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Monoclonal antibody 9A and application thereof

A technology combining molecules and heavy chains, applied in the fields of cellular immunology and molecular biology, can solve problems such as toxic side effects and poor specificity

Active Publication Date: 2018-05-04
HANGZHOU SUMGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] As one of the conventional cancer treatment methods, chemotherapy has played an important role in cancer treatment, but due to its poor specificity and clinical side effects, medical staff and researchers are seeking better treatment strategies

Method used

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  • Monoclonal antibody 9A and application thereof
  • Monoclonal antibody 9A and application thereof
  • Monoclonal antibody 9A and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Antibody Screening

[0088] In order to avoid the bias of individual immune background and ensure the diversity of the antibody library as much as possible, the peripheral blood of 100 healthy adults (half male and female) and umbilical cord blood lymphocytes of 10 neonates (half male and female) were separated by lymphocyte separation medium , a total of 2×10 9 cells. Total RNA was extracted by Trizol method, reverse transcribed into cDNA, and the variable region genes of different antibody subtypes were amplified by conventional PCR. According to the information of the antibody library vector pDF (Journal of the Academy of Military Medical Sciences, 2008, 32 (4): 305-308, 358.), the restriction site BssH II, Nhe I and the Loxp511 sequence (sequence: SGGSTITSYNVYYTKLSSSGT (SEQ The connecting peptide of ID NO: 15)) was spliced ​​into ScFv (single-chain antibody: VL-Linker (containing Loxp511 sequence)-VH, BssH II and Nhe I sites were introduced into the upst...

Embodiment 2

[0091] Example 2 Antibody expression and purification

[0092] The obtained 9A clone variable region gene was cloned into the eukaryotic expression vector pCMV-163 containing the human IgG constant region gene to construct a full antibody expression vector, and its physical map is as follows: figure 1Shown (each component of the eukaryotic expression vector pCMV-163 is a component known in the art, recombined according to the sequence shown). The whole antibody is called 9A antibody (the light chain amino acid sequence is SEQ ID NO: 11, the nucleotide sequence is SEQ ID NO: 12; the heavy chain amino acid sequence is SEQ ID NO: 13, the nucleotide sequence is SEQ ID NO: 14) . The obtained eukaryotic expression vector was transfected into CHO-S cells by using the ExpiCHOTM Expression System kit (Thermo Fisher Scientific, #A29133), and the goat anti-human IgG (KPL, KPL, #01-10-06) and horseradish enzyme-labeled goat anti-human IgG (GOAT Antihuman (HRP), Thermo Fisher Scientific,...

Embodiment 3

[0093] Example 3 Antibody Binding Activity Analysis

[0094] Coat the target antigen AXL-Fc on the ELISA plate, 1 μg / ml, overnight at 4°C; after washing with PBST, add 10% fetal bovine serum, block at 37°C for 1 hour; add different concentrations of 9A antibody, react at 37°C for 1 hour ; After washing with PBST, add horseradish peroxidase-labeled goat anti-human Fab secondary antibody (Goat Anti-human IgG (Fab') 2-HRP, Abcam), and react at 37°C for 30 minutes; repeat washing the plate 5 times with PBST, Pat dry the remaining droplets on absorbent paper as much as possible; add 100 μl TMB (eBioscience) to each well, and place in the dark at room temperature (20±5°C) for 1.5 min; add 100 μl 2N H to each well 2 SO 4 The stop solution terminated the substrate reaction, and the OD value was read at 450nm on a microplate reader to analyze the binding ability of the antibody to the target antigen AXL-Fc. The 9A antibody can specifically recognize the target antigen AXL-Fc; the rec...

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Abstract

The invention discloses a separated binding molecule. The binding molecule is a monoclonal antibody or an antigen binding segment aiming at AXL proteins. In-vitro experiments prove that the monoclonalantibody is capable of being specifically bound with the AXL proteins, has relatively strong affinity activity and is further capable of preventing and stopping diseases caused by the overexpressionof the AXL proteins. According to the research results of the invention, on one hand a new method is provided for clinical diagnosis, and on the other hand, a candidate drug is provided for the clinical treatment of diseases such as tumor relevant with overexpression of the AXL proteins.

Description

technical field [0001] The invention belongs to the fields of cellular immunology and molecular biology, and relates to a monoclonal antibody against AXL. In addition, the invention also relates to a preparation method and application of the antibody. Background technique [0002] Cancer has become a major cause of human death. According to a research data, the morbidity and mortality rate of global tumors are increasing year by year: in 2008, the incidence of cancer in the world reached 12.7 million, and it is expected to reach 20.3 million by 2030; the number of deaths caused by cancer in 2008 was 7.6 million million, which is expected to increase to 13.2 million by 2030. According to the 2015 China Cancer Statistics, based on the trend analysis of the number of cancer cases and deaths from 2000 to 2011, the results show that the number of new cancer cases in my country in 2015 is estimated to be 4.292 million and the number of deaths to be 2.814 million. With the increa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63C12N5/10G01N33/68G01N33/574G01N33/577A61K39/395A61P35/00
CPCA61K2039/505C07K16/2863C07K2317/50G01N33/574G01N33/577G01N33/6863G01N2800/085
Inventor 高婵
Owner HANGZHOU SUMGEN BIOTECH CO LTD
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