDN18.2 antibodies.
Thus, there remains a need in the art to develop a CLDN18.2 antibody and its use that has high affinity and high biological activity.
Disclosure of Invention
The invention aims to provide a CLDN18.2 antibody with high affinity and high biological activity and application thereof.
In a first aspect of the invention there is provided a heavy chain variable region of an antibody, said heavy chain variable region comprising the following three complementarity determining region CDRs:
(1) A complementarity determining region CDR1, wherein the amino acid sequence of the complementarity determining region CDR1 is shown in SEQ ID NO. 3;
(2) A complementarity determining region CDR2, wherein the amino acid sequence of said complementarity determining region CDR2 is shown in SEQ ID NO. 4 or 16; a kind of electronic device with high-pressure air-conditioning system
(3) Complementarity determining region CDR3, the amino acid sequence of said complementarity determining region CDR3 is shown in SEQ ID NO. 5 or 17.
In another preferred embodiment, the three CDRs of the heavy chain variable region are SEQ ID Nos 3, 4 and 5; or 3, 16 and 5; or 3, 4 and 17.
In another preferred embodiment, the heavy chain variable region comprises four framework regions FR from the four framework regions FR corresponding to SEQ ID NOS: 1, 11, 12 and 13.
In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 1, 11, 12 or 13.
In a second aspect of the invention there is provided an antibody heavy chain having the heavy chain variable region of an antibody according to the first aspect of the invention.
In another preferred embodiment, the constant region of the heavy chain is of human origin.
In another preferred embodiment, the constant region of the heavy chain is a heavy chain constant region of human IgG1, igG2, or the like.
In a third aspect of the invention there is provided a light chain variable region of an antibody, said light chain variable region comprising the following three complementarity determining regions CDR':
(1) A complementarity determining region CDR1', wherein the amino acid sequence of said complementarity determining region CDR1' is shown in SEQ ID NO. 8;
(2) A complementarity determining region CDR2', wherein the amino acid sequence of said complementarity determining region CDR2' is shown in SEQ ID NO. 9; a kind of electronic device with high-pressure air-conditioning system
(3) Complementarity determining region CDR3', the amino acid sequence of said complementarity determining region CDR3' is shown in SEQ ID NO. 10.
In another preferred embodiment, the light chain variable region comprises four framework regions FR from the four framework regions FR corresponding to SEQ ID NOS.6, 14 and 15.
In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 6, 14 or 15.
In a fourth aspect of the invention there is provided an antibody light chain having the light chain variable region of an antibody according to the third aspect of the invention.
In another preferred embodiment, the constant region of the light chain is of human origin.
In another preferred embodiment, the constant region of the light chain is a human Kappa, lambda, etc. light chain constant region.
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) The light chain variable region according to the third aspect of the invention.
Alternatively, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.
In another preferred embodiment, the antibody further has a heavy chain constant region and a light chain constant region.
In another preferred embodiment, the antibody has a heavy chain variable region as set forth in SEQ ID NO. 1, 11, 12 or 13; and/or the light chain variable regions as shown in SEQ ID NOS.6, 14, and 15.
In another preferred embodiment, the antibody has a heavy chain variable region as set forth in SEQ ID NO. 1, 11, 12 or 13; and/or a light chain variable region as set forth in SEQ ID NO. 15.
In another preferred embodiment, the antibody is selected from the group consisting of:
(Z1) an antibody having a heavy chain variable region shown in SEQ ID No. 1 and a light chain variable region shown in SEQ ID No. 6:
(Z2) an antibody having a heavy chain variable region shown in SEQ ID No. 11 and a light chain variable region shown in SEQ ID No. 14;
(Z3) an antibody having a heavy chain variable region shown in SEQ ID No. 11 and a light chain variable region shown in SEQ ID No. 15;
(Z4) an antibody having a heavy chain variable region shown in SEQ ID No. 12 and a light chain variable region shown in SEQ ID No. 14;
(Z5) an antibody having a heavy chain variable region shown in SEQ ID No. 12 and a light chain variable region shown in SEQ ID No. 15;
(Z6) an antibody having a heavy chain variable region shown in SEQ ID No. 13 and a light chain variable region shown in SEQ ID No. 14;
(Z7) an antibody having a heavy chain variable region shown in SEQ ID No. 13 and a light chain variable region shown in SEQ ID No. 15.
In another preferred embodiment, the heavy chain of the antibody has the amino acid sequence shown in SEQ ID No. 18, 20, 21 or 22.
In another preferred embodiment, the light chain of the antibody has the amino acid sequence shown in SEQ ID No. 19.
In another preferred embodiment, the antibody is a humanized antibody.
In another preferred embodiment, the antibody specifically binds CLDN18.2.
In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.
In another preferred embodiment, the antibody is a monoclonal antibody.
In another preferred embodiment, the antibody is a bispecific antibody.
In another preferred embodiment, the antibody is in the form of a drug conjugate.
The invention also provides antibodies corresponding to 624 having the heavy chain variable region shown in SEQ ID No. 23 and the light chain variable region shown in SEQ ID No. 24.
In a sixth aspect of the present invention, there is provided a recombinant protein having:
(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; and
(ii) Optionally a tag sequence to assist expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In a seventh aspect of the invention, there is provided an antibody preparation comprising:
(a) An antibody according to the fifth aspect of the invention; and
(b) A carrier, said carrier comprising: buffering agents, sterile water, optionally surfactants.
In another preferred embodiment, the concentration of the antibody in the formulation is 5-100mg/mL; preferably 10-70mg/mL, more preferably 20-60mg/mL.
In another preferred embodiment, the buffer is selected from the group consisting of: a citrate buffer system, a histidine buffer system, or a combination thereof.
In another preferred embodiment, the pH of the formulation is in the range of 5.0 to 7.5, preferably 5.5 to 7.
In another preferred embodiment, the formulation is an injectable formulation.
In an eighth aspect of the invention there is provided a kit comprising an antibody preparation according to the seventh aspect of the invention, and a container for containing the antibody preparation.
In a ninth aspect of the invention there is provided a CAR construct, the scFv fragment of the antigen binding region of which is a binding region that specifically binds to CLDN18.2 and which has a heavy chain variable region as described in the first aspect of the invention and a light chain variable region as described in the third aspect of the invention.
In a tenth aspect of the invention, there is provided a recombinant immune cell expressing an exogenous CAR construct according to the ninth aspect of the invention.
In another preferred embodiment, the immune cells are selected from the group consisting of: NK cells, T cells.
In another preferred embodiment, the immune cells are derived from a human or non-human mammal (e.g., a mouse).
In an eleventh aspect of the present invention, there is provided an antibody drug conjugate comprising:
(a) An antibody moiety selected from the group consisting 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, or a combination thereof; and
(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.
In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.
In a twelfth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: the heavy chain variable region according to the first aspect of the invention, the heavy chain variable region according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, the immune cell according to the tenth aspect of the invention, the antibody drug conjugate according to the eleventh aspect of the invention, or a combination thereof, the active ingredient being for use in
(a) Preparing a detection reagent or a kit;
(b) Preparing a medicament or preparation for preventing and/or treating the CLDN18.2 related diseases; and/or
(c) Preparing medicine or preparation for preventing and/or treating cancer or tumor.
In another preferred embodiment, the tumor is selected from the group consisting of: hematological tumors, solid tumors, or combinations thereof.
In another preferred embodiment, the hematological neoplasm is selected from the group consisting of: acute Myelogenous Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), hodgkin's lymphoma, or a combination thereof.
In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.
In another preferred embodiment, the tumor is a tumor that highly expresses CLDN 18.2.
In another preferred embodiment, the medicament or formulation is for the manufacture of a medicament or formulation for the prevention and/or treatment of a disease associated with CLDN18.2 (positive expression).
In another preferred embodiment, the antibody is in the form of A Drug Conjugate (ADC).
In another preferred embodiment, the detection reagent or kit is used for diagnosing CLDN18.2 related diseases.
In another preferred embodiment, the detection reagent or kit is used to detect CLDN18.2 protein in a sample.
In another preferred embodiment, the detection reagent is a detection chip.
In a thirteenth aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) An active ingredient selected from the group consisting 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, an immune cell according to the tenth aspect of the invention, an antibody drug conjugate according to the eleventh aspect of the invention, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition further comprises a second active ingredient that is anti-tumor.
In another preferred embodiment, the second active ingredient is selected from the group consisting of: cytotoxic drugs, toxins, cytokines, enzymes, antibodies, or combinations thereof.
In another preferred embodiment, the second active ingredient comprises: EGFR-targeting antibodies, HER 2-targeting antibodies.
In another preferred embodiment, the pharmaceutical composition is a liquid formulation.
In another preferred embodiment, the pharmaceutical composition is an injection.
In another preferred embodiment, the pharmaceutical composition is for the treatment of tumors.
In another preferred embodiment, the tumor is a tumor that highly expresses CLDN 18.2.
In a fourteenth 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 (b)
(2) A recombinant protein according to the sixth aspect of the invention; and/or
(3) A CAR construct according to the ninth aspect of the invention.
In a fifteenth aspect of the present invention there is provided a vector comprising a polynucleotide according to the fourteenth aspect of the present invention.
In another preferred embodiment, the carrier comprises: bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In a sixteenth aspect of the invention there is provided a genetically engineered host cell comprising a vector according to the fifteenth aspect of the invention or a polynucleotide according to the fourteenth aspect of the invention integrated in the genome.
In a seventeenth aspect of the invention, there is provided a method of detecting CLDN18.2 protein in a sample in vitro (including diagnostic or non-diagnostic), said method comprising the steps of:
(1) Contacting the sample with an antibody according to the fifth aspect of the invention in vitro;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of CLDN18.2 protein in the sample.
In an eighteenth aspect of the present invention, there is provided a detection plate comprising: a substrate (support) and a test strip comprising an antibody according to the fifth aspect of the invention or an antibody drug conjugate according to the eleventh aspect of the invention.
In a nineteenth aspect of the present invention, there is provided a kit comprising:
(1) A first container comprising an antibody according to the fifth aspect of the invention; and/or
(2) A second container comprising a second antibody against an antibody according to the fifth aspect of the invention;
Alternatively, the kit contains a detection plate according to the eighteenth aspect of the invention.
In a twentieth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:
(a) Culturing a host cell according to the fourteenth aspect of the invention under conditions suitable for expression;
(b) Isolating the recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.
In a twenty-first aspect of the present invention, there is provided a method of CLDN 18.2-related diseases, the method comprising: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, an antibody-drug conjugate of the antibody, or a CAR-T cell expressing the antibody, or a combination thereof.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows the results of an Elisa binding assay for specific binding to hybridoma supernatant cells.
FIG. 2 shows the results of flow cytometry detection. Fig. 2A shows the detection result of chimeric antibody ch429, fig. 2B shows the detection result of chimeric antibody ch623, fig. 2C shows the detection result of chimeric antibody ch624, fig. 2D shows the detection result of chimeric antibody ch782, and fig. 2E shows the detection result of reference antibody ref.
FIG. 3 shows the results of detection of the binding activity of anti-CLDN 18.2 human murine chimeric antibodies to recombinant cells. FIG. 3A shows the results of binding activity of anti-CLDN 18.2 human murine chimeric antibody to HEK 293-hCLDIN 18.1 cells; FIG. 3B shows the results of binding activity of anti-CLDN 18.2 human murine chimeric antibody to HEK 293-hCLDIN 18.2 cells.
FIG. 4 shows the results of detection of binding activity of anti-CLDN 18.2 human murine chimeric antibody to NUGC4 cells.
FIG. 5 shows the results of cell binding activity flow cytometry detection of anti-CLDN 18.2 human murine chimeric antibodies to recombinant cells.
FIG. 6 shows the results of ADCC activity of anti-CLDN 18.2 human murine chimeric antibodies against HEK 293-hCLDIN 18.2 cells.
Figure 7 shows humanized antibody binding activity to human CLDN18.1& CLDN18.2, mouse CLDN18.1& CLDN18.2, and NUGC4 cells.
Figure 8 shows the ADCC activity of the humanized antibodies.
FIG. 9 shows the results of the detection of CDC activity of humanized antibodies.
FIG. 10 shows the results of the proliferation inhibitory activity of humanized antibodies against HEK 293-hCDN18.2.
FIG. 11 shows a comparison of the key binding sites of the antibody hu782 of the invention with a reference antibody. Wherein, FIG. 11A shows the key binding site of Ref.ch175 and FIG. 11B shows the key binding site of antibody hu782 of the present invention.
FIG. 12 shows in vivo antitumor activity of the antibodies of the invention.
Detailed Description
The inventors have conducted extensive and intensive studies to unexpectedly obtain an anti-human CLDN18.2 antibody having excellent high affinity and high antitumor activity for the first time. Specifically, the inventors obtained a plurality of murine antibodies with high affinity for human CLDN18.2 for the first time through a number of screens. Based on the murine antibody, the invention further prepares chimeric antibodies and humanized antibodies, and performs mutation screening on the obtained humanized antibodies. The antibodies of the invention, particularly the mutated humanized antibodies, have similar affinity to chimeric antibodies, and are further developed into humanized monoclonal antibody medicaments for targeted therapy in the future. The antibody of the invention can effectively combine with human CLDN18.2, inhibit proliferation of various tumor cells, inhibit growth of tumor by nude mice endosome, and has excellent anti-tumor activity. The present invention has been completed on the basis of this finding.
Specifically, the inventor alternatively immunizes mice by constructing various forms of hCDN18.2 DNA as antigens, and carrying out booster immunization by using a cell strain for recombining and expressing hCDN18.2, and obtaining the mouse monoclonal antibodies mab429, mab623, mab624 and mab782 which specifically recognize hCDN18.2 through a hybridoma fusion technology.
The chimeric antibody ch782 specifically binds to hCDN18.2, can recognize clDN18.2 molecules on the cell surface of tumor cells NUGC4, and has obviously higher affinity with clDN18.2 than the reference antibody Ref.ch175.ch782 has ADCC, CDC and proliferation inhibitory activity against hcldn18.2 cells.
The chimeric antibody ch782 was humanized to obtain 4 humanized antibody combinations hu7825H 1.1L1, hu7825.2L 1, hu7825.1L 2, hu7825.2L 2, and the 4 humanized antibodies specifically recognized hCDN18.2 with comparable affinity to the ch 782.
The humanized antibody heavy chain hu782H1.1 is subjected to S60A site mutation to remove potential glycosylation sites, and V101A mutation is performed to enhance the expression level, so that the humanized antibody heavy chain hu782H 1.2-V101A is obtained. The S60A, V A site is defined according to the Kabat numbering convention.
The humanized antibodies hu782 (H1.2L2 combination), hu782-HV (H1.2-V101A L combination) have significantly better ADCC activity against tumor cells than the reference antibody ref.ch175.
And carrying out S239D/I332E mutation on amino acids 239 and 332 of a heavy chain constant region of the humanized antibody hu782 to obtain the humanized antibody hu782-DE. S239D, I E is defined by the EU numbering system.
The humanized antibody hu782-DE has significantly better ADCC activity against tumor cell NUGC4 than the reference antibody Ref.ch175, which has an EC50 value 24 times lower than that of Ref.ch175.
Humanized antibodies hu782, hu782-HV have CDC killing activity against hcldn18.2 target cells.
Humanized antibodies hu782, hu782-HV have proliferation inhibitory activity on hcldn18.2 target cells, which has a dose-dependent proliferation inhibitory activity significantly better than the reference antibody ref.ch175.
Epitope analysis found that the key sites recognized by humanized antibody hu782 included the E56, G48 sites of hcldn18.2 molecule, while the key site recognized by reference antibody ref.ch175 included the a42S, N45A, E56A, G site, confirming that there was a certain difference between the two antibody binding epitopes.
In a nude mouse tumor transplantation model, three antibodies of hu782, hu782-HV and hu782-DE can obviously transplant tumor growth, and the tumor growth inhibition effect of the antibodies is superior to that of a reference antibody Ref. Ch175, especially two antibodies of hu782 and hu 782-DE. The tumor occurrence of mice in both the hu782 and hu782-DE antibody treatment groups completely regressed, the tumor inhibition rates of hu782-HV, hu782 and hu782-DE were 34.68%, 62.88% and 65.05%, and the reference antibody Ref.ch175 was 15.5%.
Terminology
In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meanings given below, unless expressly specified otherwise herein. Other definitions are set forth throughout the application.
The term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of a reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a well known method to those skilled in the art.
As used herein, the terms "heavy chain variable region" and "V H "interchangeably used.
As used herein, the terms "light chain variable region" and "V L "interchangeably used.
As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determining region, CDR)".
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 that specifically binds CLDN18.2, such as a protein or polypeptide having a heavy chain variable region (e.g., the amino acid sequence of SEQ ID NO:1, 11, 12, or 13) and/or a light chain variable region (e.g., the amino acid sequence of SEQ ID NO:6, 14, or 15). They may or may not contain an initiating methionine.
Another preferred antibody of the invention is an antibody that specifically binds to CLDN18.2 corresponding to 624, e.g.a protein or polypeptide having a heavy chain variable region (e.g.the amino acid sequence of SEQ ID NO: 23) and/or a light chain variable region (e.g.the amino acid sequence of SEQ ID NO: 24).
CLDN18.2
The total length of the human CLDN18 contains 261 amino acids, is a four-time transmembrane protein, has four transmembrane hydrophobic regions, has two annular structures outside the cell, and is formed by encircling a transmembrane region 1 and a transmembrane region 2; the ring 2 is surrounded by a transmembrane region 3 and a transmembrane region 4. The N-terminal amino acid composition of human CLDN18.1 and CLDN18.2, the amino acid composition of transmembrane region 1 and extracellular loop 1 are completely identical, and the sequence similarity reaches 92%.
Antibodies to
As used herein, the term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed from two identical heavy chains and two identical light chains joined by an interchain disulfide bond.
Existing antibody numbering schemes include:
the Kabat protocol (Kabat et al, 1991) is based on the position of regions of high sequence variation between sequences of the same domain type, with variable domains of antibody heavy (VH) and light (V.lambda.and V.kappa.) being numbered differently.
The scheme of Chothia (Al-Lazikani, 1997) is identical to that of Kabat, but the positions of the inserts of annotations around the first VH Complementarity Determining Region (CDR) are corrected to correspond to structural loops. Likewise, the enhanced Chothia program (abhinannan and Martin, 2008) makes further structural modifications to the insertion site.
3. In contrast to these Kabat-like regimens, IMGT (Lefranc, 2003) and AHo (honeygger and plockthun, 2001) both define unique regimens of antibodies and T Cell Receptor (TCR) (vα and vβ) variable domains. Thus, equivalent residue positions can be easily compared between domain types. IMGT and AHo differ in the number of positions they annotate (128 and 149, respectively) and in the positions they consider the indels to occur.
The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or isotypes of immunoglobulins, igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α, and epsilon chains, respectively. The Ig of the same class can be further classified into different subclasses according to the amino acid composition of the heavier chain region and the number and position of disulfide bonds of the heavy chain, such as IgG can be classified into IgG1, igG2, igG3 and IgG4. Light chains are classified as either kappa chains or lambda chains depending on the constant region. Each of the five classes of Ig may have either a kappa chain or a lambda chain. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
The antibody light chains of the present invention may further comprise a light chain constant region comprising a kappa, lambda chain of human or murine origin or variants thereof.
In the present invention, the antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising IgG1, igG2, igG3, igG4 or variants thereof of human or murine origin. The sequences of the heavy and light chains of antibodies, near the N-terminus, vary widely, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged in sequence from amino terminus to finished base terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3. In example 13 of the present invention, 6 CDRs of the ch782 antibody were divided in combination with the kabat and Chothia methods.
The term "murine antibody" is herein a monoclonal antibody against CLDN18.2 prepared according to the knowledge and skill in the art. The preparation is performed by injecting the test subjects with CLDN18.2 antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In a preferred embodiment of the invention, the murine CLDN18.2 antibody or antigen binding fragment thereof may further comprise a murine kappa, lambda chain or variant light chain constant region or further comprise a murine IgG1, igG2, igG3 or variant heavy chain constant region.
The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce an immune response induced by the murine antibody.
The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting murine CDR sequences into the framework of human antibody variable regions, i.e., the framework sequences of different types of human germline antibodies. Humanized antibodies can overcome the heterologous response induced by chimeric antibodies that carry large amounts of murine protein components. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity.
The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., CLDN 18.2). Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments encompassed within the term "antigen-binding fragment of an antibody" include
(i) A Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains;
(ii)F(ab') 2 a fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge on the longer chain region;
(iii) Fd fragment consisting of VH and CH1 domains;
(iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody.
Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding.
The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding. One of the most common definitions of the 6 CDRs is provided by Kabat E.A et al, (1991) Sequences of proteins of immunological interface.
The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on a CLDN18.2 molecule). Epitopes generally comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation.
The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is present at about less than 10 -7 M, e.g. less than about 10 -8 M、10 -9 M or lO -10 Affinity (KD) binding of M or less.
The term "competitive binding" refers to an antibody that recognizes the same epitope (also referred to as an epitope) or a portion of the same epitope on the extracellular region of CLDN18.2 as the monoclonal antibody of the invention and binds to the antigen. An antibody that binds to the same epitope as the monoclonal antibody of the invention refers to an antibody that recognizes and binds to the amino acid sequence of CLDN18.2 recognized by the monoclonal antibody of the invention.
Terminology"KD" or "Kd" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the invention are administered in amounts of less than about 10 -7 M, e.g. less than about 10 -8 M、10 -9 M or 10 -10 The dissociation equilibrium constant (KD) of M or less binds CLDN18.2.
As used herein, the term "epitope" refers to a discrete, three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using DNA recombination techniques well known in the art.
As used herein, the term "monoclonal antibody" refers to an antibody secreted from a clone derived from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cells may be eukaryotic, prokaryotic or phage clonal cell lines.
In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.
In the present invention, the antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibodies of the invention to form a polypeptide. These conservatively mutated polypeptides are preferably produced by amino acid substitution according to the table below.
Initial residues Representative substitution Preferred substitution
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
Humanized antibody against CLDN18.2
The present invention provides humanized antibodies against CLDN18.2 (hereinafter referred to as CLDN18.2 antibodies). In particular, the present invention provides a high specificity and high affinity humanized antibody against CLDN18.2 comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.
Jones et al first transplanted murine monoclonal antibody heavy chain CDRs into human antibody heavy chain framework regions, then assembled the murine monoclonal antibody heavy chain into complete antibodies with murine monoclonal antibody light chains and maintained affinity similar to the original murine monoclonal antibodies, providing ideas for the development of antibody humanization technology. Queen et al in 1989 successfully constructed anti-CD 25 humanized antibodies by CDR grafting using human antibody framework regions that were humanized with murine amino acids retained at some sites in the framework regions to maintain affinity. Presta et al, 1992, reported a successful construction of humanization by CDR grafting using a human antibody subgroup consensus sequence (consensus sequence) as a template. The humanization of antibodies by the method of surface remodeling (resurfacing) was reported by Pedersen et al 1994. Hsiao et al, 1994, reported a method of humanizing CDR grafting with the framework region of the Germline sequence of a human antibody. The humanization method was successfully constructed by Jeppers et al, 1994, using phage library (shuffling library).
There are generally two choices of human framework regions in antibody humanization, one is the known mature antibody and one is the human Germline sequence. Known mature antibody framework regions typically contain somatic mutation sites that can be potentially immunogenic. Compared with the mature antibody, the human Germline sequence framework region has lower immunogenicity in theory, more flexible structure, strong plasticity and easy acceptance of different CDR regions. The human antibody Germline gene has certain bias in use frequency in human body, and the humanized antibody with Germline skeleton region with high use frequency has the advantages of low immunogenicity, high expression level, stable structure, etc.
In a preferred embodiment of the present invention, the framework region of the preferred sequence is selected for humanization by a number of experimental screens, instead of selecting the Germline sequence with the highest similarity to the murine antibody, which is compatible with both similarity and frequency of human use. The humanized antibody constructed by adopting the method has more stable structure, high expression, low immunogenicity and higher patentability.
In another preferred embodiment, the heavy chain constant region and/or the light chain constant region may be a humanized heavy chain constant region or a light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a human IgG1, igG2, or the like heavy chain constant region or a human kappa, lambda light chain constant region.
In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence having a homology of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.
The antibodies of the invention may be double-or single-chain antibodies, and may preferably be fully humanized antibodies.
The antibody derivatives of the invention may be single chain antibodies, and/or antibody fragments, such as: fab, fab ', (Fab') 2, or other antibody derivatives known in the art, and the like, as well as IgA, igD, igE, igG and any one or more of IgM antibodies or antibodies of other subtypes.
The antibodies of the invention may be humanized antibodies, CDR grafted and/or modified antibodies targeting CLDN 18.2.
In the above-described aspect of the present invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total amino acids in the original amino acid sequence.
Preparation of antibodies
Any method suitable for producing monoclonal antibodies can be used to produce the CLDN18.2 antibodies of the invention. For example, animals may be immunized with the conjugated or naturally occurring CLDN18.2 protein or fragments thereof. Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.
Any suitable form of CLDN18.2 can be used as an immunogen (antigen) for generating non-human antibodies specific for CLDN18.2 and screening the antibodies for biological activity. The immunogens may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. The immunogen may be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic (e.g., cDNA) in origin. DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, baculoviral vectors, plasmids, and non-viral vectors.
The humanized antibody may be selected from any class of immunoglobulins, including IgM, igD, igG, igA and IgE. Also, any type of light chain may be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the present invention.
The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as amplification by PCR or screening of a genomic library. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.
The term "nucleic acid molecule" refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The term "host cell" refers to a cell into which an expression vector has been introduced. 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 plant or animal cells (e.g., mammalian cells).
The steps described herein for transforming a host cell with recombinant DNA may be performed using techniques well known in the art. The transformant obtained can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultivated in conventional medium under suitable conditions.
Typically, the transformed host cell is cultured under conditions suitable for expression of the antibodies of the invention. The antibodies of the invention are then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
Antibody formulations
Antibodies have different stability in different preparation buffers, and are characterized by changes in charge heterogeneity, degradation and polymerization of antibody molecules, and the like, and the changes in quality properties are related to physicochemical properties of the antibodies, so that in the process of developing antibody drugs, preparation buffers suitable for the antibodies need to be screened according to the physicochemical properties of different antibodies. The conventional antibody preparation buffer system comprises phosphate buffer, citric acid buffer, histidine buffer and the like, and can be added with different concentrations of salt ions or excipients such as sorbitol, trehalose, sucrose and the like according to the properties of the antibody, and a proper amount of surfactant such as tween and the like so as to maintain the stability of the antibody.
Pharmaceutical composition
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding CAR-T cell as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
The antibodies of the invention may also be used for cellular therapy where the nucleotide sequence is expressed intracellularly, e.g., for chimeric antigen receptor T cell immunotherapy (CAR-T), etc.
The pharmaceutical composition of the invention can be directly used for binding to CLDN18.2 protein molecules, and thus can be used for preventing and treating diseases related to CLDN 18.2. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the monoclonal antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
Where a pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Detection application and kit
The antibodies of the invention may be used in detection applications, for example for detecting samples, thereby providing diagnostic information.
In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of an organ.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit comprising an antibody (or fragment thereof) of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, etc. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
The main advantages of the invention include:
1. the invention is resistant to
The antibody is a typical tryptophan and tyrosine embedded good structure, and the screened human VH and VL framework region template has stable structure, is well matched with a mouse monoclonal antibody CDR region, can be well matched with a light and heavy chain variable region, and has high affinity and stable structure.
2. Compared with chimeric antibody, the humanized antibody has excellent bioactivity and specificity, and has lower immunogenicity and higher expression level while retaining the affinity equivalent to CLDN 18.2.
3. The humanized antibody has remarkable in-vitro biological activity, can effectively inhibit the proliferation activity of in-vitro cultured tumor cells, and has better activity than a control antibody.
4. The humanized antibody has remarkable in vivo biological activity, has excellent inhibitory activity on tumor cells such as human gastric cancer cells and the like, and has activity superior to that of a control antibody.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Unless otherwise specified, materials and reagents used in the examples of the present invention are commercially available products.
Materials and methods
1. Reference antibody
The reference antibody used in the experiments of the present invention was derived from the 175D10 cloned antibody sequence of patent US 8168427. The 175D10 antibody is also known as IMAB362. The variable region sequence of 175D10 antibody was synthesized through complete gene synthesis, and constructed onto eukaryotic expression vectors containing constant regions of IgG1 chimeric antibody, and expression purification was performed. The reference antibody thus prepared is abbreviated herein as Ref.ch175.
The reference antibody ref.ch175 (or IMAB 362) specifically recognizes the extracellular loop 1 portion of CLDN18.2, does not bind to CLDN18.1, and kills tumor cells primarily through antibody-dependent cell-dependent cellular-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (complement dependent cytotoxicity, CDC).
Example 1: construction of cell lines stably expressing CLDN18.1 or CLDN18.2
In this example, cell lines stably expressing human CLDN18.1, human CLDN18.2, mouse CLDN18.1, mouse CLDN18.2 were constructed. The method comprises the following steps:
full-length gene fragments encoding human CLDN18.1 (NP-057453.1) and human CLDN18.2 (NP-001002026.1) were cloned into pEGFP-N2 eukaryotic expression vectors (available from Clontech) by Xhol+BamHI cleavage, designated pEGFP-N2-hCDN 18.1 and pEGFP-N2-hCDN 18.2, respectively.
HEK293 cells (from the national academy of sciences cell Bank, GNHu 43) were used at 4X 10 5 The cells were inoculated into 6-well plates, and on the next day, pEGFP-N2-hcldn18.1 and pEGFP-N2-hcldn18.2 were transfected into HEK293 cells respectively using LipoMax, and after 24 hours of transfection, the cells were passaged at a ratio of 1:10, and after cell attachment, were subjected to pressure screening at a final concentration of 300 μg/ml by adding G418 (purchased from Gibco), and subcloned to obtain HEK293 cells stably expressing human CLDN18.1 and human CLDN18.2 membrane proteins, designated HEK293-hcldn18.1 and HEK293-hcldn18.2, respectively.
Full-length gene fragments encoding mouse CLDN18.1 (NP-062789.1) and mouse CLDN18.2 (NP-001181850.1) were cloned into pcDNA3.1-P2A-EGFP eukaryotic expression vectors (available from Nanjing Style Corp) by KpnI+XbaI cleavage, designated pcDNA3.1-mCLDN18.1 and pcDNA3.1-mCLDN18.1, respectively. HEK293 cells were transfected by the same method as above to obtain cells stably expressing mouse CLDN18.1 and CLDN18.2 membrane proteins, designated HEK293-mCLDN18.1 and HEK293-mCLDN18.2, respectively.
>NP_057453.1claudin-18isoform 1precursor[Homo sapiens]SEQ ID No:25
MSTTTCQVVAFLLSILGLAGCIAATGMDMWSTQDLYDNPVTSVFQYEGLWRSCVRQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV
>NP_001002026.1claudin-18isoform 2[Homo sapiens]SEQ ID No:26
MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV
>NP_062789.1claudin-18isoform A1.1 precursor[Mus musculus]SEQ ID No:27
MATTTCQVVGLLLSLLGLAGCIAATGMDMWSTQDLYDNPVTAVFQYEGLWRSCVQQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVLGVIGILVSIFALKCIRIGSMDDSAKAKMTLTSGILFIISGICAIIGVSVFANMLVTNFWMSTANMYSGMGGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLTPDDSNFKAVSYHASGQNVAYRPGGFKASTGFGSNTRNKKIYDGGARTEDDEQSHPTKYDYV
>NP_001181850.1claudin-18isoform A2.1[Mus musculus]SEQ ID No:28
MSVTACQGLGFVVSLIGFAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGVIGILVSIFALKCIRIGSMDDSAKAKMTLTSGILFIISGICAIIGVSVFANMLVTNFWMSTANMYSGMGGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLTPDDSNFKAVSYHASGQNVAYRPGGFKASTGFGSNTRNKKIYDGGARTEDDEQSHPTKYDYV
Example 2: immunogen preparation
Since human CLDN18.2 extracellular loop 1 (CLDN 18.2 ECL 1) is identical to the amino acid sequence of mouse CLDN18.2 ECL1, it is more difficult to obtain antibodies that bind to CLDN18.2 ECL 1. Meanwhile, considering that the CLDN18.2 molecular conformation on the surface of tumor cells may be different from that on the surface of normal tissue cells, in this embodiment, various forms of DNA plasmids are designed as antigens to break through immune tolerance, so as to obtain antibodies recognizing tumor cells with high affinity.
The nucleotides encoding the full length amino acids of human CLDN18.2ECL1 and CLDN18.2 were cloned in tandem with the tetanus toxoid P2 epitopes TT830-844, P30 epitopes TT947-967, respectively, into eukaryotic expression vector PTT5 (available from Invitrogen company) designated PTT5-TT-hcldn18.2, PTT5-TT-hcldn18.2-ECL1, respectively.
The invention fuses CLDN18.2ECL1 to the Fc-C end of human IgG1, named hFc-CLDN18.2-ECL1.
TT-hCDNN18.2 nucleotide sequence SEQ ID No. 29
CAGTACATCAAGGCCAACAGCAAGTTCATCGGCATCACCGAGCTGAAGAAGCTGGGTGGTTCCAACGACATATTCAACAACTTCACCGTGAGCTTCTGGCTGCGCGTGCCGAAGGTGAGCGCCAGCCACCTGGAGCAGTACGGTGGCGGTAGCGGAATGGCCGTGACTGCCTGTCAGGGCTTGGGGTTCGTGGTTTCACTGATTGGGATTGCGGGCATCATTGCTGCCACCTGCATGGACCAGTGGAGCACCCAAGACTTGTACAACAACCCCGTAACAGCTGTTTTCAACTACCAGGGGCTGTGGCGCTCCTGTGTCCGAGAGAGCTCTGGCTTCACCGAGTGCCGGGGCTACTTCACCCTGCTGGGGCTGCCAGCCATGCTGCAGGCAGTGCGAGCCCTGATGATCGTAGGCATCGTCCTGGGTGCCATTGGCCTCCTGGTATCCATCTTTGCCCTGAAATGCATCCGCATTGGCAGCATGGAGGACTCTGCCAAAGCCAACATGACACTGACCTCCGGGATCATGTTCATTGTCTCAGGTCTTTGTGCAATTGCTGGAGTGTCTGTGTTTGCCAACATGCTGGTGACTAACTTCTGGATGTCCACAGCTAACATGTACACCGGCATGGGTGGGATGGTGCAGACTGTTCAGACCAGGTACACATTTGGTGCGGCTCTGTTCGTGGGCTGGGTCGCTGGAGGCCTCACACTAATTGGGGGTGTGATGATGTGCATCGCCTGCCGGGGCCTGGCACCAGAAGAAACCAACTACAAAGCCGTTTCTTATCATGCCTCAGGCCACAGTGTTGCCTACAAGCCTGGAGGCTTCAAGGCCAGCACTGGCTTTGGGTCCAACACCAAAAACAAGAAGATATACGATGGAGGTGCCCGCACAGAGGACGAGGTACAATCTTATCCTTCCAAGCACGACTATGTGTAA
TT-hCDN18.2-ECL 1 nucleotide sequence: SEQ ID No. 30
CAGTACATCAAGGCCAACAGCAAGTTCATCGGCATCACCGAGCTGAAGAAGCTGGGTGGTTCCAACGACATATTCAACAACTTCACCGTGAGCTTCTGGCTGCGCGTGCCGAAGGTGAGCGCCAGCCACCTGGAGCAGTACGGTGGCGGTAGCGGACGCATGGACCAGTGGAGCACCCAAGACTTGTACAACAACCCCGTAACAGCTGTTTTCAACTACCAGGGGCTGTGGCGCTCCTGTGTCCGAGAGAGCTCTGGCTTCACCGAGTGCCGGGGCTACTTCACCCTGCTGGGGCTGCCAGCCATGCTGCAGGCAGTGCGAGCCTAA
hFc-CLDN18.2-ECL1 nucleotide sequence: SEQ ID No. 31
GACAAGACCCACACATGCCCACCTTGTCCAGCTCCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCCAAGGATACACTGATGATCTCTAGGACCCCCGAGGTGACATGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCTAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTATCGGGTGGTGTCTGTGCTGACAGTGCTGCATCAGGATTGGCTGAACGGCAAGGAGTATAAGTGCAAGGTGAGCAATAAGGCCCTGCCTGCTCCAATCGAGAAGACCATCTCTAAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTACACACTGCCTCCATCCCGCGAGGAGATGACCAAGAACCAGGTGAGCCTGACATGTCTGGTGAAGGGCTTCTATCCTTCTGACATCGCTGTGGAGTGGGAGTCCAATGGCCAGCCAGAGAACAATTACAAGACCACACCCCCTGTGCTGGACAGCGATGGCTCTTTCTTTCTGTATTCCAAGCTGACCGTGGATAAGAGCAGGTGGCAGCAGGGCAACGTGTTTTCCTGTAGCGTGATGCACGAGGCCCTGCACAATCATTACACACAGAAGTCTCTGTCCCTGAGCCCTGGCAAGGGATCCGGAGGAGGCTCTGGAGGCTCCGACCAGTGGAGCACCCAGGATCTGTACAACAATCCCGTGACAGCCGTGTTCAACTATCAGGGCCTGTGGAGGTCTTGCGTGCGGGAGTCCAGCGGCTTCACCGAGTGTAGAGGCTACTTTACACTGCTGGGACTGCCTGCTATGCTGCAGGCTGTGCGCTGA
Example 3: preparation of anti-human CLDN18.2 hybridoma cell strain
Balb/c mice were purchased from Shanghai Sipuler-BiKai laboratory and all immunized mice were 4-6 week old female, standardized, disease-free, healthy, pure mice.
Since CLDN18.2 is a 4-pass transmembrane protein, in order to obtain specific antibodies that recognize tumor cells with high affinity, mouse immunization was performed by alternate immunization with various plasmids and impact immunization with cells stably expressing human CLDN18.2 membrane protein. The method comprises the following steps:
on days 1 and 14: 50 μg of eukaryotic expression vector plasmid pEGFP-N2-hCDN 18.2 encoding the full length gene of human CLDN18.2 was injected into the mouse quadriceps muscle (i.m) with the adjuvant In vivo transfection reagent In vivo In Jet PEI-Man (purchased to Polyplus, 203-10G). 50 μg of eukaryotic expression plasmid hFc-CLDN18.2-ECL1 DNA was injected into the mouse quadriceps on day 28 (i.m); 50 μg of eukaryotic expression vector plasmid PTT 5-TT-hCDN18.2 was injected into mouse quadriceps on day 42 (i.m); 50 μg of eukaryotic expression vector plasmid PTT5-TT-CLDN18.2ECL1 was injected into mouse quadriceps on day 56 (i.m); 50. Mu.g of eukaryotic expression vector plasmid PTT 5-TT-hCDN 18.2 and 50. Mu.g of eukaryotic expression vector plasmid PTT5-TT-CLDN18.2ECL1 were injected into the mouse quadriceps (i.m) on day 70.
The mice were bled from their tail vein after 14 days, serum antibody titers were determined using the cell based-elisa method, 2X 10 days prior to fusion 7 HEK 293-hCDN18.2 cells/mouse were intraperitoneally injected with the 2 mice with the highest titers of boosting antibodies. Fusion on the same day, mice eyeballs were bled and spleens were removed, single cell suspensions were prepared, and hybridoma plates were obtained by electrofusion with SP20 cells.
Figure BDA0003349820360000211
Example 4: screening of hybridoma cells that specifically bind to human CLDN18.2 but not to human CLDN18.1
In this example, the screening of hybridomas specifically bound to human CLDN18.2 but not to human CLDN18.1 hybridoma cells was performed using a cell based-ELISA method. The method comprises the following steps:
HEK 293-hCDN18.1 cells and HEK 293-hCDN18.2 cells were digested with pancreatin and then subjected to 3X 10 treatment 5 Cell densities of individual/mL were inoculated on polylysine (P4707, sigma) coated 96-well plates, 200 μl/well, respectively, and cultured overnight; removing the supernatant when the cell confluence reaches more than 90%, and fixing for 15min at room temperature by using 10% neutral formalin solution; after 3 washes with PBS, the cells were blocked with 5% skim milk overnight at 4 ℃. When detecting hybridoma, adding the supernatant of the same well onto HEK 293-hCDN18.1 cell and HEK 293-hCDN18.2 cell coating plate, adding 80 μl/well, incubating at 37deg.C for 1H, washing with PBST 3 times, adding 100 μl/well 1:5000 diluted goat anti-mouse IgG (H+L) -HRP, incubating at 37deg.C for 45min, and developing color for 30min Into 50 μl/well 2M H 2 SO 4 The color development was terminated and the microplate reader read OD450.
Through screening ten thousands of clones, the most obtained multiple hCDN18.2 specific antibodies, and the optimal 4 strains are shown in the following table:
Figure BDA0003349820360000221
example 5: murine monoclonal antibody subtype detection
Subtype detection was performed on clones specifically binding to CLDN18.2 using subtype detection kit (SBA Clonotyping System-HRP kit, available from southern biotech, 5300-05).
The detection results are as follows:
clone numbering Heavy chain subtype Light chain subclass
mab429 IgM Kappa
mab623 IgM Kappa
mab624 IgG2b Kappa
mab782 IgG2a Kappa
Example 6: cloning and identification of variable region coding sequence of anti-human CLDN18.2 murine monoclonal antibody
Since IgM subtype antibodies exist in the screened mouse monoclonal antibodies, for further evaluation of the activity, the variable region sequences of the mouse monoclonal antibodies are obtained by using a RACE-PCR method, and chimeric antibodies are constructed.
The total mRNA of the hybridoma obtained in example 4 was extracted using RNA extraction kit TakaRa MiniBEST Universal RNA Extration Kit (Takara, 9767), and the murine antibody variable region sequence was obtained by PCR using SMART RACE 5'/3' kit (Clontech, 634858), as follows:
624 antibody heavy chain variable region amino acid sequence (SEQ ID No: 23):
QVQLQQPGAELVRPGASVKLSCKASGYSFTSYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARTSYGNSFAYWGQGTLVTVSA
624 antibody light chain variable region amino acid sequence (SEQ ID No: 24):
DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIK
782 antibody heavy chain variable region nucleotide sequence (SEQ ID No: 2):
GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTTATAGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACACTACAGTGATAACACGAACTACAGCCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGGATCTACTATGGTAACTCGTTTGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA
782 antibody heavy chain variable region amino acid sequence (SEQ ID No: 1):
DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYIHYSDNTNYSPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARIYYGNSFVYWGQGTLVTVSA
782 antibody light chain variable region nucleotide sequence (SEQ ID No: 7):
GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGACAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAACAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATTTTTATCCGTACACGTTCGGGGGGGGGACCAAGCTGGAAATAAAA
782 antibody light chain variable region amino acid sequence (SEQ ID No: 6):
DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCQNDYFYPYTFGGGTKLEIK
example 7: construction and expression of anti-CLDN 18.2 human murine chimeric antibodies
Primers were designed according to the primer design principle of the homologous recombination method, and the murine antibody heavy and light chain variable region gene fragments obtained in example 6 were amplified, respectively linked to human heavy and light chain constant region sequences, and constructed into an expression vector PTT5 (purchased from Invitrogen).
Plasmid extraction is carried out by using a commercial plasmid large extraction kit, 293FV suspension cells are transfected by using a PEI transfection method, the supernatant is collected after 7 days of transfection, and chimeric antibodies are obtained by using protein A affinity chromatography.
Example 8: anti-CLDN 18.2 human murine chimeric antibody specificity assay
After digestion of HEK293 cells stably transfected with human CLDN18.2 and HEK293 cells stably transfected with human CLDN18.1 with pancreatin, respectively, 5X 10 cells were prepared 5 Mu.l of each cell/100. Mu.l of hybridoma supernatant was added, incubated on ice for 1 hour, washed 2 times with FACS buffer, diluted 1:400 goat anti-mouse IgG-PE secondary antibody was added, incubated on ice for 45min, washed 2 times with FACS buffer, added 300. Mu.l of loading buffer, transferred to BD flow tubes and analyzed by BD FACS calibur. The average fluorescence intensity MFI (MedianFluorescence Intensity) was analyzed using Flowjo software.
The results are shown in FIG. 2 and the following table.
Antibody numbering HEK293-hCLDN18.2 HEK293-hCLDN18.1 P/N
ch429 1219 2.26 539.4
ch623 235 2.05 114.6
ch624 1420 2.27 625.6
ch782 4179 2.86 1461.2
Ref.ch175 2618 2.48 1055.6
Note that: P/N is the MFI of the antibody binding to HEK 293-hCDN 18.2 cells divided by the MFI of HEK 293-hCDN 18.1 cells binding to them, indicating their specificity.
The results showed that ch429, ch623, ch624, and ch782 specifically bound to human CLDN18.2 and did not bind to human CLDN 18.1. Wherein the MFI of chimeric antibody ch782 is higher than that of reference antibody ref.ch175.
Example 9: anti-CLDN 18.2 human mouse chimeric antibody and recombinant cell binding activity detection
HEK 293-hCDN18.1 and HEK 293-hCDN18.2 cells are inoculated on a 96-well plate coated with polylysine (P4707, sigma) according to a certain cell density after being digested by pancreatin, and are cultured overnight; removing the supernatant when the cell confluence reaches more than 90%, and fixing the supernatant for 15min at room temperature by using 10% formalin solution; after one wash with PBS, the mixture was blocked with 5% skim milk overnight at 4 ℃. The candidate chimeric antibody and the reference antibody Ref.ch175 were diluted 3-fold from 1. Mu.g/ml to 10 gradients, 100. Mu.l/well was added to the cell-coated 96-well plate, incubated at 37℃for 1 hour, washed 3 times with PBST, added with 1:2000 dilution of murine anti-human IgG Fc-HRP, incubated at 37℃for 1 hour, washed 3 times with PBST, developed with TMB developing solution for 15 minutes, developed with 2M H 2 SO 4 And (5) terminating. OD450-OD630 values were read and the EC50 was calculated by fitting with GraphPad Prism 8 software. The results are shown in FIG. 3, which shows that none of ch429, ch623, ch624, ch782 and the reference antibody Ref.ch175 bind to HEK 293-hCDN18.1 cells, specifically to HEK293-HCLDN18.2 cells, and that the relative binding activities of ch429 and ch782 relative to the reference antibody Ref.ch175 were 257.5% and 179.9%, respectively. The results are shown in the following table:
antibody numbering EC50(ng/ml) Relative binding Activity
ch429 12.06 257.5％
ch623 91.81 33.8％
ch624 39.17 79.3％
ch782 17.26 179.9％
Ref.ch175 31.05 100.0％
Example 10: anti-CLDN 18.2 human murine chimeric antibodies and NUGC4 cell binding activity assays
NUGC4 cells (purchased from Nanjing Bai Biotechnology Co., ltd.) were plated onto 96-well plates by the same method as in example 9, candidate chimeric antibody and reference antibody Ref.ch175 were diluted 3-fold from 100. Mu.g/ml to 10 gradients, 100. Mu.l/well was added to the cell-coated 96-well plates, incubated at 37℃for 1 hour, washed 3 times with PBST, 1:2000 diluted murine anti-human IgG Fc-HRP, incubated at 37℃for 1 hour, washed 3 times with PBST, developed for 15 minutes with TMB developing solution, and developed with 2M H 2 SO 4 And (5) terminating. OD450-OD630 values were read and the EC50 was calculated by fitting with GraphPad Prism 8 software.
The results are shown in FIG. 4 and the following table.
Antibody numbering EC50(μg/ml)
ch429 5.25
ch623 -
ch624 -
ch782 0.57
Ref.ch175 -
EC50 of chimeric antibodies ch782 and ch429 were 0.57 μg/ml and 5.25 μg/ml, respectively, and the read-out value of ch782 at high concentration (od450=2.3) was higher than that of found ch429 (od450=1.2); while none of ch623, ch624 and reference antibody ref.ch175 calculated EC50.
This indicates that the binding activity of the ch782 antibody to NUGC4 cells endogenously expressing the human CLDN18.2 protein is best, significantly stronger than the reference antibody ref.
Example 11: detection of HEK 293-hCDN18.2 cell binding Activity of anti-CLDN 18.2 human mouse chimeric antibody
The chimeric antibodies ch429, ch624, ch782 and Ref.ch175 were subjected to 3-fold gradient dilution from 10. Mu.g/ml concentration in accordance with the method of example 8, and a flow-through experiment was performed. EC50 was calculated by fitting a graph with MFI as ordinate using GraphPad Prism8 software.
The results are shown in FIG. 5 and the following table.
Antibody numbering EC50(μg/ml)
ch429 0.52
ch624 -
ch782 0.81
Ref.ch175 0.85
The chimeric antibodies ch429, ch782 and the reference antibody Ref.ch175 can be combined with HEK 293-hCDN18.2 cells, and the EC50 is respectively 0.52 mug/ml, 0.81 mug/ml and 0.85 mug/ml, but the combination activity of ch624 is obviously weaker.
Furthermore, at high concentrations the MFI of ch782 binding to HEK 293-hCDN 18.2 cells is higher than Ref. Ch175.
Example 12: reporter gene detection of ADCC activity of anti-CLDN 18.2 antibody human-mouse chimeric antibody on HEK 293-hCLDIN 18.2 cells
Antibody Fc-terminal mediated ADCC effect is an important indicator for evaluating anti-tumor activity of antibodies. ADCC is an antibody-dependent cell-mediated cytotoxicity whose mechanism of action is that after binding of an antibody to a tumor surface antigen, the Fc region of the antibody binds to immune effector cell receptors, primarily Fc receptor-targeted recognition on NK cells, to directly lyse and kill tumor cells.
In the embodiment, a reporter gene method is adopted to detect the ADCC biological activity of the antibody, engineering Jurkat cells which stably express the FcgammaRIIIa receptor are selected as effector cells, a Bio-GloTM Luciferase Assay System kit is selected to detect the chemiluminescent signal of luciferase, and the function of the Fc effector of the antibody is quantitatively detected by measuring the activation of an NFAT signal path.
HEK 293-hCDN18.2 cells constructed in example 1 were digested and then subjected to 2.5X10-th 5 Diluting with/ml, spreading 100 μl/well into a white opaque cell culture plate, standing at 37deg.C for 24 hr, removing supernatant, and adding 25 μl/well of analysis medium containing 0.1% BSA; dilution of Jurkat/NFAT-luc+FcgammaRIIIa cells to 6X 10 6 Mu.l of Jurkat/NFAT-luc+FcgammaRIIIa cell suspension was added to each well, and then 3-fold serial dilutions were made starting at a final concentration of 6. Mu.g/mL by adding 25. Mu.l of antibody dilution to each well, for a total of 10 dilution gradients; placing the cell plates into a constant temperature incubator, culturing for 6h under induction, balancing the 96-well plates at room temperature for at least 15min, adding 75 mu LBio-GloTM Luciferase Assay Reagent (the staining solution needs to be protected from light) into each of all the reaction wells by using a continuous sample adding gun, reacting for 5min at room temperature, and detecting the value of the Lumineancence optical signal by using a multifunctional enzyme-labeling instrument. The fluorescence values should be plotted against antibody concentration using GraphPad Prism 8 software fit to calculate EC50 values.
The results are shown in FIG. 6 and the following table.
Antibody numbering EC50(ng/ml)
ch782 47.64
Ref.ch175 36.46
The results showed that, after gradient dilution of ch782 with reference antibody ref.ch175, the target ratio was effective e:t=6: 1, after incubation for 6h at 37 ℃, it was found that both ch782 and ref.ch175 induced stronger ADCC activity.
Example 13: humanization of anti-CLDN 18.2 antibody ch782
In order to reduce the immunogenicity of the antibody and reduce the effect of the antibody on human anti-mouse antibodies (HAMA) to be generated after in vivo, in this example, the preferred antibody ch782 was humanized. Specifically, humanized sequences were designed for the mab782 antibody variable region sequence of example 6. Coding heavy chain amino acid sequences according to a kabat coding system, carrying out sequence homology comparison through NCBIIgblast (https:// www.ncbi.nlm.nih.gov/igblast /), selecting a human antibody sequence with higher homology as a framework, carrying out murine antibody CDR transplantation, and carrying out back mutation on part of core amino acids of a framework region.
Blast search 782 antibody heavy chain and human germline homologous sequence:
IGHV4-38-2*02 71.1％(69/97)
IGHV4-61*01 71.4％(70/98)
IGHV4-61*08 71.4％(70/98)
blast search 782 antibody light chain and human germline homology sequence:
IGKV4-1*01 79.2％(80/101)
IGKV3-7*02 65.3％(66/101)
IGKV3-7*04 65.3％(66/101)
782 antibody the FR and CDR sequences in the heavy chain variable region are defined according to kabat as follows:
Figure BDA0003349820360000271
782 antibody the FR and CDR sequences in the light chain variable region are defined as follows according to kabat:
Figure BDA0003349820360000272
In the invention, IGHV4-61 with higher antibody heavy chain humanization selection homology is used as a template for CDR region replacement, a CDR1 site defined by Chothia is simultaneously included during heavy chain CDR1 transplantation, and I48M/V67I/V71R3 sites are subjected to back mutation to obtain hu782H1.1.
The S60 site is found to have potential glycosylation sites through post-translational modification analysis, is mutated into S60A by a point mutation mode, is named hu7825H 1.2, and is mutated into A at the 101 st amino acid V so as to optimize the expression quantity, and is named hu782H 1.2-V101A
In the invention, the humanized antibody light chain is respectively replaced by CDR regions by taking V kappa I consensus sequence as a template to obtain hu782L1; CDR region replacement is carried out by taking IGKV4-1 as a template to obtain hu782L2
After the above sequences are optimized by codons, complete gene synthesis is carried out, and the complete gene synthesis is cloned to an expression vector containing a heavy and light chain constant region for expression purification.
hu782H1.1(SEQ ID No:11)
QVQLQESGPGLVKPSETLSLTCTVSGYSITSGYSWHWIRQPPGKGLEWMGYIHYSDNTNYSPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARIYYGNSFVYWGQGTLVTVSS
hu782H1.2(SEQ ID No:12)
QVQLQESGPGLVKPSETLSLTCTVSGYSITSGYSWHWIRQPPGKGLEWMGYIHYSDNTNY
Figure BDA0003349820360000282
PSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARIYYGNSFVYWGQGTLVTVSS
hu782H1.2-V101A(SEQ ID No:13)
QVQLQESGPGLVKPSETLSLTCTVSGYSITSGYSWHWIRQPPGKGLEWMGYIHYSDNTNYAPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARIYYGNSF
Figure BDA0003349820360000283
YWGQGTLVTVSS
hu782L1(SEQ ID No:14)
DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNDYFYPYTFGQGTKVEIK
hu782L2(SEQ ID No:15)
DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPLLIYWASTRESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQNDYFYPYTFGGGTKLEIK
Example 14: detection of specific binding activity of humanized antibody to CLDN18.2
The binding activity of the humanized antibodies to human CLDN18.1 and CLDN18.2, mouse CLDN18.1 and CLDN18.2, and NUGC4 cells was examined using the chimeric antibody binding activity evaluation method described in the above examples.
The results are shown in FIG. 7 and the following table.
Figure BDA0003349820360000281
Figure BDA0003349820360000291
The results showed that both humanized antibodies were able to bind specifically to human CLDN18.2 and that no significant decrease in binding activity was seen compared to chimeric antibody ch782.
Furthermore, unexpectedly, the EC50 value of the binding of the 4 humanized antibodies to the cells of tumor cell NUGC4 was significantly better than that of chimeric antibody ch782.
Example 15: humanized antibody ADCC Activity detection
One of the main mechanisms of in vivo activity of the antibody of the present invention is ADCC functional activity, and a human IgG1 antibody (hIgG 1) binds to fcγr mainly through the antibody hinge region and CH2 region, thereby exerting ADCC effect. In order to enhance the ADCC activity of the humanized antibody, in this example, the S239D/I332E (DE) mutation was introduced into the Fc of the humanized antibody heavy chain hu7825H 1.2 to enhance its affinity for FcgammaR, thereby achieving the objective of ADCC enhancement. After expression and purification, ADCC of wild-type and DE mutant human antibodies was evaluated by reporter gene method.
Figure BDA0003349820360000292
The method comprises the following steps: following the specific procedure in example 12, target cells HEK 293-hCDN18.2 and NUGC4 were each grown according to 2.5X10 5 Diluting with/ml, spreading 100 μl/well into a white opaque cell culture plate, standing at 37deg.C for 24 hr, removing supernatant, and adding 25 μl/well of analysis medium containing 0.1% BSA; dilution of Jurkat/NFAT-luc+FcgammaRIIIa cells to 6X 10 6 The 96-well plate was removed at each well by adding 25. Mu.L of Jurkat/NFAT-luc+FcgammaRIIIa cell suspension, and then adding 25. Mu.L of antibody dilution to each well to give a final concentration of 6. Mu.g/mL for 3-fold serial dilutions, for a total of 10 dilution gradients; placing the cell plate into a constant temperature incubator, culturing for 6 hr under induction, balancing the 96-well plate at room temperature for at least 15min, and adding 75 μl of Bio-GloTM Luciferase Assay R to each of all the reaction wells using a continuous sample adding gunThe stain is required to be protected from light, and reacts for 5min at room temperature, and the stain is placed in a multifunctional enzyme-labeled instrument to detect the value of the Luminescence optical signal. The fluorescence values should be plotted against antibody concentration using GraphPad Prism 8 software fit to calculate EC50 values.
Figure BDA0003349820360000301
* nd: no detection was performed; * NA: the signal is weak and is not calculated.
The results showed that, for recombinant cells highly expressing human CLDN18.2, both chimeric antibody ch782 and humanized antibodies hu782, hu782-HV were effective in generating ADCC activity comparable to that of the reference antibody (EC 50: 47.64ng/ml, 64.03ng/ml, 70.94ng/ml, respectively), and that the ADCC activity of hu782-DE mutated in Fc primer S239D/I332E was significantly improved (FIG. 8).
The same results were also observed on NUGC4 cells endogenously expressing human CLDN18.2, and ADCC activity on NUGC4 cells by the candidate antibodies ch782 and hu782 obtained in the present invention was superior to that of the reference antibody ref.ch175.
Next, ADCC activity was examined using NUGC4 cells stably transformed with hCLDIN 18.2 as target cells, and the results showed that the ADCC activities of hu782-DE and hu782-HV-DE were significantly superior to those of the control antibody Ref. Ch175, with EC50 values of 6.87ng/ml and 11.48ng/ml, respectively (FIG. 8C).
Example 16: humanized antibody CDC Activity assay
CDC is a complement dependent cell-mediated cytotoxic effect whose mechanism of action is that a mixture of antigen and antibody that bind complement eventually perforates the cell membrane, altering the osmotic pressure of the cell, ultimately leading to cell lysis. In this example, HEK 293-hCDN 18.2 cells were used as target cells, baby rabbit complement serum was used as a supplement, and CCK-8 cell proliferation-toxicity assay kit was used to detect antibody CDC effects.
HEK 293-hCDN18.2 cells of example 1 were used as target cells and diluted to 2.0X10 after digestion 5 The cells are paved in 96-hole transparent blackbody culture plates at the volume of 100 mu L per hole per mL, and the cells are subjected to static culture for about 4 hours until the cells are completely attached. Will growThe medium was blotted dry and 50 μl of assay medium was added to each well. 50 mu L of diluted antibody is added into each hole of the reaction hole, 50 mu L of young rabbit complement diluted according to the ratio of 1:15 is added, the final volume of each hole is 150 mu L, the volume of each hole sample is 1/3 of the total volume, the initial concentration is 3 times of the target concentration, and the target concentration is 6 mu g/mL. Placing the cell plate into a constant temperature incubator, performing induced culture for 12h, sucking and removing the culture medium, adding 100 μl of fresh analysis culture medium into each well, adding 10 μl of CCK-8 solution into each well at room temperature, and placing in 5% CO 2 The incubator reacts for 4 hours at 37 ℃ in the dark. The OD value is detected by a multifunctional enzyme labeling instrument with the wavelength of 450 nm. EC50 values were calculated using GraphPad Prism 8 software fit mapping.
The results are shown in the following table,
Figure BDA0003349820360000311
the results show that the CDC effect (EC 50 is 30.9ng/ml, 49.46ng/ml, 47.58ng/ml and 40.89 ng/ml) equivalent to that of the reference antibody Ref.ch175 can be induced on HEK 293-hCDN18.2 cells by ch782, hu782 and hu782HV in the invention.
EXAMPLE 17 detection of the inhibition of proliferation of HEK 293-hCDN18.2 cells by humanized antibodies
HEK 293-hCLDIN 18.2 cells in logarithmic growth phase were taken. Target cells were digested, counted and diluted to 1.0X10 cells 5 Each mL was plated in 96-well clear black-body plates at a volume of 100. Mu.L per well; and (3) standing and culturing for about 4 hours until the cells are completely attached. 100. Mu.L of diluted antibody was added to each well of the reaction well, the final volume of each well was 200. Mu.L, and the 96-well plate was placed in 5% CO 2 Incubator, culture induced at 37deg.C for 96 hr, absorbing and removing culture medium, adding 100 μl fresh analysis culture medium per well, adding 10 μl CCK-8 solution per well at room temperature, and placing in 5% CO 2 The incubator reacts for 4 hours at 37 ℃ in the dark. The OD value is detected by a multifunctional enzyme labeling instrument with the wavelength of 450 nm. Results calculation of cell proliferation inhibition ratio = 1- (OD 450) Antibodies to /OD450 Blank space )。
The result shows that the proliferation inhibition effect of the antibody hu782 of the invention on target cells at low concentration (1 mug/ml) is better than that of a reference antibody Ref.ch175 (18.3%vs. 7.3%) at high concentration (30 mug/ml), and the proliferation inhibition effect is enhanced to show a dose-dependent effect along with the increase of the concentration of the antibody, and the cell proliferation inhibition rates of ch782, hu782 and hu782-HV reach 67.8%, 76% and 63.2% respectively at the concentration of 30 mug/ml; significantly better than the reference antibody ref.
EXAMPLE 18 humanized antibody binding site analysis to hCDN18.2 protein
To investigate the fact that the antibodies of the invention bind to the hcldn18.2 protein as a key amino acid site not binding to the hcldn18.1 protein, in this example, the amino acid difference site between hcldn18.2 ECL1 and hcldn18.1 ECL1 and its nearby amino acids were subjected to alanine mutation on the pEFGP-N2-hcldn18.2 plasmid constructed in example 1 by means of point mutation, wherein a42 was mutated to the allelic amino acid S in hcldn18.1 ECL1 (Q29A, S31A, N37A, A42S, N45A, Q47A, G48A, S52A, E A, G A, L A, G a). The plasmids with successful mutation and wild type plasmids were transfected into 293Fv cells, respectively, as described in example 1, and after 48 hours the cells were taken for flow detection.
Hu782, ref.ch175 and isotype control (Isotypectrol hIgG 1) were diluted to 10. Mu.g/ml and incubated on ice with transfected cells above for 1 hour, FACS buffer washed 2 times, goat anti-mouse IgG-PE secondary antibody diluted 1:400 was added, incubated on ice for 45min, FACS buffer washed 2 times, 300. Mu.l of loading buffer was added, transferred to BD flow tubes and analyzed by BD FACS calibur. The average fluorescence intensity MFI (Median Fluorescence Intensity) was analyzed using Flowjo software and the results determined that this site was considered to be the key amino acid site for affecting antibody binding when the MFI of transfected cells after mutation was reduced by more than 50% relative to wild type.
As shown in FIG. 11, the effect of the A42S, N45A, E A mutation on the binding of the reference antibody Ref.ch175 is larger, while the effect of the A42S mutation on the binding of hu782 is not larger, the effect of the N45A mutation on the binding of hu782 is smaller, and the effect of the E56A mutation on the binding of hu782 is larger. Meanwhile, according to the invention, the fact that the reference antibodies Ref.ch175 and hu782 can not be combined after the mutation of the same amino acid locus G48A of hCDN18.2ECL 1 and hCDN18.1ECL 1 is unexpectedly discovered.
EXAMPLE 19 evaluation of humanized antibody Activity in vivo
In this example, BALB/c nude mice were subcutaneously transplanted with tumor cells to establish CDX animal models, and in vivo efficacy evaluation of humanized antibodies was assessed.
NUGC4-hcldn18.2 stable transgenic cell lines were constructed as described in example 1 for expansion culture. Selecting 4-6 female SPF-grade BALB/c nude mice, adaptively raising the nude mice for 3-4 days after 4-6 weeks of age, and preparing seed tumors. Recombinant NUGC 4-hCLDIN 18.2 cells in logarithmic growth phase are digested, washed 2 times with serum-free 1640 medium and diluted to 2.5X10 7 Per ml, mice were inoculated subcutaneously with 0.2ml for a total of 5X 10 in the right armpit 6 Individual cells/individual. Tumors grow to 70-100 mm after 1 week of inoculation 3 Random granule design grouping was performed with 6 groups each. The blank model group was an IgG1 isotype control. The experimental group was dosed at 10mg/kg twice a week, with intraperitoneal injection and intravenous injection alternating, and tumor volume and mouse weight data were recorded before each dose.
Tumor volume calculation: v (mm) 3 )＝(W 2 xL)/2, w is the tumor proximal midline short diameter, L is the tumor proximal midline long diameter.
When the tumor volume is larger than 2000mm 3 Mice were sacrificed by cervical dislocation when weight loss was greater than 20% or interference with important physiology or tumor ulcer necrosis was observed.
The tumor inhibition (TGI) was calculated as follows:
Figure BDA0003349820360000321
vt-v0= (average tumor volume on day t of the dosing group) - (average tumor volume on day of grouping for this group)
Vct-vc0= (average tumor volume on day t of administration group) - (average tumor volume on day of grouping for the group)
In NUGC4-hcldn18.2 mice subcutaneously transplanted tumor model, it was observed that the humanized antibody of the present invention and its mutant showed a certain efficacy against tumor growth activity. At endpoint, tumor volumes for each group are shown in the following table.
Figure BDA0003349820360000331
* CR, number of animals with complete tumor regression/total number of animals in the group
The tumor inhibition rates of the humanized antibody hu782 and mutants hu782-HV and hu782-DE are obviously higher than that of the Ref.ch175 group, and especially the tumor inhibition rates of the hu782 and ADCC enhanced mutant antibody hu782-DE of the humanized antibody are respectively 62.88% and 65.05%. In addition, complete tumor regression was observed in 1 mouse in 6 mice in the hu782 group, and in 2 mice in the hu782-DE group, but not in the Ref.ch175 combination hu782-HV group.
The results show that the in vivo efficacy effect of the antibodies (particularly the humanized antibodies) of the invention is significantly better than that of the reference antibody Ref.ch175.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
<110> Shanghai biological products research all Limited company
<120> anti-CLDN 18.2 monoclonal antibody and use thereof
<130> P2021-2047
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Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 19
<211> 220
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Phe Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210> 20
<211> 448
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly
20 25 30
Tyr Ser Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile His Tyr Ser Asp Asn Thr Asn Tyr Ala Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Val Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 21
<211> 448
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 21
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly
20 25 30
Tyr Ser Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile His Tyr Ser Asp Asn Thr Asn Tyr Ala Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 22
<211> 448
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 22
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly
20 25 30
Tyr Ser Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
35 40 45
Met Gly Tyr Ile His Tyr Ser Asp Asn Thr Asn Tyr Ala Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 23
<211> 118
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 23
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Thr Ser Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 24
<211> 113
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 25
<211> 261
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 25
Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu
1 5 10 15
Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly
35 40 45
Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg
50 55 60
Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
100 105 110
Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser
115 120 125
Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly
145 150 155 160
Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe
165 170 175
Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met
180 185 190
Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala
195 200 205
Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly
210 215 220
Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile
225 230 235 240
Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser
245 250 255
Lys His Asp Tyr Val
260
<210> 26
<211> 261
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 26
Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile
1 5 10 15
Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly
35 40 45
Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg
50 55 60
Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
100 105 110
Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser
115 120 125
Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly
145 150 155 160
Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe
165 170 175
Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met
180 185 190
Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala
195 200 205
Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly
210 215 220
Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile
225 230 235 240
Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser
245 250 255
Lys His Asp Tyr Val
260
<210> 27
<211> 264
<212> PRT
<213> mice (Mus musculus)
<400> 27
Met Ala Thr Thr Thr Cys Gln Val Val Gly Leu Leu Leu Ser Leu Leu
1 5 10 15
Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asp Asn Pro Val Thr Ala Val Phe Gln Tyr Glu Gly
35 40 45
Leu Trp Arg Ser Cys Val Gln Gln Ser Ser Gly Phe Thr Glu Cys Arg
50 55 60
Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Val Ile Gly Ile Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Asp Asp Ser
100 105 110
Ala Lys Ala Lys Met Thr Leu Thr Ser Gly Ile Leu Phe Ile Ile Ser
115 120 125
Gly Ile Cys Ala Ile Ile Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Ser Gly Met Gly Gly
145 150 155 160
Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala
165 170 175
Ala Leu Phe Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly
180 185 190
Val Met Met Cys Ile Ala Cys Arg Gly Leu Thr Pro Asp Asp Ser Asn
195 200 205
Phe Lys Ala Val Ser Tyr His Ala Ser Gly Gln Asn Val Ala Tyr Arg
210 215 220
Pro Gly Gly Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Arg Asn
225 230 235 240
Lys Lys Ile Tyr Asp Gly Gly Ala Arg Thr Glu Asp Asp Glu Gln Ser
245 250 255
His Pro Thr Lys Tyr Asp Tyr Val
260
<210> 28
<211> 264
<212> PRT
<213> mice (Mus musculus)
<400> 28
Met Ser Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile
1 5 10 15
Gly Phe Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly
35 40 45
Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg
50 55 60
Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Val Ile Gly Ile Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Asp Asp Ser
100 105 110
Ala Lys Ala Lys Met Thr Leu Thr Ser Gly Ile Leu Phe Ile Ile Ser
115 120 125
Gly Ile Cys Ala Ile Ile Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Ser Gly Met Gly Gly
145 150 155 160
Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala
165 170 175
Ala Leu Phe Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly
180 185 190
Val Met Met Cys Ile Ala Cys Arg Gly Leu Thr Pro Asp Asp Ser Asn
195 200 205
Phe Lys Ala Val Ser Tyr His Ala Ser Gly Gln Asn Val Ala Tyr Arg
210 215 220
Pro Gly Gly Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Arg Asn
225 230 235 240
Lys Lys Ile Tyr Asp Gly Gly Ala Arg Thr Glu Asp Asp Glu Gln Ser
245 250 255
His Pro Thr Lys Tyr Asp Tyr Val
260
<210> 29
<211> 942
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
cagtacatca aggccaacag caagttcatc ggcatcaccg agctgaagaa gctgggtggt 60
tccaacgaca tattcaacaa cttcaccgtg agcttctggc tgcgcgtgcc gaaggtgagc 120
gccagccacc tggagcagta cggtggcggt agcggaatgg ccgtgactgc ctgtcagggc 180
ttggggttcg tggtttcact gattgggatt gcgggcatca ttgctgccac ctgcatggac 240
cagtggagca cccaagactt gtacaacaac cccgtaacag ctgttttcaa ctaccagggg 300
ctgtggcgct cctgtgtccg agagagctct ggcttcaccg agtgccgggg ctacttcacc 360
ctgctggggc tgccagccat gctgcaggca gtgcgagccc tgatgatcgt aggcatcgtc 420
ctgggtgcca ttggcctcct ggtatccatc tttgccctga aatgcatccg cattggcagc 480
atggaggact ctgccaaagc caacatgaca ctgacctccg ggatcatgtt cattgtctca 540
ggtctttgtg caattgctgg agtgtctgtg tttgccaaca tgctggtgac taacttctgg 600
atgtccacag ctaacatgta caccggcatg ggtgggatgg tgcagactgt tcagaccagg 660
tacacatttg gtgcggctct gttcgtgggc tgggtcgctg gaggcctcac actaattggg 720
ggtgtgatga tgtgcatcgc ctgccggggc ctggcaccag aagaaaccaa ctacaaagcc 780
gtttcttatc atgcctcagg ccacagtgtt gcctacaagc ctggaggctt caaggccagc 840
actggctttg ggtccaacac caaaaacaag aagatatacg atggaggtgc ccgcacagag 900
gacgaggtac aatcttatcc ttccaagcac gactatgtgt aa 942
<210> 30
<211> 327
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
cagtacatca aggccaacag caagttcatc ggcatcaccg agctgaagaa gctgggtggt 60
tccaacgaca tattcaacaa cttcaccgtg agcttctggc tgcgcgtgcc gaaggtgagc 120
gccagccacc tggagcagta cggtggcggt agcggacgca tggaccagtg gagcacccaa 180
gacttgtaca acaaccccgt aacagctgtt ttcaactacc aggggctgtg gcgctcctgt 240
gtccgagaga gctctggctt caccgagtgc cggggctact tcaccctgct ggggctgcca 300
gccatgctgc aggcagtgcg agcctaa 327
<210> 31
<211> 870
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
gacaagaccc acacatgccc accttgtcca gctccagagc tgctgggagg accaagcgtg 60
ttcctgtttc cacccaagcc caaggataca ctgatgatct ctaggacccc cgaggtgaca 120
tgcgtggtgg tggacgtgtc ccacgaggac cccgaggtga agttcaactg gtacgtggac 180
ggcgtggagg tgcataatgc taagaccaag cccagggagg agcagtacaa cagcacctat 240
cgggtggtgt ctgtgctgac agtgctgcat caggattggc tgaacggcaa ggagtataag 300
tgcaaggtga gcaataaggc cctgcctgct ccaatcgaga agaccatctc taaggccaag 360
ggccagccca gagagcctca ggtgtacaca ctgcctccat cccgcgagga gatgaccaag 420
aaccaggtga gcctgacatg tctggtgaag ggcttctatc cttctgacat cgctgtggag 480
tgggagtcca atggccagcc agagaacaat tacaagacca caccccctgt gctggacagc 540
gatggctctt tctttctgta ttccaagctg accgtggata agagcaggtg gcagcagggc 600
aacgtgtttt cctgtagcgt gatgcacgag gccctgcaca atcattacac acagaagtct 660
ctgtccctga gccctggcaa gggatccgga ggaggctctg gaggctccga ccagtggagc 720
acccaggatc tgtacaacaa tcccgtgaca gccgtgttca actatcaggg cctgtggagg 780
tcttgcgtgc gggagtccag cggcttcacc gagtgtagag gctactttac actgctggga 840
ctgcctgcta tgctgcaggc tgtgcgctga 870

Claims (10)

1. A heavy chain variable region of an anti-human CLDN18.2 antibody comprising the following three complementarity determining region CDRs:
(1) A complementarity determining region CDR1, wherein the amino acid sequence of the complementarity determining region CDR1 is shown in SEQ ID NO. 3;
(2) A complementarity determining region CDR2, wherein the amino acid sequence of said complementarity determining region CDR2 is shown in SEQ ID NO. 4 or 16; a kind of electronic device with high-pressure air-conditioning system
(3) Complementarity determining region CDR3, the amino acid sequence of said complementarity determining region CDR3 is shown in SEQ ID NO. 5 or 17.
2. An anti-human CLDN18.2 antibody heavy chain having the heavy chain variable region of the antibody of claim 1.
3. A light chain variable region of an anti-human CLDN18.2 antibody comprising the following three complementarity determining regions CDR':
(1) A complementarity determining region CDR1', wherein the amino acid sequence of said complementarity determining region CDR1' is shown in SEQ ID NO. 8;
(2) A complementarity determining region CDR2', wherein the amino acid sequence of said complementarity determining region CDR2' is shown in SEQ ID NO. 9; a kind of electronic device with high-pressure air-conditioning system
(3) Complementarity determining region CDR3', the amino acid sequence of said complementarity determining region CDR3' is shown in SEQ ID NO. 10.
4. An anti-human CLDN18.2 antibody light chain having the light chain variable region of the antibody of claim 3.
5. An anti-human CLDN18.2 antibody, said antibody having:
(1) The heavy chain variable region of claim 1; and/or
(2) A light chain variable region according to claim 3;
alternatively, the antibody has: the heavy chain of claim 2; and/or the light chain of claim 4.
In another preferred embodiment, the antibody comprises: murine antibodies, chimeric antibodies, humanized antibodies.
6. The antibody of claim 5, wherein the antibody has a heavy chain variable region as set forth in SEQ ID NOs 1, 11, 12, or 13; and/or the light chain variable region as shown in SEQ ID NO. 6, 14 or 15.
7. A recombinant protein, said recombinant protein having:
(i) A heavy chain variable region according to claim 1, a heavy chain according to claim 2, a light chain variable region according to claim 3, a light chain according to claim 4, or an antibody according to claim 5; and
(ii) Optionally a tag sequence to assist expression and/or purification.
8. An antibody preparation, wherein the antibody preparation comprises:
(a) The anti-human CLDN18.2 antibody of claim 5; and
(b) A carrier, said carrier comprising: buffering agents, sterile water, optionally surfactants.
9. An antibody drug conjugate, comprising:
(a) An antibody moiety selected from the group consisting of: 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 a combination thereof; and
(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.
10. Use of an active ingredient, characterized in that the active ingredient is selected from the group consisting of: 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, the recombinant protein of claim 7, the antibody drug conjugate of claim 9, or a combination thereof, the active ingredient being for use in
(a) Preparing a detection reagent or a kit;
(b) Preparing a medicament or preparation for preventing and/or treating the CLDN18.2 related diseases; and/or
(c) Preparing medicine or preparation for preventing and/or treating cancer or tumor.
CN202111333547.XA 2021-11-11 2021-11-11 anti-CLDN 18.2 monoclonal antibody and application thereof Pending CN116102649A (en)

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MX2020009326A (en) * 2018-03-08 2020-10-08 Phanes Therapeutics Inc Anti-claudin 18.2 antibodies and uses thereof.
MX2020009514A (en) * 2018-03-14 2020-12-07 Beijing Xuanyi Pharmasciences Co Ltd Anti-claudin 18.2 antibodies.
EP3808376A4 (en) * 2018-06-17 2021-09-01 L&L Biopharma Co., Ltd. Antibody targeting cldn18.2, bispecific antibody, adc, and car, and applications thereof
CN110606891B (en) * 2018-06-17 2022-12-06 上海健信生物医药科技有限公司 Antibody molecule aiming at human CLDN18.2, antigen binding fragment and medical application thereof
EP3826612A4 (en) * 2018-07-25 2022-09-14 Accurus Biosciences, Inc. Novel cldn 18.2-specific monoclonal antibodies and methods of use thereof
CN112707965A (en) * 2019-09-30 2021-04-27 和铂医药(苏州)有限公司 Antibody targeting CLDN18.2 and preparation method and application thereof

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