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EGFR antibody and application thereof

An antibody, bispecific antibody technology, applied in applications, antibodies, anti-tumor drugs, etc., can solve problems such as limited indications

Pending Publication Date: 2020-11-03
白先宏
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although high expression and / or abnormal activity of EGFR are the driving factors of many tumors, the scope of indications for monoclonal antibody therapy is relatively limited, which is related to drug activity and patient tolerance

Method used

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  • EGFR antibody and application thereof
  • EGFR antibody and application thereof
  • EGFR antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1 Mutant library design

[0076] Taking the heavy chain of Nimotuzumab as the parental sequence, the design scheme carries out biased mutations in its complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3. The mutation frequency refers to Sidhu SS et al. (J.Mol.Biol.(2004 )338, 299–310). Specifically, according to the 31st-35th amino acid (CDR1), 37th amino acid (secondary structure transition amino acid), 51-55 (CDR2, part) and 101-105 (CDR3, part) in the Kobat antibody sequence amino acid coding rules Assign the coding nucleotide sequence as listed in the table, and its theoretical amino acid diversity and frequency are also the same Figure 10 shown.

[0077] *Amino acid species are indicated by single letters in standard nomenclature.

[0078] **V=A, C or G, R=A or G, D=A, G or T, M=A or C, K=G or T, N=A, G, C or T.

[0079] The fixed sequence peptide of the variable region is also reverse-translated into a nucleotide sequence, which is spli...

Embodiment 2

[0084] Synthesis of embodiment 2 mutant library

[0085] 2.1, DNA fragment ligation

[0086] Composition system Total oligonucleotides (1 µl each) 13μL T4 DNA ligase 2.5μL 10x T4 DNA Ligase Reaction Buffer 4.5μL water 30μL total capacity 50μL Reaction conditions Ligation overnight at 16°C heat inactivation Heat at 60°C for 30 minutes

[0087] 2.2. Construction of antibody fragment mutation library

[0088] The DNA encoding the variable region of the light chain was obtained by PCR amplification, and the PCR reaction system was carried out with the heavy chain gene library of the mutant antibody by the method of overlapping PCR as follows.

[0089]

[0090] Take 1 μL of the product for electrophoresis and use Axygen AP-PCR-250G to purify the recovered fragments, and the concentration of the recovered fragments is measured to be 152 ng / μL.

[0091] 2.3. Digest the constructed ScFv mutant antibody library DNA a...

Embodiment 3

[0096] Embodiment 3 mutant library transformation Escherichia coli

[0097] 3.1 Preparation of electroporation competent

[0098] Pick TG1 monoclonal clones from the M9 plate and insert them into 40ml 2TY medium, culture at 37OC 250rpm overnight.

[0099] Inoculate 500ml of 2×YT medium with 5ml of the bacterial liquid, and shake at 250rpm at 37°C.

[0100] OD of TG1 bacterial solution 600 About 0.5 or so, take it out from the shaker, and ice bath for 1 hour.

[0101] Centrifuge the bacterial solution at 2200×g for 12 minutes at 4°C.

[0102] Discard the supernatant and resuspend the pellet with 500ml pre-cooled deionized water. Centrifuge at 2200×g, 4°C for 12 minutes.

[0103] Discard the supernatant and resuspend the pellet with 250ml pre-cooled deionized water. Centrifuge at 2200×g, 4°C for 12 minutes.

[0104] Discard the supernatant and resuspend the pellet with 30ml pre-cooled 10% glycerol. Centrifuge at 2200×g, 4°C for 12 minutes.

[0105] Discard the supernata...

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Abstract

The invention provides an EGFR antibody and an application thereof. Specifically, the invention discloses an anti-EGFR antibody or the antigen binding fragment thereof with higher affinity and higherADCC killing activity, which is obtained by constructing a mutation library for screening on the basis of the Nimotuzumab, and the anti-EGFR antibody or the antigen binding fragment thereof is used for cancer treatment.

Description

technical field [0001] The disclosure belongs to the field of biotechnology, and specifically, the disclosure provides an EGFR antibody and applications thereof. Background technique [0002] Epidermal growth factor receptor EGFR is a member of the cell surface tyrosine kinase family, which is widely expressed on the surface of epidermis, pluripotent cells and nerve tissues, and plays a key role in the process of development and differentiation. EGFR, or HER1, c-erbB-1, is a type I transmembrane glycoprotein with a molecular weight of 170kDa (MLlrich et al., Nature, Vol.309,418-25,1986), and its ligands include EGF, transforming growth factor TGFa , EGF-like growth factor with heparin binding activity and various other hormones and cell growth factors (Singh, A. and Harris, R., 2005, Cell μLar Signaling 17:1183-1193). Ligand binding causes EGFR dimerization and phosphorylation, and induces tyrosine kinase signaling cascade reaction, DNA synthesis and cell division, so EGFR ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCC07K16/2863A61P35/00A61K2039/505C07K2317/56
Inventor 白先宏宋其峰曾彭越高志慧张会文李理想
Owner 白先宏
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