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Single-chain antibody of target EGFR dimerized interface and application of antibody

A single-chain antibody and interface technology, which is applied in applications, antibodies, antibody mimics/stents, etc., can solve the problems of large molecular weight, impact on tumor suppression effect, poor penetration, etc., and achieve small molecular weight, friendly production environment, The effect of high production efficiency

Active Publication Date: 2019-01-04
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Traditional monoclonal antibodies are composed of 2 identical heavy chains and 2 identical light chains, with relatively large molecular weight and poor penetration. In the treatment of solid tumors, the tumor inhibitory effect is greatly affected

Method used

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  • Single-chain antibody of target EGFR dimerized interface and application of antibody
  • Single-chain antibody of target EGFR dimerized interface and application of antibody
  • Single-chain antibody of target EGFR dimerized interface and application of antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1 Construction of genes for single-chain antibodies and optimization of codons

[0127] 1. Acquisition of EGFR dimerization interface targeting monoclonal antibody light chain gene and heavy chain gene

[0128] 1. Experimental operation

[0129] The inventor's previous research has obtained the monoclonal antibody Antidimer 5G9 that can target the dimerization interface of EGFR, and preserved its hybridoma cells.

[0130] Hybridoma cells targeting the EGFR dimerization interface were cultured, cells were collected, and RNA was extracted by Trizol method. After reverse transcription, use universal degenerate primers to amplify the heavy chain variable region and light chain variable region genes of the EGFR dimerization interface monoclonal antibody Antidimer 5G9 respectively, and clone them into the cloning vector pMD18-T vector by AT cloning superior. Sent to the sequencing company for sequencing analysis. The sequence was further optimized for Pichia pasto...

Embodiment 2

[0141] Example 2 Construction of Fusion Gene Recombinant Expression Vector of Single-chain Antibody EGFR-scFv

[0142] 1. Experimental operation

[0143] 1. PCR amplification of single chain antibody

[0144] Escherichia coli containing the fusion gene of the single-chain antibody EGFR-scFv (EGFR-scFv gene) was inoculated into 2ml of LB medium (containing 0.1% AMP), and cultured at 37°C and 180rpm for 14-16h to amplify the bacteria. Using the amplified Escherichia coli containing the single-chain antibody gene to extract the plasmid DNA therein, use it as a template strand for PCR amplification for PCR amplification.

[0145] The upstream primer for PCR amplification is: 5'-GACA CTCGAG AAGAGAGAGGCTGAGGC-3', the downstream primer is: 5'-ACTC TCTAGA TCAGTGGTGGTGGTGG-3'. Increased 5' upstream of the EGFR-scFv gene wxya Restriction site, 3' downstream increase wxya Restriction sites, and add protective bases at both ends.

[0146] PCR amplification system preparation: ...

Embodiment 3

[0171] Extraction and yeast transformation of embodiment 3 recombinant plasmids

[0172] 1. Experimental operation

[0173] 1. Extraction of recombinant plasmids

[0174] Shake 100ml of the recombinant, extract the pGAPZαA-EGFR-scFv plasmid (about 20 μg) with a small extraction kit, and linearize the recombinant plasmid with BlnI.

[0175] Enzyme cutting system:

[0176]

[0177] Enzyme digested in a water bath at 37°C for 2 hours, and samples were taken for gel electrophoresis detection. The resulting enzyme digestion supplies were desalted with phenol-chloroform. method:

[0178] (1) with ddH 2 O Make up 500 μl of the digested plasmid, add 500 μl of phenol-chloroform-isoamyl alcohol and mix by inverting up and down. Centrifuge at 4°C and 10,000rpm for 5min, carefully absorb the supernatant, and transfer to a 2ml EP tube.

[0179] (2) Add 3M NaAc to the sample at a ratio of 10:1 between the sample and NaAc (pH5.2), then add 2 times the volume of -20°C pre-cooled abs...

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Abstract

The invention discloses a single-chain antibody of a target EGFR dimerized interface and application of the antibody. Fusion protein contains an amino acid sequence in which heavy-chain variable-region protein, connecting peptide and light-chain variable-region protein are sequentially connected with one another from 5' to 3', wherein the amino acid sequence of the heavy-chain variable-region protein is shown as SEQ ID NO:1, and the amino acid sequence of the light-chain variable-region protein is shown as SEQ ID NO:3. Genes of the single-chain antibody of the target EGFR dimerized interface and Pichia pastoris engineering bacteria are constructed and optimized, and the single-chain antibody is obtained through fermentation and separation purification, that is to say that the single-chainantibody of the target EGFR dimerized interface is prepared efficiently and successfully by means of a genetic engineering technology. The antibody is low in relative molecular mass, high in penetrability, more excellent in tumor inhibition effect during solid tumor treatment and capable of effectively inhibiting abnormal expression of EGFR to prevent the proliferation and growth of tumor cells; afoundation is laid for later development of novel inhibitors of the EGFR, and the antibody has a high application value.

Description

technical field [0001] The present invention relates to the technical field of treating tumors with abnormal expression of EGFR, and more specifically, relates to a single-chain antibody targeting the dimerization interface of EGFR and its application. Background technique [0002] Epidermal growth factor receptor (EGFR for short) is a member of the tyrosine kinase type I receptor family, and other members of this family include HER2, HER3 and HER4. The EGFR receptor is divided into three parts, namely the extracellular ligand-binding region, the transmembrane immobilization region and the intracellular tyrosine kinase region. After the specific ligand binds to the EGFR receptor, it causes the receptor to change from an inactive monomer state to an activated dimerization state, and EGFR forms a dimer with itself or other members of the family, thereby activating the intracellular Tyrosine kinases, the tyrosine residues in the intracellular segment are autophosphorylated or ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13A61K39/395A61P35/00
CPCA61K39/395A61P35/00C07K16/2863C07K2317/56C07K2317/622C07K2319/00
Inventor 赵林李黄金蔡金艳郭土敬
Owner GUANGDONG PHARMA UNIV
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