WO2020038404A1

WO2020038404A1 - Anti-human claudin 18.2 monoclonal antibody and application thereof

Info

Publication number: WO2020038404A1
Application number: PCT/CN2019/101785
Authority: WO
Inventors: 吴燕燕; 崔文俊; 胡琛霏
Original assignee: 瑞阳(苏州)生物科技有限公司
Priority date: 2018-08-22
Filing date: 2019-08-21
Publication date: 2020-02-27

Abstract

Provided are an anti-human claudin 18.2 monoclonal antibody and application thereof. The antibody has three heavy chain complementarity-determining regions (heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3) and three light chain complementarity-determining regions (light chain CDR1, light chain CDR2, and light chain CDR3).

Description

Anti-human claudin 18.2 monoclonal antibody and its application

Technical field

The invention relates to the field of biomedicine, in particular to an anti-human claudin 18.2 monoclonal antibody and application thereof.

Background technique

The Claudins family is a tightly-linked backbone protein between epithelial cells and participates in the formation of tightly-connected cells. Tightly-connected cells maintain the polar arrangement of cells and barrier functions. The abnormal expression of these cells leads to the destruction of tightly-connected structures and functions, which is the basis of many diseases. Studies have shown that the Claudins family is abnormally expressed in a variety of epithelial-derived tumors, and is closely related to the occurrence and development of these tumors. hot spot.

The human Claudin18 gene has two different first exons, so two subtypes, Claudin 18.1 and Claudin 18.2, can be produced. The N-terminal 69 amino acids of these two molecules have different structures and are located in the first extracellular region of the cyclic structure. The two subtypes of Claudin18 are transcribed and amplified in different tissues respectively. Among them, Claudin 18.1 is mainly expressed in lung tissue, while Claudin 18.2 is specifically expressed in gastric tissue.

In 2006, Sanada et al found that the expression of claudin-18 gene was down-regulated in 57% of gastric cancer. According to immunohistochemical analysis, claudin-18 was expressed on the membrane of normal gastric mucosa and duodenal Pancreatic cells, but in some intestinal In 90% and 90% of gastric adenomas, the expression of claudin-18 is reduced. At the same time, the decrease in expression of intestinal gastric cancer is more common than other gastric cancers. It is speculated that it may be involved in the occurrence of early gastric cancer. Survival analysis showed that the decrease of claudin-18 expression in gastric cancer is related to the poor prognosis of advanced patients. It is considered that the decrease of claudin-18 expression is a factor of poor prognosis in patients with gastric cancer.

In 2008, Sahin and other studies confirmed that Claudin 18.2 expression was present in 77% of primary gastric adenocarcinoma tissues, and it was extremely important that 56% of the tissues expressed more than 60%. Consistent with the above findings, the expression of Claudin 18.2 protein is down-regulated in intestinal gastric cancer, while the expression of Claudin 18.2 protein is higher in diffuse gastric cancer. However, Claudin 18.2 protein is not expressed in normal tissues such as pancreas, esophagus, and ovary, while Claudin 18.2 protein can be expressed in large amounts in corresponding tumor tissues. Therefore, Claudin 18.2 protein can be used as a target for clinical tumor diagnosis and treatment. However, no antibody has been developed that targets this protein.

Technical solutions

The invention provides an anti-human claudin 18.2 monoclonal antibody with good specificity, high affinity and stability.

The first object of the present invention is to provide the amino acid sequence of an anti-human claudin 18.2 monoclonal antibody, which is as follows:

An anti-human claudin 18.2 monoclonal antibody with three heavy chain complementarity determining regions (heavy chain CDR1, heavy chain CDR2, heavy chain CDR3) and three light chain complementarity determining regions (light chain CDR1, light chain CDR2, light chain Chain CDR3),

The heavy chain CDR1 is selected from: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID: NO: 49, SEQ ID: NO: 55, SEQ ID: NO: 61, SEQ ID: NO: 67, SEQ ID: NO: 73 or a homology greater than 50% sequence;

The heavy chain CDR2 is selected from: SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID: NO: 50, SEQ ID: NO: 56, SEQ ID: NO: 62, SEQ ID: NO: 68, SEQ ID: NO: 74 or a homology with one of which is greater than 50% sequence;

The heavy chain CDR3 is selected from: SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID: NO: 51, SEQ ID: NO: 57, SEQ ID: NO: 63, SEQ ID: NO: 69, SEQ ID: NO: 75 or a homology with one of which is greater than 50% sequence;

The light chain CDR1 is selected from: SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID: NO: 52, SEQ ID: NO: 58, SEQ ID: NO: 64, SEQ ID: NO: 70, SEQ ID: NO: 76 or a homology with one that is greater than 50% sequence;

The light chain CDR2 is selected from: SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID: NO: 53, SEQ ID: NO: 59, SEQ ID: NO: 65, SEQ ID: NO: 71, SEQ ID: NO: 77 or a homology with one of which is greater than 50% sequence;

The light chain CDR3 is selected from: SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID: NO: 54, SEQ ID: NO: 60, SEQ ID: NO: 66, SEQ ID: NO: 72, SEQ ID: NO: 78 or a homology with one of which is greater than 50% sequence.

Preferably, the heavy chain CDR1 is SEQ ID NO: 1, the heavy chain CDR2 is SEQ ID NO: 2, the heavy chain CDR3 is SEQ ID NO: 3, and the light chain CDR1 is SEQ ID NO: 4, and the light chain CDR2 is SEQ ID NO: 5, light chain CDR3 is SEQ ID NO: 6; or heavy chain CDR1 is SEQ ID NO: 7, heavy chain CDR2 is SEQ ID NO: 8, heavy chain CDR3 is SEQ ID NO: 9, and light chain CDR1 is SEQ ID NO: 10, light chain CDR2 is SEQ ID NO: 11, light chain CDR3 is SEQ ID NO: 12; or heavy chain CDR1 is SEQ ID NO: 13, heavy chain CDR2 is SEQ ID NO: 14, heavy chain CDR3 Is SEQ ID NO: 15, and light chain CDR1 is SEQ ID NO: 16, light chain CDR2 is SEQ ID NO: 17, light chain CDR3 is SEQ ID NO: 18; or heavy chain CDR1 is SEQ ID NO: 19, heavy Chain CDR2 is SEQ ID NO: 20, heavy chain CDR3 is SEQ ID NO: 21, and light chain CDR1 is SEQ ID NO: 22, light chain CDR2 is SEQ ID NO: 23, and light chain CDR3 is SEQ ID NO: 24; Or the heavy chain CDR1 is SEQ ID NO: 25, the heavy chain CDR2 is SEQ ID NO: 26, the heavy chain CDR3 is SEQ ID NO: 27, and the light chain CDR1 is SEQ ID NO: 28, and the light chain CDR2 is SEQ ID NO: 29. The light chain CDR3 is SEQ ID NO: 30; or the heavy chain CDR1 is SEQ ID NO: 31, and the heavy chain CDR2 is SEQ I DNO: 32, heavy chain CDR3 is SEQ ID NO: 33, and light chain CDR1 is SEQ ID NO: 34, light chain CDR2 is SEQ ID NO: 35, light chain CDR3 is SEQ ID NO: 36; or heavy chain CDR1 Is SEQ ID NO: 37, heavy chain CDR2 is SEQ ID NO: 38, heavy chain CDR3 is SEQ ID NO: 39, and light chain CDR1 is SEQ ID NO: 40, light chain CDR2 is SEQ ID NO: 41, light chain CDR3 is SEQ ID NO: 42; or heavy chain CDR1 is SEQ ID NO: 43, heavy chain CDR2 is SEQ ID NO: 44, heavy chain CDR3 is SEQ ID NO: 45, and light chain CDR1 is SEQ ID NO: 46, Light chain CDR2 is SEQ ID NO: 47, light chain CDR3 is SEQ ID NO: 48; or heavy chain CDR1 is SEQ ID NO: 49, heavy chain CDR2 is SEQ ID NO: 50, and heavy chain CDR3 is SEQ ID NO: 51 And light chain CDR1 is SEQ ID NO: 52, light chain CDR2 is SEQ ID NO: 53, light chain CDR3 is SEQ ID NO: 54; or heavy chain CDR1 is SEQ ID NO: 55, and heavy chain CDR2 is SEQ ID NO : 56, heavy chain CDR3 is SEQ ID NO: 57, and light chain CDR1 is SEQ ID NO: 58, light chain CDR2 is SEQ ID NO: 59, light chain CDR3 is SEQ ID NO: 60; or heavy chain CDR1 is SEQ ID NO: 61, heavy chain CDR2 is SEQ ID NO: 62, heavy chain CDR3 is SEQ ID NO: 63, and light chain CDR1 SEQ ID NO: 64, light chain CDR2 is SEQ ID NO: 65, light chain CDR3 is SEQ ID NO: 66; or heavy chain CDR1 is SEQ ID NO: 67, heavy chain CDR2 is SEQ ID NO: 68, heavy chain CDR3 Is SEQ ID NO: 69, and light chain CDR1 is SEQ ID NO: 70, light chain CDR2 is SEQ ID NO: 71, light chain CDR3 is SEQ ID NO: 72; or heavy chain CDR1 is SEQ ID NO: 73, heavy Chain CDR2 is SEQ ID NO: 74, heavy chain CDR3 is SEQ ID NO: 75, and light chain CDR1 is SEQ ID NO: 76, light chain CDR2 is SEQ ID NO: 77, and light chain CDR3 is SEQ ID NO: 78.

Preferably, the heavy chain variable region comprises the aforementioned heavy chain CDR1, CDR2 and CDR3; the light chain variable region comprises the aforementioned light chain CDR1, CDR2 and CDR3.

Preferably, the amino acid sequence of the heavy chain variable region is selected from the group consisting of: SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID: NO: 91, SEQ ID: NO: 93, SEQ ID: NO: 95, SEQ ID: NO: 97, SEQ ID: NO: 99, SEQ ID: NO: 101 or SEQ ID: NO: 103;

Preferably, the amino acid sequence of the light chain variable region is selected from the group consisting of: SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86; SEQ ID NO: 88, SEQ ID NO: 90, SEQ One of ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, or SEQ ID NO: 104.

As a preference,

The heavy chain variable region is SEQ ID NO: 79 and the light chain variable region is SEQ ID NO: 80; or

The heavy chain variable region is SEQ ID NO: 81 and the light chain variable region is SEQ ID NO: 82; or

The heavy chain variable region is SEQ ID NO: 83 and the light chain variable region is SEQ ID NO: 84; or

The heavy chain variable region is SEQ ID NO: 85 and the light chain variable region is SEQ ID NO: 86; or

The heavy chain variable region is SEQ ID NO: 87 and the light chain variable region is SEQ ID NO: 88; or

The heavy chain variable region is SEQ ID NO: 89 and the light chain variable region is SEQ ID NO: 90; or

The heavy chain variable region is SEQ ID NO: 91 and the light chain variable region is SEQ ID NO: 92; or

The heavy chain variable region is SEQ ID NO: 93 and the light chain variable region is SEQ ID NO: 94; or

The heavy chain variable region is SEQ ID NO: 95 and the light chain variable region is SEQ ID NO: 96; or

The heavy chain variable region is SEQ ID NO: 97 and the light chain variable region is SEQ ID NO: 98; or

The heavy chain variable region is SEQ ID NO: 99 and the light chain variable region is SEQ ID NO: 100; or

The heavy chain variable region is SEQ ID NO: 101 and the light chain variable region is SEQ ID NO: 102; or

The heavy chain variable region is SEQ ID NO: 103 and the light chain variable region is SEQ ID NO: 104.

Preferably, the sequence of the amino acid in the variable region of the heavy chain is a similar sequence having at least 50% homology with the sequence a, wherein the sequence a is SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID One of NO: 101 or SEQ ID NO: 103.

Preferably, the amino acid sequence of the light chain variable region is a similar sequence having at least 50% homology with sequence b, which is SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86; SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID Either NO: 102 or SEQ ID: NO: 104.

The amino acid sequence of the heavy chain includes heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, and light chain CDR1, light chain CDR2, and light chain CDR3; heavy chain CDR1 is SEQ ID NO: 1, and heavy chain CDR2 is SEQ ID NO: 2, The heavy chain CDR3 is SEQ ID NO: 3, and the light chain CDR1 is SEQ ID NO: 4, the light chain CDR2 is SEQ ID NO: 5, and the light chain CDR3 is SEQ ID NO: 6; or the heavy chain CDR1 is SEQ ID NO: 7. The heavy chain CDR2 is SEQ ID NO: 8, the heavy chain CDR3 is SEQ ID NO: 9, and the light chain CDR1 is SEQ ID NO: 10, the light chain CDR2 is SEQ ID NO: 11, and the light chain CDR3 is SEQ ID NO. : 12; or heavy chain CDR1 is SEQ ID NO: 13, heavy chain CDR2 is SEQ ID NO: 14, heavy chain CDR3 is SEQ ID NO: 15, and light chain CDR1 is SEQ ID NO: 16, and light chain CDR2 is SEQ ID NO: 17, light chain CDR3 is SEQ ID NO: 18; or heavy chain CDR1 is SEQ ID NO: 19, heavy chain CDR2 is SEQ ID NO: 20, heavy chain CDR3 is SEQ ID NO: 21, and light chain CDR1 Is SEQ ID NO: 22, light chain CDR2 is SEQ ID NO: 23, light chain CDR3 is SEQ ID NO: 24; or heavy chain CDR1 is SEQ ID NO: 25, heavy chain CDR2 is SEQ ID NO: 26, heavy chain CDR3 is SEQ ID NO: 27, and light chain CDR1 is SEQ ID NO: 28, and light chain CDR2 is SEQ ID. NO: 29, the light chain CDR3 is SEQ ID NO: 30; or the heavy chain CDR1 is SEQ ID NO: 31, the heavy chain CDR2 is SEQ ID NO: 32, the heavy chain CDR3 is SEQ ID NO: 33, and the light chain CDR1 is SEQ ID NO: 34, light chain CDR2 is SEQ ID NO: 35, light chain CDR3 is SEQ ID NO: 36; or heavy chain CDR1 is SEQ ID NO: 37, heavy chain CDR2 is SEQ ID NO: 38, heavy chain CDR3 Is SEQ ID NO: 39, and light chain CDR1 is SEQ ID NO: 40, light chain CDR2 is SEQ ID NO: 41, light chain CDR3 is SEQ ID NO: 42, or heavy chain CDR1 is SEQ ID NO: 43, heavy Chain CDR2 is SEQ ID NO: 44, heavy chain CDR3 is SEQ ID NO: 45, and light chain CDR1 is SEQ ID NO: 46, light chain CDR2 is SEQ ID NO: 47, and light chain CDR3 is SEQ ID NO: 48; Or the heavy chain CDR1 is SEQ ID NO: 49, the heavy chain CDR2 is SEQ ID NO: 50, the heavy chain CDR3 is SEQ ID NO: 51, and the light chain CDR1 is SEQ ID NO: 52, and the light chain CDR2 is SEQ ID NO: 53. The light chain CDR3 is SEQ ID NO: 54; or the heavy chain CDR1 is SEQ ID NO: 55, the heavy chain CDR2 is SEQ ID NO: 56, the heavy chain CDR3 is SEQ ID NO: 57, and the light chain CDR1 is SEQ ID NO: 58, light chain CDR2 is SEQ ID NO: 59, light chain CDR3 is SEQ ID NO: 60; or heavy chain CDR1 Is SEQ ID NO: 61, heavy chain CDR2 is SEQ ID NO: 62, heavy chain CDR3 is SEQ ID NO: 63, and light chain CDR1 is SEQ ID NO: 64, light chain CDR2 is SEQ ID NO: 65, light chain CDR3 is SEQ ID NO: 66; or heavy chain CDR1 is SEQ ID NO: 67, heavy chain CDR2 is SEQ ID NO: 68, heavy chain CDR3 is SEQ ID NO: 69, and light chain CDR1 is SEQ ID NO: 70, Light chain CDR2 is SEQ ID NO: 71, light chain CDR3 is SEQ ID NO: 72; or heavy chain CDR1 is SEQ ID NO: 73, heavy chain CDR2 is SEQ ID NO: 74, and heavy chain CDR3 is SEQ ID NO: 75 And the light chain CDR1 is SEQ ID NO: 76, the light chain CDR2 is SEQ ID NO: 77, and the light chain CDR3 is SEQ ID NO: 78.

Preferably, the amino acid sequence of the antibody includes the heavy chain variable region and the light chain variable region described above.

A second object of the present invention is to provide a nucleic acid sequence of an anti-human claudin 18.2 monoclonal antibody, specifically as follows:

A nucleic acid molecule comprising a nucleic acid sequence capable of encoding a heavy chain CDR and / or a light chain CDR. The heavy chain CDR1 is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, and SEQ ID NO: 19.SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 61, SEQ ID NO: 67, One of SEQ ID NO: 73 or a similar sequence having a homology of greater than 50% with one of them;

A nucleic acid molecule, characterized in that it contains a nucleic acid sequence capable of encoding a heavy chain variable region and / or a light chain variable region, and the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, or SEQ ID NO: 103;

The amino acid sequence of the light chain variable region is selected from the group consisting of: SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86; SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92. One of SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, or SEQ ID NO: 104.

The present invention also provides a vector containing any of the nucleic acid molecules described above.

The present invention also provides a host cell containing the amino acid sequence, nucleic acid molecule, or vector described above.

The present invention also provides a conjugate containing the antibody according to any one of the above.

The present invention also provides a composition containing a main component and an auxiliary component, the main component containing the antibody or the nucleic acid molecule or the vector, the host cell, or the couple One or more of the combinations, the auxiliary ingredients are selected from pharmaceutically acceptable carriers or excipients, and optionally other biologically active substances.

The other biologically active substances include, but are not limited to, other antibodies, fusion proteins, or drugs (for example, antitumor drugs such as radiotherapy and chemotherapy drugs).

The invention also provides the application of the above-mentioned antibodies, nucleic acid molecules, vectors, host cells, conjugates, and compositions in the preparation of drugs or detection reagents for treating diseases.

The disease is a malignant tumor, a cardiovascular disease or an inflammatory disease.

The present invention also provides a diagnostic reagent or kit containing the antibody according to any one of the above. The diagnostic reagent or kit is used to diagnose a disease related to claudin 18.2 in vitro (e.g., a cell or tissue) or in vivo (e.g., a human or animal model).

The present invention is further described below: In the present invention, unless otherwise stated, scientific and technical terms used herein have meanings commonly understood by those skilled in the art. Furthermore, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory procedures used herein are terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below. The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked to each other by a disulfide bond, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region and a heavy chain constant region.

In the present invention, "homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences at the most preferred alignment.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and can be implemented in accordance with the contents of the description, the following describes in detail the preferred embodiments of the present invention and the accompanying drawings as follows.

Embodiments of the invention

The invention discloses a monoclonal antibody and its application, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters for implementation. In particular, it should be noted that all similar replacements and modifications will be apparent to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments. Obviously, relevant persons can obviously modify or appropriately modify and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the present invention. Invention technology.

The monoclonal antibody provided by the present invention and the raw materials and reagents used in the application can be purchased from the market.

The following further describes the present invention in combination with the embodiments:

Example:

Antigen preparation

GST-ECD protein preparation

The partial sequence of the first extracellular domain of human claudin 18.2 was constructed on the pGEX-KG vector (antigen sequence 1), and the constructed expression vector was transferred into E. coli strain TG1, and cultured with LB medium and induced with IPTG Method to express antigenic protein. The collected E.coli TG1 bacterial cells were broken by a homogenizer, Glutathione Sepharose 4Fast Flow (GE, Cat # 17-5132-01) was used for affinity purification, and HiTrap Capto Q (GE, Cat # 11001302 ) The column was subjected to secondary purification and the protein was dialyzed into PBS, pH 7.4 to obtain an antigen protein with a purity of> 85%.

DNA immunogen preparation

The partial or full-length sequence of the first extracellular domain of claudin 18.2 was constructed on the pcDNA3.1 vector as an immunogen (antigen sequence 2, antigen sequence 3).

Construction of Human Claudin 18.1 and Human Claudin 18.2 Stable Cell Lines

The full-length sequences of human claudin 18.1 and human claudin 18.2 were cloned into the pcDNA3.1 (-) vector, and the constructed expression vector was transferred into HEK293F cells using Lipofectamine ^TM 3000 Transfection Reagent (Thermo, Cat # L3000150) reagent. 400 μg / ml G418 (Gibco, Cat # 10131-027) was selected. After Q-PCR and FACS screening and verification, stable expression of HEK293F_human claudin 18.1 and HEK293F_human claudin 18.2 cell lines were obtained.

The full-length sequence of human claudin 18.2 was cloned into an adenoviral vector, and the constructed expression vector was transferred into CHO-K1 cells using Lipofectamine ^TM 3000 Transfection Reagent (Thermo, Cat # L3000150) reagent, and 5 μg / mL puromycin (Gibco, Cat # 10131-027). After Q-PCR and FACS screening and verification, stable expression of CHO-K1_human claudin 18.2 cell line was obtained.

2. Animal immunity

In order to obtain high-affinity and high-specificity antibodies against human claudin 18.2, a series of antigen combinations (DNA immunity, protein immunity, and cell line whole cell immunity) were designed in this experiment to immunize several Balb / c mice and C57BL / 6 cells. Mice are immunized using the classic immunization schedule, which triggers an immune response in mice. Mouse serum was collected, and GST-ECD protein cell line was used as an antigen. ELISA method or CHO-K1_human claudin 18.2 cell line was used for FACS to determine the serum titer. The corresponding mice with high titer were selected for fusion.

3. Hybridoma fusion and screening

Before fusion, adjust the state of mouse myeloma SP2 / 0 to ensure that its growth density does not exceed 1x10 ^6. Animals will be exempted 3 days in advance. SP2 / 0 cells will be prepared one day in advance. The number of plates will be 2.0x10 ⁴ cells / well. Electrofusion instrument fusion to ensure that the ratio of the number of splenocytes or lymph node cells to SP2 / 0 cells is between 10: 1 and 5: 1, and the number of splenocytes spread per well does not exceed 1x105, and the supernatant is harvested 7 days after fusion The medium was changed, and the collected supernatant was tested for cell binding activity by ELISA binding method and flow cytometry to obtain hybridoma cell lines that were positive in all three screening experiments.

A. ELISA binding method

Dilute GST-ECD with a coating solution of PBS pH 7.4 to 0.5 μg / ml to coat a 96-well plate, add 100 μl to each well, block and add the hybridoma culture supernatant and incubate. After washing, add Peroxidase-AffiniPure Goat Anti- Mouse IgG, FcγFragment Specific (minXHu, Bov, HrsSrProt), wash and add chromogenic substrate and incubate. Read the absorbance of OD450 after stopping the reaction.

B. Cell ELISA test

Coat a 96-well plate with CHO-K1_human claudin 18.2 cell line, 1 × 10 ⁵ cells / well. After the 96-well plate is closed, the primary antibody is the hybridoma supernatant, positive antibody, isotype control and PBS, and the primary antibody is incubated and washed. Cells were incubated with Peroxidase-AffiniPure Goat Anti-Mouse IgG, FcγFragment Specific (min X Hu, Bov, Hrs Sr Prot) as secondary antibodies. After washing, they were incubated with a chromogenic substrate. After the reaction was terminated, the absorbance of OD450 was read.

C. Flow cytometry to determine the cell binding activity of the hybridoma supernatant

The HEK293F_human claudin 18.1, HEK293F_human claudin 18.2 cell line and CHO-K1_human claudin 18.2 cell line were subjected to flow cytometry experiments with the hybridoma supernatant. The human clone was specifically cloned as the target clone.

50 μl of 2.5 × ^{10 5} cells were used for each reaction. The primary antibody was the hybridoma supernatant, positive antibody, isotype control and PBS. After incubation with the primary antibody, the cells were washed, and Goat anti-Mouse IgG iFlour 647 (GenScript, 3 μg / ml) was used. Incubate as a secondary antibody and read the fluorescence signal by flow cytometry after washing.

4. Preparation and identification of human and mouse chimeric antibodies

A. Acquisition of murine anti-genes

The total RNA of hybridoma cells was extracted by Trizol (Ambion 15596-026) kit, and then the total RNA was reverse transcribed using Takara PrimeScript 1 ^st Strand cDNA synthesis kit to prepare cDNA, which were amplified using degenerate primers, respectively. The heavy and light chain variable region sequences of an antibody.

B. Construction of human and mouse chimeric antibodies

The murine anti-light chain variable region sequence was spliced with human IgG kappa CL region sequence to obtain the full-length light chain sequence, which was constructed on the pcDNA3.1 vector containing the nitrogen-terminal signal peptide by whole gene synthesis to obtain a light chain expression vector.

The murine anti-heavy chain variable region sequence was spliced with the CH1, CH2 and CH3 region sequences of human IgG4S228P to obtain the full-length heavy chain sequence. Through the gene synthesis, the heavy chain was constructed on the pcDNA3.1 vector containing the nitrogen terminal signal peptide. Expression vector.

C. Expression and purification of human and mouse chimeric antibodies

HEK293F cells were transiently transfected by co-transfection of light and heavy chain expression vectors. The 293F cells were cultured for 7 days after transfection, and the culture supernatant was collected. Then the chimeric antibody in the supernatant was purified by protein A affinity chromatography column and dialyzed into PBS pH 7.4. The concentration was determined by A280 method and analyzed by SDS-PAGE.

D. Determination of the binding ability of human and mouse chimeric antibodies to cells by flow cytometry

Determination of the binding ability of human and mouse chimeric antibodies to HEK293F_human claudin 18.1 and HEK293F_human claudin 18.2 cell lines

100 μl 2.0x10 ⁷ cells / ml cells were used for each reaction. The primary antibody was purified human-mouse chimeric antibody, positive antibody (IMAB362 and Anti-Claudin18antibody [34H14L15] (abcam, Cat # ab203563)), isotype control and 1 % BSA in PBS, after primary antibody incubation, wash cells, and use Goat anti-human IgG Fc (FITC) (abcam, Cat # ab97224) and goat Anti-Rabbit IgG H & L (Alexa

488) (abcam, Cat # ab150077) was incubated as a secondary antibody, and the fluorescence signal was read by a flow cytometer after washing.

F. Determination of binding ability of human and mouse chimeric antibodies to cancer cell lines by flow cytometry

For each reaction, 100 μl of 1.0 × ^{10 7} cells / ml cells were used. The primary antibody was human-mouse chimeric antibody, positive antibody (IMAB362), isotype control and 1% BSA in PBS. After the primary antibody was incubated, the cells were washed, and Goat anti-human IgG Fc (FITC) (abcam, Cat # ab97224) was incubated as a secondary antibody, and the fluorescence signal was read by flow cytometry after washing.

5. Humanization of murine antibodies

Analyze the sequence of the variable region of the mouse antibody and compare it with the human germline gene. Select the sequence of the framework region of the human light and heavy chain germline gene with the highest homology to the sequence of the mouse antibody framework region as the basic skeleton, or select the framework. Specific amino acids in the region were restored for mutation, and then the CDR region sequence of the mouse antibody was grafted with the framework region sequence of the selected human germline gene, and the light and heavy chain variable region sequences of the humanized antibody were synthesized by gene synthesis after splicing The expression vectors were constructed on the basis of human IgG4 heavy chain Fc and human IgG kappa CL. The constructed humanized antibody light and heavy chain vectors were used for transient expression of HEK293F in one-to-one pair. After affinity purification and dialysis of antibodies in the culture supernatant of transient cells, the concentrations were determined by A280 method and analyzed by SDS-PAGE. After purity and purity, the affinity with HEK293F_human claudin 18.2 cell line was determined by FACS method.

6. ADCC experiments for antibodies

The HEK293F_human claudin 18.2 cell line was used to determine the humanized antibody of the invention after FPLC purification to mediate complement-dependent cytotoxicity.

Effector cells PBMC (including effector cells such as NK cells and monocytes) are derived from fresh blood samples provided by healthy blood donors. Fresh blood is diluted 4 times with DPBS (Gibco, 14190-144), and Ficoll-Paque Plus (GE, Healthcare) density gradient centrifugation method to extract effector cell PBMC. As a target cell, a HEK293F_human claudin 18.2 cell line stably expressing CLDN18A2 was used. The medium (RPMI1640medium (Gibco, 11835-030) + 1% FBS (Gibco, 10099-141)) was used to dilute the concentration of the antibody to be detected and the control antibody to a final concentration of 10ug / ml, and then diluted 5 times in 8 gradients. Into a 96-well cell U-shaped culture plate (Corning, 3799). Add 40 μl / well of previously diluted target cells (1E4 cells / well) to a 96-well culture plate. Add 40 μl / well of diluted effector cells (2.5E5 cells / well) to a 96-well plate with effector cell: target cell ratio (25: 1). The effector cells and the target cells were respectively added with a medium as a control, and after mixing, the cells were cultured for 6 hours at 37 ° C and 5% CO ₂ . After incubation, centrifuge at 1500 rpm / min (Eppendorf, 5810R) for 10 minutes. Pipette 70 μl / well of the supernatant into a 96-well cell culture plate (Corning, 3599). The LDH (Roche, 4743940001) kit was used to test OD492 / OD620 using a multifunctional microplate reader (Thermo, MULTISKAN FC). The percentage of cytotoxicity was calculated as follows: Cytotoxicity (%) = 100 * (OD sample–OD Target cell only-ODPBMC Only) / (OD Target cell lysis-OD Target cell only) *%.

7. Antibody CDC experiment

As a target cell, a HEK293F_human claudin 18.2 cell line stably expressing CLDN18A2 was used. The medium (RPMI1640medium (Gibco, 11835-030) + 1% FBS (Gibco, 10099-141)) was used to dilute the concentration of the antibody to be detected and the control antibody to a final concentration of 50ug / mL, and then diluted 5 times in 8 gradients. Complement (Quidel, A113) was diluted to a final concentration of 1:50 (V / V). Add 40 μl / well of pre-diluted antibody to a 96-well cell culture plate (Corning, 3917). Add 40 μl / well of previously diluted target cells (1E4 cells / well) to a 96-well culture plate. Add 40 μl / well of diluted complement to a 96-well plate. The effector cells and complement were respectively added with a medium as a control, and after mixing, the cells were cultured for 6 hours at 37 ° C and 5% CO ₂ . After incubation, add 50 μl / well

Luminescent Cell Viability Assay (Promega, G7571) into a 96-well cell culture plate. The chemiluminescence value was tested using a multifunctional microplate reader (BioTek, Synergy 2). The percentage of cytotoxicity was calculated as follows: Cytotoxicity (%) = 100 * (1- (RLU Sample-RLUPNHS) / (RLU Cell + PNHS- RLUPNHS)) *%.

8. Epitope mapping

The epitope mapping method of the antigen recognized by the antibody in this invention is performed according to the "epitope mapping method" in "Methods of Molecular Biology" by Johan Rockberg (ISBN978-1-4939-7839-7).

In short, the peptides of the epitope-mapped overlapping peptide library were designed according to the antigen sequence and synthesized in GenScript. The peptides were coated with a high affinity ELISA plate, and first buffered with Na2CO ₃ / NaHCO ₃ , pH 9.59. The peptide was diluted with solution and coated at 37 ° C for about 2 hours. The coating solution in the well was discarded, and then the peptide was diluted with coating buffer with PBS, pH 7.4 into the well, and then coated twice at 37 ° C for about 2 hours. The coating solution in the well was discarded, and finally the polypeptide diluted with ddH ₂ O was added to the well and coated for a third time at 4 ° C. overnight. Wash with PBST containing 0.1% Tween-20 and block with 3% BSA / PBST. Dilute the antibody, positive control, and isotype of this invention with 0.5% BSA / PBST as well as 0.5% BSA / PBST as the primary antibody, incubate at 37 ° C for 2 hours, and wash with PBST containing 0.1% Tween-20; use 0.5% BSA / PBST A goat anti-human Fc fragment antibody (Solarbio, Cat # SE101) coupled with an HRP group was diluted as a secondary antibody, incubated at 37 ° C for 1 hour, and washed with PBST containing 0.1% Tween-20. TMB was developed (Solarbio, Cat # PR1200) and determine the OD450 absorbance.

9. Species binding characteristics and affinity determination of humanized antibodies

The species-binding properties of the humanized antibody were detected by flow cytometry according to the method in Example 4. The pcDNA3.1 (+) vector was used to construct CLDN18A2 expression vectors for mice, rats, rabbits and orangutans. The expression vectors were transiently transfected into HEK293F cells using ExpiFectamine ^™ 293 Transfection Kit (gibico, Cat # A14524). Samples were taken for measurement 48 hours after dyeing. Blank HEK293F cells were used as negative controls, and HEK293F cell lines stably expressing human CLDN18A1 and human CLDN18A2 were used as positive controls for flow cytometry analysis.

100 μl of 2.0 × ^{10 7} cells / ml cells were used for each reaction, and the primary antibody was the humanized antibody of the present invention purified by FPLC, and the positive antibody (IMAB362 and Anti-Claudin18 antibody [34H14L15] (abcam, Cat # ab203563)), Isotype control and 1% BSA in PBS, after primary antibody incubation, cells were washed, and Goat anti-human IgG Fc (FITC) (abcam, Cat # ab97224) and goat Anti-Rabbit IgG H & L (Alexa

10. Inhibition of proliferation

As a detection cell, a HEK293F_human claudin 18.2 cell line stably expressing CLDN18A2 was used. The medium (FreeStyle ^™ 293 Expression Medium (Gibco, 12338-018)) was used to dilute the concentration of the antibody to be detected and the isotype control antibody to a final concentration of 15 ug / ml, and then diluted 8.16 times by 8 gradients. Add 50 μl / well of pre-diluted antibody to a 96-well cell culture plate (Corning, 3610). Add 50 μl / well of previously diluted cells ((5E3 cells / well /) to a 96-well culture plate. After mixing, incubate for 7 days at 37 ° C and 5% CO _2. Add 50 μl / well after incubation

Luminescent Cell Viability Assay (Promega, G7571) into a 96-well cell culture plate. The chemiluminescence value was tested using a multifunctional microplate reader (BioTek, Synergy 2). The percentage of cytotoxicity was calculated as follows: Cytotoxicity (%) = 100 * (RLUCell only-RLU Sample) / RLU Cell only *%.

11. Endocytosis

As a detection cell, a HEK293F_human claudin 18.2 cell line stably expressing CLDN18A2 was used. Dilute the concentration of the antibody to be detected and the isotype control antibody to 40 nM using a medium (FreeStyle ^TM 293 Expression Medium (Gibco, 12338-018)), 120ul Fab-ZAP (Advanced Targeting Systems, IT-51) + 120uL antibody or Saporin (Advanced Targeting Systems, PR-1), mix, incubate at 37 ° C, 5% CO2 for 1 hour, and then dilute 8 gradients by 2.5 times. Add 50 μl / well of pre-diluted antibody to a 96-well cell culture plate (Corning, 3610). Add 50 μl / well of previously diluted cells (5E3 cells / well /) to a 96-well culture plate. After mixing, the cells were cultured for 7 days at 37 ° C and 5% CO ₂ . After incubation, add 50 μl / well

Luminescent Cell Viability Assay (Promega, G7571) into a 96-well cell culture plate. Use a multifunctional microplate reader (BioTek, Synergy 2) to test the chemiluminescence value, use a 4-parameter equation to fit the curve, and use the chemiluminescence value Lum to plot the antibody concentration or the percentage of cytotoxicity as follows: Cytotoxicity (%) = 100 * (RLUCell only-RLU Sample) / RLU Cell only *%.

12. Effect of antibodies in mouse models

A. Treatment of delayed overexpressing CLDN18A2 tumors in mice

Fifty female BALB / c nude mice were inoculated with human gastric cancer cell NUGC-4 subcutaneously in the right armpit. When the tumor volume grew to a suitable size, 36 tumor-bearing mice with a tumor volume of 37.54 to 124.64 mm3 were selected and randomly divided into 6 groups: 1- negative control group, 2- positive control group (0.2mg / head), 3-RB0011-FK-13 group (0.2mg / head), 4-RB0011-FK-1 group (0.2mg / head), 5- In the RB0011-FK-2 group (0.2 mg / head) and 6-186CG4-mut group (0.2 mg / head), 6 animals in each group were administered twice a week after grouping for a total of 4 weeks by intravenous injection. General clinical observations were performed twice a day after the first dose, and the tumor diameter was measured and measured twice a week. At D29, animals were euthanized, tumor tissues were stripped and weighed, and tumor volume, relative tumor volume (RTV), and relative tumor volume were calculated. Tumor proliferation rate (T / C%).

result

1. Preparation and identification of human and mouse chimeric antibodies

a. Flow cytometry to determine the binding ability of human and mouse chimeric antibodies and cells

One weakly positive clone (170B3) and two strong positive clones (180G8 and 186F7) were screened specifically against HEK293F_human claudin 18.2.

Table 1 Binding of each clone to HEK293F_human claudin 18.2

Table 2.Specific binding of each clone to HEK293F_human claudin 18.2

b. Flow cytometry to determine the binding ability of human and mouse chimeric antibodies to cancer cell lines

Two strong positive clones (180G8 and 186F7) obtained from the screening were able to bind NUGC-4

抗体antibody	同型对照Isotype	182GB182GB	186F7186F7
MFIMFI	371371	1330013300	1000010000

c. FACS method for measuring the affinity of human and mouse chimeric antibodies and HEK293F_human claudin 18.2 cell line

抗体antibody	170B3170B3	180GB180GB	186F7186F7
EC50(nM)EC50 (nM)	12331233	13.7113.71	6.6596.659

2. ADCC effect of humanized antibodies

The two humanized antibodies and the HEK293F_human claudin 18.2 cell line showed ADCC effects, with EC50s of 0.019 μg / ml and 0.003 μg / ml, respectively. See attached picture 1.

3. The CDC effect of humanized antibodies

The two humanized antibodies and the HEK293F_human claudin 18.2 cell line did not show a significant CDC effect, see FIG. 2.

4. Epitope mapping

5. Species binding characteristics and affinity determination of humanized antibodies (EC50 (nM))

e. Proliferation inhibition

The two humanized antibodies did not show a significant proliferation inhibitory effect on the HEK293F_human claudin 18.2 cell line. See Figure 3.

6. Inhibition of proliferation

Two humanized antibodies showed a certain endocytosis effect on HEK293F_human claudin 18.2 cell line. See Figure 4.

7. Effect of antibodies in mouse models

a. Treatment of delayed overexpressing CLDN18A2 tumors in mice

This experiment successfully established a subcutaneous transplanted tumor model of human gastric cancer NUGC-4 in BALB / c nude mice. Under the conditions of this experiment, positive control substances, test substances RB0011-FK-13, RB0011-FK-1, RB0011-FK-2, and 186CG4-mut were administered at a dose of 2 mg / head twice a week for a total intravenous administration. The drug had inhibitory effects on the growth of human gastric cancer NUGC-4 BALB / c subcutaneously transplanted tumors in nude mice for 4 weeks. See Figure 5 and Figure 6.

Claims

The anti-human claudin 18.2 monoclonal antibody has three heavy chain CDRs and three light chain CDRs, which is characterized by:

The heavy chain CDR1 is selected from: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID: NO: 49, SEQ ID: NO: 55, SEQ ID: NO: 61, SEQ ID: NO: 67, SEQ ID: NO: 73 or a homology greater than 50% sequence;

The heavy chain CDR2 is selected from: SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID: NO: 50, SEQ ID: NO: 56, SEQ ID: NO: 62, SEQ ID: NO: 68, SEQ ID: NO: 74 or a homology with one of which is greater than 50% sequence;

The heavy chain CDR3 is selected from: SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID: NO: 51, SEQ ID: NO: 57, SEQ ID: NO: 63, SEQ ID: NO: 69, SEQ ID: NO: 75 or a homology with one of which is greater than 50% sequence;

The light chain CDR1 is selected from: SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID: NO: 52, SEQ ID: NO: 58, SEQ ID: NO: 64, SEQ ID: NO: 70, SEQ ID: NO: 76 or a homology with one that is greater than 50% sequence;

The light chain CDR2 is selected from: SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID: NO: 53, SEQ ID: NO: 59, SEQ ID: NO: 65, SEQ ID: NO: 71, SEQ ID: NO: 77 or a homology with one of which is greater than 50% sequence;

The light chain CDR3 is selected from: SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID: NO: 54, SEQ ID: NO: 60, SEQ ID: NO: 66, SEQ ID: NO: 72, SEQ ID: NO: 78 or a homology with one of which is greater than 50% sequence.
The antibody of claim 1, wherein the heavy chain variable region comprises the heavy chain CDR1, CDR2, and CDR3; and the light chain variable region comprises the light chain CDR1, CDR2, and CDR3.
The antibody according to claim 2, characterized in that the amino acid sequence of the variable region of the heavy chain is selected from the group consisting of: SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 85 : 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 or SEQ ID NO: 103 One of

The amino acid sequence of the light chain variable region is selected from the group consisting of: SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86; SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92. One of SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, or SEQ ID NO: 104.
The antibody according to claim 3, wherein

The heavy chain variable region is SEQ ID NO: 79 and the light chain variable region is SEQ ID NO: 80; or the heavy chain variable region is SEQ ID NO: 81 and the light chain variable region Is SEQ ID NO: 82; or the heavy chain variable region is SEQ ID NO: 83 and the light chain variable region is SEQ ID NO: 84; or the heavy chain variable region is SEQ ID NO: 85 And the light chain variable region is SEQ ID NO: 86; or the heavy chain variable region is SEQ ID NO: 87 and the light chain variable region is SEQ ID NO: 88; or the heavy chain may be The variable region is SEQ ID NO: 89 and the light chain variable region is SEQ ID NO: 90; or the heavy chain variable region is SEQ ID NO: 91 and the light chain variable region is SEQ ID NO: 92; or the heavy chain variable region is SEQ ID NO: 93 and the light chain variable region is SEQ ID NO: 94; or the heavy chain variable region is SEQ ID NO: 95 and the light chain The variable region is SEQ ID NO: 96; or the heavy chain variable region is SEQ ID NO: 97 and the light chain variable region is SEQ ID NO: 98; or the heavy chain variable region is SEQ ID NO: 99 and the light chain variable region is SEQ ID NO: 100; or the heavy chain variable region is SEQ ID NO: 101 and the light chain variable region SEQ ID NO: 102; or the heavy chain variable region is SEQ ID NO: 103 and the light chain variable region SEQ ID NO: 104.
The antibody according to claim 2, characterized in that the sequence of the amino acid in the variable region of the heavy chain is a similar sequence having at least 50% homology with the sequence a, and the sequence a is SEQ ID NO: 79, SEQ ID NO: 81, SEQ IDNO: 83, SEQ IDNO: 85; SEQ IDNO: 87, SEQ IDNO: 89, SEQ IDNO: 91, SEQ IDNO: 93, SEQ IDNO: 95, SEQ IDNO: 97. One of SEQ ID NO: 99, SEQ ID NO: 101, or SEQ ID NO: 103.
The antibody according to claim 2, characterized in that the amino acid sequence of the light chain variable region is a similar sequence having at least 50% homology with sequence b, and the sequence b is SEQ ID NO: 80, SEQ ID NO: 82, SEQ IDNO: 84, SEQ IDNO: 86; SEQ IDNO: 88, SEQ IDNO: 90, SEQ IDNO: 92, SEQ IDNO: 94, SEQ IDNO: 96, SEQ IDNO: 98. One of SEQ ID NO: 100, SEQ ID NO: 102, or SEQ ID NO: 104.
The antibody according to claim 1, wherein the heavy chain CDR1 is SEQ ID NO: 1, the heavy chain CDR2 is SEQ ID NO: 2, the heavy chain CDR3 is SEQ ID NO: 3, and the light chain CDR1 is SEQ ID NO: 4, light chain CDR2 is SEQ ID NO: 5, light chain CDR3 is SEQ ID NO: 6; or heavy chain CDR1 is SEQ ID NO: 7, heavy chain CDR2 is SEQ ID NO: 8, and heavy chain CDR3 is SEQ ID NO: 9, and light chain CDR1 is SEQ ID NO: 10, light chain CDR2 is SEQ ID NO: 11, light chain CDR3 is SEQ ID NO: 12; or heavy chain CDR1 is SEQ ID NO: 13, and heavy chain CDR2 Is SEQ ID NO: 14, heavy chain CDR3 is SEQ ID NO: 15, and light chain CDR1 is SEQ ID NO: 16, light chain CDR2 is SEQ ID NO: 17, light chain CDR3 is SEQ ID NO: 18; or The chain CDR1 is SEQ ID NO: 19, the heavy chain CDR2 is SEQ ID NO: 20, the heavy chain CDR3 is SEQ ID NO: 21, and the light chain CDR1 is SEQ ID NO: 22, and the light chain CDR2 is SEQ ID NO: 23, Light chain CDR3 is SEQ ID NO: 24; or heavy chain CDR1 is SEQ ID NO: 25, heavy chain CDR2 is SEQ ID NO: 26, heavy chain CDR3 is SEQ ID NO: 27, and light chain CDR1 is SEQ ID NO: 28. The light chain CDR2 is SEQ ID NO: 29, and the light chain CDR3 is SEQ ID NO: 30; or the heavy chain CDR 1 is SEQ ID NO: 31, heavy chain CDR2 is SEQ ID NO: 32, heavy chain CDR3 is SEQ ID NO: 33, and light chain CDR1 is SEQ ID NO: 34, light chain CDR2 is SEQ ID NO: 35, light Chain CDR3 is SEQ ID NO: 36; or heavy chain CDR1 is SEQ ID NO: 37, heavy chain CDR2 is SEQ ID NO: 38, heavy chain CDR3 is SEQ ID NO: 39, and light chain CDR1 is SEQ ID NO: 40 Light chain CDR2 is SEQ ID NO: 41, light chain CDR3 is SEQ ID NO: 42; or heavy chain CDR1 is SEQ ID NO: 43, heavy chain CDR2 is SEQ ID NO: 44, and heavy chain CDR3 is SEQ ID NO: 45, and the light chain CDR1 is SEQ ID NO: 46, the light chain CDR2 is SEQ ID NO: 47, the light chain CDR3 is SEQ ID NO: 48; or the heavy chain CDR1 is SEQ ID NO: 49, and the heavy chain CDR2 is SEQ ID NO: 50, heavy chain CDR3 is SEQ ID NO: 51, and light chain CDR1 is SEQ ID NO: 52, light chain CDR2 is SEQ ID NO: 53, light chain CDR3 is SEQ ID NO: 54; or heavy chain CDR1 is SEQ ID NO: 55, heavy chain CDR2 is SEQ ID NO: 56, heavy chain CDR3 is SEQ ID NO: 57, and light chain CDR1 is SEQ ID NO: 58, light chain CDR2 is SEQ ID NO: 59, light chain CDR3 Is SEQ ID NO: 60; or CDR1 of the heavy chain is SEQ ID NO: 61, and CDR2 of the heavy chain is SEQ ID NO: 62; Chain CDR3 is SEQ ID NO: 63, and light chain CDR1 is SEQ ID NO: 64, light chain CDR2 is SEQ ID NO: 65, light chain CDR3 is SEQ ID NO: 66; or heavy chain CDR1 is SEQ ID NO: 67 , Heavy chain CDR2 is SEQ ID NO: 68, heavy chain CDR3 is SEQ ID NO: 69, and light chain CDR1 is SEQ ID NO: 70, light chain CDR2 is SEQ ID NO: 71, and light chain CDR3 is SEQ ID NO: 72; or heavy chain CDR1 is SEQ ID NO: 73, heavy chain CDR2 is SEQ ID NO: 74, heavy chain CDR3 is SEQ ID NO: 75, and light chain CDR1 is SEQ ID NO: 76, and light chain CDR2 is SEQ ID NO: 77, light chain CDR3 is SEQ ID NO: 78.
The antibody according to claim 1, characterized in that the amino acid sequence of the antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3 and a light chain CDR1, a light chain CDR2, and a light chain CDR3; the heavy chain CDR1 is SEQ ID NO: 1. The heavy chain CDR2 is SEQ ID NO: 2, the heavy chain CDR3 is SEQ ID NO: 3, and the light chain CDR1 is SEQ ID NO: 4, the light chain CDR2 is SEQ ID NO: 5, and the light chain CDR3 is SEQ ID NO : 6; or heavy chain CDR1 is SEQ ID NO: 7, heavy chain CDR2 is SEQ ID NO: 8, heavy chain CDR3 is SEQ ID NO: 9, and light chain CDR1 is SEQ ID NO: 10, and light chain CDR2 is SEQ ID NO: 11, light chain CDR3 is SEQ ID NO: 12; or heavy chain CDR1 is SEQ ID NO: 13, heavy chain CDR2 is SEQ ID NO: 14, heavy chain CDR3 is SEQ ID NO: 15, and light chain CDR1 Is SEQ ID NO: 16, light chain CDR2 is SEQ ID NO: 17, light chain CDR3 is SEQ ID NO: 18; or heavy chain CDR1 is SEQ ID NO: 19, heavy chain CDR2 is SEQ ID NO: 20, heavy chain CDR3 is SEQ ID NO: 21, and light chain CDR1 is SEQ ID NO: 22, light chain CDR2 is SEQ ID NO: 23, light chain CDR3 is SEQ ID NO: 24; or heavy chain CDR1 is SEQ ID NO: 25, The heavy chain CDR2 is SEQ ID NO: 26, and the heavy chain CDR3 is SEQ ID NO: 27 And the light chain CDR1 is SEQ ID NO: 28, the light chain CDR2 is SEQ ID NO: 29, the light chain CDR3 is SEQ ID NO: 30; or the heavy chain CDR1 is SEQ ID NO: 31, and the heavy chain CDR2 is SEQ ID NO: 32. The heavy chain CDR3 is SEQ ID NO: 33, and the light chain CDR1 is SEQ ID NO: 34, the light chain CDR2 is SEQ ID NO: 35, and the light chain CDR3 is SEQ ID NO: 36; or the heavy chain CDR1 is SEQ ID NO: 37, heavy chain CDR2 is SEQ ID NO: 38, heavy chain CDR3 is SEQ ID NO: 39, and light chain CDR1 is SEQ ID NO: 40, light chain CDR2 is SEQ ID NO: 41, and light chain CDR3 is SEQ ID NO: 42; or heavy chain CDR1 is SEQ ID NO: 43, heavy chain CDR2 is SEQ ID NO: 44, heavy chain CDR3 is SEQ ID NO: 45, and light chain CDR1 is SEQ ID NO: 46, light chain CDR2 Is SEQ ID NO: 47, light chain CDR3 is SEQ ID NO: 48; or heavy chain CDR1 is SEQ ID NO: 49, heavy chain CDR2 is SEQ ID NO: 50, heavy chain CDR3 is SEQ ID NO: 51, and light Chain CDR1 is SEQ ID NO: 52, light chain CDR2 is SEQ ID NO: 53, light chain CDR3 is SEQ ID NO: 54; or heavy chain CDR1 is SEQ ID NO: 55, and heavy chain CDR2 is SEQ ID NO: 56, The heavy chain CDR3 is SEQ ID NO: 57, the light chain CDR1 is SEQ ID NO: 58, and the light chain CDR2 is SEQ ID NO: 59, light chain CDR3 is SEQ ID NO: 60; or heavy chain CDR1 is SEQ ID NO: 61, heavy chain CDR2 is SEQ ID NO: 62, heavy chain CDR3 is SEQ ID NO: 63, and light chain CDR1 is SEQ ID NO: 64, light chain CDR2 is SEQ ID NO: 65, light chain CDR3 is SEQ ID NO: 66; or heavy chain CDR1 is SEQ ID NO: 67, heavy chain CDR2 is SEQ ID NO: 68, heavy chain CDR3 Is SEQ ID NO: 69, and light chain CDR1 is SEQ ID NO: 70, light chain CDR2 is SEQ ID NO: 71, light chain CDR3 is SEQ ID NO: 72; or heavy chain CDR1 is SEQ ID NO: 73, heavy Chain CDR2 is SEQ ID NO: 74, heavy chain CDR3 is SEQ ID NO: 75, and light chain CDR1 is SEQ ID NO: 76, light chain CDR2 is SEQ ID NO: 77, and light chain CDR3 is SEQ ID NO: 78.
A nucleic acid molecule, characterized in that it comprises a nucleic acid sequence capable of encoding a heavy chain CDR and / or a light chain CDR, and the heavy chain CDR1 is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 61, SEQ ID NO: 67, one of SEQ ID NO: 73 or a similar sequence with a homology greater than 50%;

The heavy chain CDR2 is selected from: SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID: NO: 50, SEQ ID: NO: 56, SEQ ID: NO: 62, SEQ ID: NO: 68, SEQ ID: NO: 74 or a homology with one of which is greater than 50% sequence;

The heavy chain CDR3 is selected from: SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID: NO: 51, SEQ ID: NO: 57, SEQ ID: NO: 63, SEQ ID: NO: 69, SEQ ID: NO: 75 or a homology with one of which is greater than 50% sequence;

The light chain CDR1 is selected from: SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID: NO: 52, SEQ ID: NO: 58, SEQ ID: NO: 64, SEQ ID: NO: 70, SEQ ID: NO: 76 or a homology with one that is greater than 50% sequence;

The light chain CDR2 is selected from: SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID: NO: 53, SEQ ID: NO: 59, SEQ ID: NO: 65, SEQ ID: NO: 71, SEQ ID: NO: 77 or a homology with one of which is greater than 50% sequence;

The light chain CDR3 is selected from: SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID: NO: 54, SEQ ID: NO: 60, SEQ ID: NO: 66, SEQ ID: NO: 72, SEQ ID: NO: 78 or a homology with one of which is greater than 50% sequence.
A nucleic acid molecule, characterized in that it contains a nucleic acid sequence capable of encoding a heavy chain variable region and / or a light chain variable region, and the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, or SEQ ID NO: 103;

The amino acid sequence of the light chain variable region is selected from the group consisting of: SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86; SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92. One of SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, or SEQ ID NO: 104.
A vector, characterized in that it contains the nucleic acid molecule according to any one of claims 9 to 10.
A host cell, characterized in that the host cell contains the amino acid sequence according to any one of claims 1 to 8, the nucleic acid molecule according to any one of claims 9 to 10, or the vector according to claim 13. .
A conjugate comprising the antibody according to any one of claims 1 to 8.
A composition comprising a main component and an auxiliary component, the main component containing the antibody according to any one of claims 1 to 8 or the nucleic acid molecule according to claims 9 to 10 or claim 11 One or more of the carrier, the host cell according to claim 12, or the conjugate according to claim 13, the auxiliary component is selected from a pharmaceutically acceptable carrier or excipient, and optionally Other biologically active substances.
The antibody according to any one of claims 1 to 8, the nucleic acid molecule according to claims 9 to 10, the vector according to claim 11, the host cell according to claim 12, or the conjugate according to claim 13. Or use of the composition according to claim 14 in the preparation of a medicament or a detection reagent for treating a disease.
The antibody according to any one of claims 1 to 8, the nucleic acid molecule according to claims 9 to 10, the vector according to claim 11, the host cell according to claim 12, or the conjugate according to claim 13. Or use of the composition according to claim 14 in the preparation of a medicament or a detection reagent for treating a disease, which is a malignant tumor, a cardiovascular disease or an inflammatory disease.
A kit comprising the antibody according to any one of claims 1 to 8.