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EGFR and CD47 targeting bispecific fusion protein, and preparation method and application thereof

A fusion protein and bispecific antibody technology, applied in the biological field, can solve the problems of inability to eliminate and kill tumor cells, macrophage incapacity, etc., and achieve the effect of enhancing anti-tumor immune response and good anti-tumor effect

Active Publication Date: 2017-12-12
TAIZHOU MABTECH PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, the widespread expression of CD47 on the surface of various lymphoma and solid tumor cells inhibits the phagocytosis of tumor cells by macrophages
This also leads to the inability of macrophages to clear and kill tumor cells in tumor patients.

Method used

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  • EGFR and CD47 targeting bispecific fusion protein, and preparation method and application thereof
  • EGFR and CD47 targeting bispecific fusion protein, and preparation method and application thereof
  • EGFR and CD47 targeting bispecific fusion protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of novel bispecific antibody fusion protein Bi-SP

[0043] Using the "Knob-in-hole" antibody heavy chain constant region mutation technology, the Fc segment of the Pan side of the anti-EGFR and the Fc segment of the SIRPα mutant (SIRPα-Fc for short) are mutated, so that the Pan side of the anti-EGFR is compatible with CD47 One side of the receptor SIRPα mutant-Fc segment (SIRPα-Fc for short) can be correctly paired and assembled into the structure of the bispecific antibody fusion protein. The structure of the novel bispecific antibody fusion protein Bi-SP is as follows: figure 1 shown.

[0044] The Fc segment mutant Hole has the base sequence of SEQ ID NO:1 and the amino acid sequence described in EQ ID NO:2.

[0045] The Fc segment mutant Knobs has the base sequence of SEQ ID NO:3 and the amino acid sequence described in EQ ID NO:4.

[0046] The anti-EGFR Pan light chain of the mutation as described above has the base sequence of SEQ ID NO:5, ...

Embodiment 2

[0050] Example 2: Expression and purification of novel bispecific antibody fusion protein Bi-SP

[0051] The verified correct cloned gene was connected to the expression vector, and then transformed into CHO cells for the expression of the bispecific antibody fusion protein. Using Dot Blotting, CHO cell monoclonals with higher expression levels can be screened, expanded, and the culture supernatant harvested. Using Protein A affinity chromatography technology, the bispecific soluble antibody fusion protein Bi-SP with a purity of more than 90% was purified. SDS-PAGE results see figure 2 , lane 1 is molecular weight standard, lane 2 is Pan, lane 3 is SIRPα mutant, and lane 4 is Bi-SP.

experiment example 1

[0052] Experimental Example 1: In vitro biological function research of Bi-SP

[0053] 1. Obtain lung cancer H292 cells (referred to as H292-R) and epidermal cancer A431 cells (referred to as A431-R) resistant to cetuximab

[0054] Cetuximab was added to the culture medium of H292 cells and A431 cells to a concentration of 1 μg / mL, and the culture was continued for 6 months. The successful cultivation of drug-resistant cell lines was verified by in vitro and in vivo growth inhibition tests.

[0055] 2. In vitro biological function research of Bi-SP

[0056] (1) The molecular weight and purity of Bi-SP were characterized by SDS-PAGE.

[0057] (2) Analyze the binding activity of Bi-SP to EGFR-positive 231 cells and CD47-positive HL-60 cells by flow cytometry (FACS) ( image 3 , Figure 4 ).

[0058] (3) The dissociation constant of Bi-SP was determined by Biacore analysis.

[0059] (4) The ability of Bi-SP to promote macrophage phagocytosis was determined by flow cytometry....

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Abstract

The invention relates to a novel EGFR and CD47 targeting bispecific antibody fusion protein, and a preparation method and application thereof. The novel bispecific antibody fusion protein Bi-SP provided by the invention has a function of simultaneously binding EGFR and CD47; and at the same time of binding the EGFR, the bispecific antibody fusion protein can block or prevent the binding of the CD47 and SIRPalpha, reactivate the phagocytosis of macrophages and enhance an antitumor immune response, thereby achieving a better antitumor effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a novel bispecific antibody fusion protein targeting EGFR and CD47, a preparation method and application thereof. Background technique [0002] Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase that plays an important role in tumor progression. Excessive EGFR activation is associated with tumorigenesis and metastasis. It has been reported that 60%-80% of colon cancers have overexpression of EGFR. In addition, EGFR is also an important marker for predicting colorectal cancer and breast cancer. More importantly, EGFR is located on the surface of cancer cells, so it is an ideal target for the development of targeted antibodies. Antibodies can specifically bind to the extracellular domain III domain of EGFR, which not only prevents ligand binding, but also prevents dimerization of extended domains on domain II (Li S, et ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/85A61K39/395A61P35/00A61K38/17
CPCA61K38/1774A61K39/39558A61K2039/505C07K14/70503C07K16/40C07K2319/30A61K2300/00
Inventor 郭亚军寇庚钱卫珠郭怀祖徐进
Owner TAIZHOU MABTECH PHARM CO LTD
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