EGFR-CD3 bifunctional antibody and application thereof

An EGFR-CD3, bifunctional antibody technology, applied in applications, antibodies, anti-tumor drugs, etc., can solve the problems of increasing the risk of nervous system complications, host rejection, etc., to promote differentiation and proliferation, and reduce immunogenicity. Effect

Active Publication Date: 2020-10-30
广东安普泽生物医药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its clinical use can cause host rejection, and the use of this drug in children may increase

Method used

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  • EGFR-CD3 bifunctional antibody and application thereof
  • EGFR-CD3 bifunctional antibody and application thereof
  • EGFR-CD3 bifunctional antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Antibody Preparation of EGFR-CD3 Bifunctional Antibody

[0036] According to the amino acid sequence matching of the heavy chain and light chain of the EGFR-CD3 bifunctional antibody, the heavy chain Cetuxi-H1L1, Cetuxi-H2L2, Cetuxi-H3L3, Cetuxi-H4L1, Cetuxi-H2L1, and Cetuxi-H3L2 of the bifunctional antibody were artificially synthesized, respectively. and light chain Cetuxi-L cDNA and constructed into pcDNA3.1 vector. The light chain plasmid and the heavy chain plasmid of the bifunctional antibody identified by sequencing were co-transfected into 293F cells at a weight ratio of 1:1.

[0037] Electroporation conditions: 4mm click cup, voltage 250v, 5mm, cells 1×10 8 . The electroporated cells were placed at 37° C., 5% CO2, and cultured at 130 rpm / min for 7 days. After the cells were cultured for 7 days, the supernatant was collected by centrifugation. The supernatant was centrifuged at 6000 rpm for 10 min, and the final concentration of 200 mM NaCl and 50 ...

Embodiment 2

[0038] Example 2: Detection of binding activity of EGFR-CD3 bifunctional antibody to CD3 protein on the surface of Jurkat cells

[0039] The cell surface protein binding ability of each EGFR-CD3 bifunctional antibody (Cetuxi-OKT11, Cetuxi-OKT22, Cetuxi-OKT33, Cetuxi-OKT41, Cetuxi-OKT21 and Cetuxi-OKT32) was detected by flow cytometry. The binding ability of EGFR-CD3 bifunctional antibody to CD3 protein on the surface of Jurkat cells was detected. The negative control of the experiment was set up as human IgG antibody, the positive control was humanized OKT clone antibody (produced by Sino Biological), and the experimental group was each expressed and purified EGFR-CD3 bifunctional antibody. Set detection cells to 1×10 6 Each tube was added with 2 μg / ml of each antibody and incubated on ice for 30 min. After washing the cells by centrifugation, Alexa 488 fluorescein-labeled mouse anti-human secondary antibody (diluted 1:200) was added and incubated on ice for 30 min. After t...

Embodiment 3

[0041] Example 3: EGFR-CD3 bifunctional antibody detection of EGFR protein binding activity on the surface of epithelial tumor cell A431 cells

[0042] The cell surface protein binding ability of each bifunctional antibody was detected by flow cytometry. The binding ability of the bifunctional antibody to the EGFR protein on the surface of A431 cells was detected. The negative control of the experiment was set as human IgG antibody, the positive control was human-mouse chimeric antibody (Erbitux monoclonal antibody, namely cetuximab), and the experimental group was each expressed and purified bifunctional antibody. Set detection cells to 1×10 6 Each tube was added with 2 μg / ml of each antibody and incubated on ice for 30 min. After washing the cells by centrifugation, Alexa488 fluorescein-labeled mouse anti-human secondary antibody (1:200 dilution) was added and incubated on ice for 30 min. After the incubation is complete, wash the cells by centrifugation. Fluorescent lab...

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PUM

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Abstract

The invention provides an EGFR-CD3 bifunctional antibody. The bifunctional antibody comprises two same light chains and two same recombinant heavy chains, wherein the amino acid sequence of each lightchain is as shown in SEQ ID NO. 8, each recombinant heavy chain is formed by connecting an EGFR antibody heavy chain and a single-chain antibody scFv of CD3 protein through a linker sequence; the amino acid sequence of the EGFR antibody heavy chain is as shown in SEQ ID NO. 7; the amino acid sequence of the linker sequence linker is as shown in SEQ ID NO. 9; the amino acid sequence of the single-chain antibody scFv of the CD3 protein is shown as any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6. The EGFR-CD3 bifunctional antibody can be specifically combined with human CD3 protein and EGFR, the immunogenicity of the murine antibody can be reduced, T cells can be effectively activated, the binding of human EGFR and EGFR can be blocked, the bifunctional antibody can be anchored on the surface of tumor cells by binding with EGFR, and can activate T cells by binding with CD3 so as to achieve the tumor cell killing purpose.

Description

technical field [0001] The invention relates to antibodies, in particular to bifunctional antibodies capable of blocking the combination of human EGF and EGFR and activating T cells. Background technique [0002] CD3 protein is a stimulating signal molecule expressed on the surface of T cells, which plays a very important role in T cell activation. CD3 molecule and TCR form a complex receptor molecule, activate tyrosine kinase, and promote the phosphorylation of tyrosine (Y) in the CD3 molecular immunoreceptor tyrosine activation motif. Phosphorylated tyrosine (pY) further phosphorylates downstream tyrosine-containing proteins, thereby causing a cascade reaction of kinase activation, regulating the target genes of T cell proliferation and activation, causing gene expression and transcription, and T cells from resting The state transitions to a proliferating and activated state. CD3 molecule has the functions of stabilizing the structure of TCR and transmitting activation s...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00
CPCC07K16/2863C07K16/2809A61P35/00A61K2039/505C07K2317/622C07K2317/51C07K2317/31
Inventor 叶才果王笑非汪波
Owner 广东安普泽生物医药股份有限公司
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