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EGFR/PD-1 double-targeted antibody, preparation method and application thereof

A dual-targeting antibody and PD-1 technology, applied in the biological field, can solve problems that need to be improved

Active Publication Date: 2018-12-07
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, even for tumor-targeting antibodies with better efficacy, the response rate and efficacy still need to be improved, and most patients gradually develop tolerance during treatment.

Method used

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  • EGFR/PD-1 double-targeted antibody, preparation method and application thereof
  • EGFR/PD-1 double-targeted antibody, preparation method and application thereof
  • EGFR/PD-1 double-targeted antibody, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. [Cetuximab and 5C4 antibody variable region gene cloning]

[0036] Referring to the patents (PCT / US96 / 09847 and PCT / JP2006 / 309606), the heavy chain variable region gene and light chain variable region gene of Cetuximab and 5C4 were synthesized respectively. Wherein the antibody signal peptide amino acid sequence MGWSCIILFLVATATGVHS. The correct cloned fragments were obtained by sequencing. SEQ ID NO: 2 shows the amino acid sequence of the heavy chain variable region of Cetuximab, its nucleotide sequence is SEQ ID NO: 1; SEQ ID NO: 3 is the nucleotide sequence of the 5C4 heavy chain variable region, its amino acid sequence is SEQ ID NO: 4; SEQ ID NO: 6 shows the amino acid sequence of the 5C4 light chain variable region, its nucleotide sequence is SEQ ID NO: 5; SEQ ID NO: 24 is the amino acid sequence of the Cetuximab light chain variable region, Its nucleotide sequence is SEQ ID NO:23.

Embodiment 2

[0037] Example 2. [Cloning of human antibody CL, heavy chain CH1, and Fc region]

[0038] Healthy human lymphocytes were separated with lymphocyte separation medium (product of Dingguo Biotechnology Development Co., Ltd.), and total RNA was extracted with Trizol reagent (product of Life Company). According to the literature (Cloned human and mouse kappaimmunoglobulin constant and J region genes conserve homology in functional segments. Hieter PA, Max EE, Seidman JG, Maizel JV Jr, Leder P. Cell. 1980 Nov; 22(1Pt1):197-207.) and literature (The nucleotide sequence of a human immunoglobulin Cgamma1gene. Ellison JW, Berson BJ, Hood LE .Nucleic Acids Res.1982Jul 10; 10(13):4071-9.) Using RT-PCR reaction to amplify the antibody light chain constant region, heavy chain constant region CH1 and Fc region genes. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector. After sequencing verification, it was confirmed that the correct clo...

Embodiment 3

[0039] Example 3. [Construction of antibody Fc region knob mutants]

[0040] Taking the Fc region of the antibody obtained in Example 2, the overlap PCR method was used to introduce mutation points: T366W, S354C and point mutation N297Q. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector. After sequencing verification, it was confirmed that the correct clone was obtained. SEQ ID NO:14 shows the amino acid sequence of Fc-knob, and its nucleotide sequence is SEQ ID NO:13.

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PUM

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Abstract

The invention provides an epidermal growth factor receptor / programmed death-1 (EGFR / PD-1) double-targeted antibody, a preparation method thereof and an application thereof in preparation of anti-tumordrugs. The EGFR / PD-1 double-targeted antibody is prepared by taking a Cetuximab antibody and a 5C4 antibody as parents and adopting a knob-into-hole technology. The double-targeted antibody has a better anti-tumor effect than the combined use of the Cetuximab antibody and the 5C4 antibody, and has a huge application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention discloses an EGFR / PD-1 dual-targeting antibody, its preparation method and its application in the preparation of anti-tumor drugs. Background technique [0002] Tumor, especially malignant tumor, is a disease that seriously endangers human health in the world today, and its fatality rate ranks at the forefront of the fatality rate of various diseases. And in recent years, its incidence has shown an obvious upward trend. The curative effect of malignant tumors is poor, the rate of advanced metastasis is high, and the prognosis is often poor. Although conventional treatment methods such as radiotherapy, chemotherapy and surgery are currently used clinically, although the pain is relieved to a large extent and the survival time is prolonged, these methods have great limitations, and the curative effect is difficult to further improve. [0003] Antibody-targeted dru...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00
CPCC07K16/2818C07K16/2863A61K2039/505C07K2317/31
Inventor 赵磊胡毅张帆崔月倩
Owner GENERAL HOSPITAL OF PLA
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