Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Affinity mature binding protein of EGFR and application

A technology for binding proteins and affinity, applied in the field of affinity mature binding proteins and applications, which can solve the problems of poor permeability, large molecular weight, and inability to effectively remove small lesions.

Active Publication Date: 2021-04-13
JINAN UNIVERSITY
View PDF4 Cites 2 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Affinity mature binding protein of EGFR and application
  • Affinity mature binding protein of EGFR and application
  • Affinity mature binding protein of EGFR and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of affinity maturation binding protein library

[0057] (1) Using the wild-type binding protein aEG4D9 (with the nucleotide sequence shown in SEQ ID NO: 12) as a template to construct a mutant library. Mismatch-prone PCR was performed according to the instructions of the GeneMorph II Random Mutagenesis Kit (GeneMorph II, Stratagene). The binding protein DNA is amplified by error-prone PCR and the SfiI restriction site is introduced, and the primers are errorF1 (with the nucleotide sequence shown in SEQ ID NO: 16) and errorR1 (with the nucleotide sequence shown in SEQ ID NO: 17 acid sequences).

[0058] (2) The above PCR products were purified by agarose gel electrophoresis.

[0059] (3) After separating the bands of the correct size, follow Gel Extraction Kit manual operation gel recovery.

[0060] (4) The recovered DNA product is ready for use.

[0061] (5) Add SfiI enzyme to the mismatch-prone PCR product and the pComb3XSS phagemid vector ...

Embodiment 2

[0070] Example 2 preparation of helper phage

[0071] (1) Take out the glycerol-preserved TG1 Escherichia coli clone strain from the -80°C refrigerator, spread it on a TYE plate without antibiotics by the method of four-section line, and culture it upside down overnight at 37°C.

[0072] (2) Pick a TG1 monoclonal strain from the plate and inoculate it into 5 mL of 2×TY (without antibiotics) liquid medium, and culture overnight at 37° C. and 230 rpm.

[0073] (3) Transfer the TG1 bacterial solution to 5 mL of 2×TY antibiotic-free body culture medium at a volume ratio of 1:100, and culture it at 37°C and 230 rpm for 2 hours (bacterial solution OD 600 = around 0.5).

[0074] (4) Take 200 μL TG1 bacterial solution (OD 600 =0.5 or so) into a 1.5mL centrifuge tube, add 10μL KM13 helper phage (1×10 13 pfu / mL), placed in preheated 37°C water bath for 30min.

[0075] (5) Prepare soft agar, let it cool to about 40°C, pour the TG1 bacterial solution treated in step (4), mix, and then...

Embodiment 3

[0080] Example 3 Amplification of affinity maturation binding protein library

[0081] (1) Thaw 5 mL of bacteria containing the binding protein library on ice, transfer to 500 mL of LB liquid medium (containing 0.1% ampicillin by mass volume ratio and 1% glucose by mass volume ratio), and culture in a shaking table at 37°C for 2.5 hours .

[0082] (2) The quantity added is 2×10 12 The pfu helper phage KM13 was incubated at 37°C for 30min.

[0083] (3) Centrifuge at 3200 g for 10 min, and discard the supernatant.

[0084] (4) Resuspend with fresh LB liquid medium (containing 0.1% ampicillin by mass volume ratio and 1% glucose by mass volume ratio). Incubate on a shaker at 25°C for 20 hours.

[0085] (5) Centrifuge at 3200 g for 20 min, and collect the supernatant.

[0086] (6) 20% (w / v) PEG / NaCl solution was added to precipitate the phage in the solution, and placed on ice for 4 hours.

[0087] (7) Centrifuge at 3200 g for 30 min to collect the phage precipitate.

[0088...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Titeraaaaaaaaaa
Login to View More

Abstract

The invention discloses an affinity mature binding protein of an anti-human EGFR and an application. The affinity mature binding protein is an aEG22C4 affinity mature binding protein with an amino acid sequence shown as SEQ ID NO: 4, or a binding protein formed by combining at least one of an aEG12E2 affinity mature binding protein with an amino acid sequence shown as SEQ ID NO: 2, an aEG13E8 affinity mature binding protein with an amino acid sequence shown as SEQ ID NO: 3 and an aEG11A6 affinity mature binding protein with an amino acid sequence shown as SEQ ID NO: 1 with the aEG22C4 affinity mature binding protein. The affinity mature binding protein has the advantages of high affinity, high specificity, low immunogenicity, better stability, simple structure, easiness in engineering transformation and the like, and can be better applied to 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 after 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 a variety of 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 many cancers. The overexpression of EGFR in cancer cells leads to the continuous activation of EGFR / EGF signaling pathway, thereby promoting the proliferation, migration and invasion of cancer cells. It can also promote the secretion of VEGF factors and induce the formation of blood vessels in the cancer tissue microenvironment. ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K16/28C12N15/13C12N15/70A61K39/395A61P35/00A61P37/00C12R1/19
CPCC07K16/2863C07K16/005C12N15/70A61P35/00A61P37/00C07K2317/569C07K2317/92C07K2317/565C07K2317/567C07K2317/94C07K2317/14A61K2039/505
Inventor 魏星陈涛
Owner JINAN UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com