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Affinity mature binding protein of EGFR and its application

A protein-binding and affinity technology, applied in the field of affinity-matured binding proteins, can solve the problems of ineffective removal of small lesions, poor permeability, large molecular weight, etc., and achieve the effects of easy transformation, good stability, and application promotion.

Active Publication Date: 2022-06-24
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the monoclonal antibody targeting EGFR has achieved good results in the clinical treatment of non-small cell lung cancer, but because it is a chimeric antibody, it has certain immunogenicity, and because of its large molecular weight and poor penetrability, it cannot Effective removal of microscopic lesions

Method used

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  • Affinity mature binding protein of EGFR and its application
  • Affinity mature binding protein of EGFR and its application
  • Affinity mature binding protein of EGFR and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of Affinity Mature Binding Protein Library

[0039] (1) A mutant library was constructed using the wild-type binding protein aEG4D9 (having the nucleotide sequence shown in SEQ ID NO: 12) as a template. Mismatch-prone PCR was performed according to the instructions of the GeneMorph II Random Mutagenesis Kit (GeneMorph II, Stratagene). The binding protein DNA was amplified by mismatch-prone PCR and the SfiI restriction site was introduced. The primers were errorF1 (with the nucleotide sequence shown in SEQ ID NO: 16) and errorR1 (with the nucleotide sequence shown in SEQ ID NO: 17) acid sequence).

[0040] (2) The above PCR product was purified by agarose gel electrophoresis.

[0041] (3) After separating the bands of the correct size, follow the Gel Extraction Kit instructions operate gel recovery.

[0042] (4) The recovered DNA product is ready for use.

[0043] (5) SfiI enzyme was added to the mismatch-prone PCR product and pComb3XSS phage...

Embodiment 2

[0052] Example 2 Preparation of helper phage

[0053] (1) Take out the TG1 Escherichia coli cloned strain stored in glycerol from the -80°C refrigerator, spread it on a TYE plate without antibiotics by the method of four-district streak, and invert overnight at 37°C.

[0054] (2) Pick a TG1 monoclonal strain from the plate and inoculate it into 5 mL of 2×TY (antibiotic-free) liquid medium, and culture at 37° C. and 230 rpm overnight.

[0055] (3) The TG1 bacterial solution was transferred to 5 mL of 2×TY antibiotic-free body medium at a volume ratio of 1:100, and cultured at 37°C and 230 rpm for 2 h (the bacterial solution OD 600 = about 0.5).

[0056] (4) Take 200 μL of TG1 bacterial solution (OD 600 = about 0.5) into a 1.5 mL centrifuge tube, add 10 μL KM13 helper phage (1×10 13 pfu / mL), put it into preheated 37°C water for 30min.

[0057] (5) After preparing soft agar and cooling it to about 40°C, pour the TG1 bacterial solution treated in step (4), mix, and then pour t...

Embodiment 3

[0062] Example 3 Amplification of Affinity Matured Binding Protein Libraries

[0063] (1) Thaw 5 mL of the bacteria containing the binding protein library on ice, transfer it to 500 mL of LB liquid medium (containing 0.1% ampicillin and 1% glucose by weight), and culture at 37°C for 2.5 hours on a shaker .

[0064] (2) The number of additions is 2×10 12 The pfu helper phage KM13 was incubated at 37°C for 30 min.

[0065] (3) Centrifuge at 3200g for 10min, and discard the supernatant.

[0066] (4) Resuspend with fresh LB liquid medium (containing 0.1% ampicillin by weight and 1% glucose by weight). Shaker at 25°C for 20h.

[0067] (5) Centrifuge at 3200g for 20min, and collect the supernatant.

[0068] (6) Add 20% (w / v) PEG / NaCl solution to precipitate the phage in the solution, and place on ice for 4 hours.

[0069] (7) Centrifuge at 3200g for 30min, and collect the phage pellet.

[0070] (8) Resuspend the obtained phage pellet in sterile PBS.

[0071] (9) The prepared...

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Abstract

The invention discloses an anti-human EGFR affinity maturation binding protein and application thereof. The affinity maturation binding protein is an aEG11A6 affinity maturation binding protein whose amino acid sequence is shown in SEQ ID NO:1; or an aEG12E2 affinity maturation binding protein whose amino acid sequence is shown in SEQ ID NO:2, and whose amino acid sequence is such as SEQ ID NO:3 The shown aEG13E8 affinity maturation binding protein and amino acid sequence are the binding protein formed by combining at least one of the aEG22C4 affinity maturation binding protein shown in SEQ ID NO: 4 with the aEG11A6 affinity maturation binding protein. The affinity-matured binding protein has high affinity, high specificity and low immunogenicity, good stability, simple structure, and easy engineering transformation, etc., and can be better applied to the development of binding protein drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an affinity maturation binding protein of human EGFR and its application. Background technique [0002] The term cancer was proposed in more than 400 BC, and it has not been conquered for more than 2,000 years. Today, the threat to human health is becoming more and more obvious. In recent years, studies have found that epidermal growth factor (EGFR) is overexpressed in various solid tumor cells and plays an important role in the occurrence and development of cancer. EGFR overexpression is positively associated with poor prognosis and drug resistance in a variety of cancers. The overexpression of EGFR in cancer cells leads to the continuous activation of the EGFR / EGF signaling pathway, thereby promoting the proliferation, migration and invasion of cancer cells, and can also promote the secretion of VEGF factors and induce the formation of blood vessels in the cancer tissue microenviron...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/70A61K39/395A61P35/00A61P37/00C12R1/19
CPCC07K16/2863C07K16/005C12N15/70A61P35/00A61P37/00C07K2317/569C07K2317/92C07K2317/565C07K2317/567C07K2317/14C07K2317/94A61K2039/505
Inventor 魏星陈涛
Owner JINAN UNIVERSITY