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Antibody targeting AXL protein, antigen binding fragment thereof, preparation method and application thereof

A technology for binding fragments and antibodies, applied in biochemical equipment and methods, antibodies, receptors/cell surface antigens/cell surface determinants, etc., can solve problems such as low blood half-life, easy drug resistance, single site, etc. , to achieve the effect of strong affinity, precise and effective tumor killing effect, strong binding ability and specificity

Active Publication Date: 2021-09-28
SHANGHAI SINOBAY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinical studies have shown that ADCs derived from existing AXL antibodies are prone to drug resistance due to their single site targeting tumor antigens, and their low blood half-life leads to increased toxicity

Method used

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  • Antibody targeting AXL protein, antigen binding fragment thereof, preparation method and application thereof
  • Antibody targeting AXL protein, antigen binding fragment thereof, preparation method and application thereof
  • Antibody targeting AXL protein, antigen binding fragment thereof, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0258] Example 1: Construction and screening of natural human antibody phage display library

[0259] AXL antigen preparation: Human recombinant AXL protein was purchased from novoprotein, Cat. No.: C02B.

[0260] AXL fully human antibody screening was entrusted to Sanyou Biomedical (Shanghai) Co., Ltd. A total of four rounds of screening were carried out, and the clones obtained in the fourth round were selected for ELISA screening of positive clones by ELISA. Finally, 71 positive clones capable of binding to hAXL-ECD protein were screened out in 2304 clones. After sequencing analysis and ELISA combination, the sequences of 4 clones were finally selected to construct full-length antibodies for further experiments. The specific implementation method is as follows:

[0261] 1.1 Sequencing and analysis of positive clones

[0262] After completing the preliminary screening, number 71 positive clones capable of binding to hAXL-ECD protein, pipette 2 μL of bacterial liquid in...

Embodiment 2

[0269] Example 2 Construction, expression and purification of full-length antibodies

[0270] In this example, the two Fab antibodies obtained in Example 1 with better blocking activity on binding to hAXL-ECD were constructed as human IgG1 subtype, in which the light chains were all of κ type, and the antibody type was fully human antibody.

[0271] 2.1 Plasmid construction

[0272] From the antibody-containing strains obtained by screening, the antibody light and heavy chain variable region fragments were amplified by PCR, and constructed into the eukaryotic expression vector plasmid AbVec-hIgKappa containing the light and heavy chain constant region fragments respectively by homologous recombination Or AbVec-hIgG1 WT ( FIG. 2 ), which constitutes the complete antibody light and heavy chain full-length genes, encodes the antibody heavy chain and light chain amino acid sequences shown in SEQ ID NO1-8.

[0273] The constructed vectors containing the full-length genes of the ...

Embodiment 3

[0277] Example 3 Affinity Test for Specific Binding of Candidate Antibodies to hAXL-ECD

[0278] The specific binding affinity of the candidate antibody to hAXL-ECD was detected by flow cytometry. A total of four clones, Hu001-5, Hu001-7, Hu001-11 and Hu001-14, were detected under acidic (PH=6.0) and neutral (PH=7.4) conditions, respectively.

[0279] Culture CHO-AXL cells (constructed by Shanghai Xinwan Biotechnology Co., Ltd.) in cell culture flasks. The medium is DMEM / F-12 medium (10% FBS, 1% penicillin / streptomycin), at 37°C, CO 2After culturing in the incubator for 48 hours, the supernatant was discarded, the cells were collected by digestion with 0.25% trypsin, and the cells were centrifuged at 800 g for 3 minutes. Discard the supernatant and resuspend the cells in 2% FBS wash buffer (10 6 / mL). Adjust the pH of the buffer solution and divide the cells into pH6.0 and pH7.4 groups, add primary antibodies (Hu001-5 antibody, Hu001-7 antibody, Hu001-11 antibody, Hu001-...

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Abstract

The invention provides an antibody targeting AXL protein or an antigen binding fragment thereof, a preparation method and application thereof. The invention also provides isolated polynucleotides encoding the antibodies or antigen binding fragments thereof, and vectors comprising the isolated polynucleotides. The AXL-targeting antibody provided by the invention can be combined with AXL protein with high affinity, can mediate ADCC to kill tumors, or can be used as a target recognition domain of CART cells to play an anti-tumor role, can be used for preventing or treating tumors, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of biological immunity, and specifically relates to an antibody capable of specifically binding to the AXL protein and an antigen-binding fragment thereof. The invention also relates to a preparation method and application of the antibody and an antigen-binding fragment thereof. Background technique [0002] AXL is a 104-140kDa transmembrane protein that belongs to the TAM subfamily of mammalian receptor tyrosine kinases (RTKs) and has transforming ability (Paccez et al., 2014). The AXL ectodomain consists of two membrane-distal N-terminal immunoglobulin (Ig)-like domains (Ig1 and Ig2 domains) combined with two membrane-proximal fibronectin type III (FNIII) repeats (FN1 and FN2 domains) ( Paccez et al., 2014). The AXL protein is encoded and expressed by the gene AXL (UFO, ARK, Tyro7 or JTK11), the receptor is a member of the Tyro-3 family of tyrosine kinases, and the binding ligand Gas6 (homologous to the a...

Claims

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Application Information

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IPC IPC(8): C07K16/40C12N5/10A61K39/00A61K39/395A61K45/06A61K47/68A61K48/00A61P35/00
CPCC07K16/40C12N5/0636C07K14/7051A61K47/6801A61K45/06A61K39/39558A61K39/0011A61K48/005A61K48/0008A61P35/00C12Y207/10001A61K2039/505C07K2317/56C07K2317/565C07K2317/52C12N2510/02A61K2300/00
Inventor 徐建青张晓燕丁相卿廖启彬
Owner SHANGHAI SINOBAY BIOTECH CO LTD
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