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Monoclonal antibody specially combined with AXL

A monoclonal antibody, species-specific technology, applied in the fields of cellular immunology and molecular biology, which can solve problems such as elevated

Active Publication Date: 2018-03-06
SUMGEN MAB BEIJING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the concentration of soluble AXL (sAxl) in the serum of hepatocellular carcinoma (HCC), advanced fibrosis (F3 stage) or liver cirrhosis (F4 stage) is specifically increased; while in chronic viral hepatitis, autoimmune hepatitis, cholestatic Elevated sAxl levels were not detected in patients with liver disease or chronic liver disease (CLD) such as non-alcoholic fatty liver disease

Method used

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  • Monoclonal antibody specially combined with AXL
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  • Monoclonal antibody specially combined with AXL

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Hybridoma Technology Preparation of Monoclonal Antibodies Specifically Binding to AXL

[0085]Mammalian cells - Chinese hamster ovary cells CHO-K1 ( CCL-61TM) expressed human AXL protein (human AXL protein extracellular segment-human IgG1Fc segment fusion protein, named AXL-Fc, wherein the sequence information of AXL refers to NP_068713, and the sequence of human IgG1Fc fragment refers to AEO21920.1) routinely immunized with Balb / c mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). On days 0, 14 and 28, Balb / c was treated with 100 μg soluble AXL-Fc in the presence of Freund's complete adjuvant (first injection) or Freund's incomplete adjuvant (second and third injection). Mice were injected subcutaneously. Splenocytes from mice were fused with mouse myeloma cells SP2 / 0 (ATCC) using conventional hybridoma technology protocols (Salhi et al., Biochem. J. 2004). Cells were cultured in plates containing HAT medium (10 per well 5 cells) for...

Embodiment 2

[0086] Example 2 Monoclonal Antibody Recognition Target Antigen

[0087] 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 3D3 antibodies, react at 37°C for 1 hour ; After washing with PBST, add horseradish peroxidase-labeled goat anti-human Fab secondary antibody (GoatAnti-human IgG (Fab') 2HRP, Abcam), and react at 37°C for 30 minutes; repeat washing the plate 5 times with PBST, and place the plate on absorbent paper. Pat dry the remaining droplets 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 3D3 antibody can specifically recognize...

Embodiment 3

[0088] Example 3 Monoclonal antibody gene transfer

[0089] The variable domain of the mouse anti-AXL antibody was cloned from the hybridoma cells obtained in Example 1. RNA was prepared using the RNA extraction kit TRIzol Reagent (Life technologies). The cDNA encoding the antibody gene was prepared using a reverse transcription kit (Beijing Quanshijin Biotechnology Co., Ltd.) according to the manufacturer's instructions. Using the primers in Table 1, the antibody variable region gene was amplified by PCR, the T-easy vector was cloned, and the antibody variable region gene was obtained by sequencing. The obtained variable region gene was analyzed online at http: / / www.abysis.org / . Among the obtained antibody clones, the sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the amino acids of the CDR1, CDR2, and CDR3 regions are The sequences are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; the light chain variable region sequence is SEQ ID NO: 8, and the ...

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Abstract

The invention discloses a monoclonal antibody specially combined with AXL or an antigen combined segment thereof. The monoclonal antibody can be specifically combined with an AXL protein. The antibodydisclosed by the invention can be used for preventing and treating diseases induced by overexpression of the AXL protein, and has a wide application prospect clinically.

Description

technical field [0001] The invention belongs to the fields of cellular immunology and molecular biology, and relates to a monoclonal antibody specifically binding to AXL. Background technique [0002] AXL (Ark, Ufo and Tyro7) belongs to the receptor tyrosine kinase TAM family, which also includes Mer and Tyro3. The combination of AXL and the ligand Gas 6 can activate the tyrosine kinase activity of AXL, thereby activating its downstream signal transduction pathways such as PI3K / AKT, MAPK / ERK, and participating in the process of cell survival, proliferation, migration and adhesion. [0003] AXL is low / weakly expressed in various normal tissues such as brain, heart, skeletal muscle and some monocytes, but is highly / superhighly expressed in various tumors, and is associated with invasion, metastasis and poor prognosis, such as breast cancer, Colon cancer, prostate cancer, lung cancer, stomach cancer, ovarian cancer, endometrial cancer, kidney cancer, hepatocellular carcinoma, ...

Claims

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Application Information

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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 SUMGEN MAB BEIJING BIOTECH CO LTD
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