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Anti-Claudin 18_2 antibody and application thereof

An antibody and antigen technology, applied in the fields of application, antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problem of loose structure, incomplete protein glycosylation, and easy invasion and metastasis of gastric cancer cells And other issues

Active Publication Date: 2020-03-06
NANJING SANHOME PHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the structure of gastric cancer cells is loose, the cells are prone to invasion and metastasis, and the glycosylation of CLDN18.2 protein expressed on them is incomplete, which may lead to target exposure (WO2004 / 047863)

Method used

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  • Anti-Claudin 18_2 antibody and application thereof
  • Anti-Claudin 18_2 antibody and application thereof
  • Anti-Claudin 18_2 antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] The preparation of embodiment 1 positive control antibody

[0112] 1. Preparation of positive control antibody IMAB362-similar-hG1:

[0113] According to the 175D10 clone (IMAB362) antibody sequence (ie, SEQ ID NO 11 and SEQ ID NO 12 in this disclosure) provided by the patent application WO2007 / 059997, Nanjing GenScript Co., Ltd. was entrusted to perform codon optimization and then synthesized in pcDNA3. 1(+) vector. The constructed plasmids include pcDNA3.1-hG1 and pcDNA3.1-hK. The plasmid was co-transfected into expiCHO cells at a ratio of 1:1 for transient expression at 110 rpm, 37°C, 8% CO 2 After culturing for 4 days, transfer to a shaker at 32°C to continue culturing for 8-10 days. Centrifuge at 4500rpm for 25 minutes, harvest the protein supernatant and purify by ProteinA affinity chromatography: wash the AKTA chromatography system and chromatography column with 0.1M NaOH solution for 30min, rinse with ultrapure water; column volume; set the flow rate at 2ml / ...

Embodiment 2

[0118] Example 2 CLDN18.2 antigen gene synthesis and expression vector construction

[0119] The fusion protein sequence design expressing human CLDN18.2 antigen is shown in Table 1 below:

[0120] Table 1 CLDN18.2 sequence design

[0121]

[0122] The fusion protein sequence of the human CLDN18.2 antigen designed above was optimized for eukaryotic codons, and a restriction site such as NotI and tPA signal peptide sequence were added to the 5' end of the gene, and a restriction site such as XhoI was added to the 3' end of the gene and a Myc-His fusion protein tag. The above sequences of SEQ ID NO 1, 3, 5, and 6 were synthesized and constructed into the pcDNA3.1(+) vector to obtain a DNA plasmid expressing human CLDN18.2 antigen. Plasmids include: pcDNA3.1-loop1, pcDNA3.1-TML, pcDNA3.1-CpG-loop1 and pcDNA3.1-CLDN18.2-FL.

[0123] The aforementioned sequences of SEQ ID NO 7 and 9 were optimized and synthesized by Treasure Bioengineering (Dalian) Co., Ltd. (hereinafter refe...

Embodiment 3

[0125] Example 3 Expression of CLDN18.2 antigenic protein

[0126] 1. Transient transfection of plasmid DNA in vitro

[0127] The plasmid amplified and cultured in Example 2 was transiently transfected into HEK293T cells in vitro (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, hereinafter referred to as Shanghai Cell Institute), and identified at the protein level. The specific experimental steps are as follows: the day before transfection, press 3×10 5 piece / cm 2 The seeding density of HEK293T cells was spread in six-well plates, cultured (medium composition: 10% fetal bovine serum DMEM medium + 1% penicillin-streptomycin) overnight, so that the cells were completely attached on the day of transfection, and Make the cell abundance reach 70%-90% (the size of each hole in a six-well plate is 9cm 2 , cells overgrow about 2-2.5×10 6 per well); use PEI cationic transfection reagent (sigma) or liposome (Lipo3000, Thermo) to mix with DNA plasmid to prep...

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Abstract

The invention relates to the technical field of antibody drugs, in particular to an anti-Claudin 18.2 antibody or antigen binding fragment thereof as well as a pharmaceutical composition containing the anti-Claudin 18.2 antibody or antigen binding fragment and application thereof. The antibody has the remarkable binding activity and affinity with Claudin 18.2 and has the high-efficiency in-vitro ADCC killing activity on tumor cells. The anti-Claudin 18.2 antibody or antigen binding fragment thereof can be applied to preparation of anti-tumor drugs and has the broad market prospects.

Description

technical field [0001] The present invention relates to the technical field of antibody medicines, in particular to an anti-Claudin18.2 antibody or an antigen-binding fragment thereof, a pharmaceutical composition containing an anti-Claudin18.2 antibody or an antigen-binding fragment thereof, and applications thereof. Background technique [0002] Gastric cancer is the fifth most common type of cancer in the world, and its mortality rate is the third highest. Currently, chemotherapy is the standard first-line treatment for advanced or recurrent gastric cancer. More than 90% of cancer patients will receive ordinary chemotherapy and targeted chemotherapy, but chemotherapy generally cannot cure cancer, it can only prolong the survival period of patients and improve the quality of life, and it is easy to produce drug resistance. Biological targeted therapy usually targets tumor-specific antigens or tumor-associated antigens, and relies on the binding or blocking of antibodies t...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13A61K39/395A61P35/00
CPCC07K16/18A61P35/00C07K2317/56C07K2317/52C07K2317/24C07K2317/92C07K2317/732A61K2039/505C07K16/28C07K2317/565C07K2317/55
Inventor 王勇赵立文陆臻桢孙世超庞玉红魏坚周秋华韩璐薇宋其峰
Owner NANJING SANHOME PHARMACEUTICAL CO LTD
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