Anti-human PD-1 protein antibody, and coding gene and application thereof

A technology of PD-1 and encoding genes, applied in the field of genetic engineering, can solve the problems of poor prognosis of tumor patients and achieve the effect of preventing and treating tumor diseases with high affinity

Inactive Publication Date: 2017-01-18
杭州贝颐药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] High expression of PD-1 and PD-L1 in these tumor cell

Method used

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  • Anti-human PD-1 protein antibody, and coding gene and application thereof
  • Anti-human PD-1 protein antibody, and coding gene and application thereof
  • Anti-human PD-1 protein antibody, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of fully human single-chain antibody library for ribosome display

[0034] The DNA sequence construction process of fully human antibody libraries (T7-VH-Vκ-Cκ and T7-VH-Vλ-Cκ) for ribosome display, such as figure 2 shown.

[0035] The specific construction process is as follows:

[0036] 1. Amplify human antibody heavy chain and light chain variable region DNA

[0037] Using polyA+RNA (purchased from Clontech) from human bone marrow, human fetal liver, human spleen and human peripheral blood leukocytes as templates, using oligo(dT) and random primers (random primers), using reverse transcriptase kit (purchased from Clontech), poly A+ RNA was reverse transcribed into cDNA according to the method guide provided by the Clontech kit.

[0038] Using the above cDNA as a template, use a series of primers that recognize the human antibody heavy chain variable region (VH) and light chain variable region (VL) genes to perform PCR amplification to obtai...

Embodiment 2

[0103] Example 2 Ribosome display screening of fully human single-chain antibodies that bind to PD-1 protein

[0104] The process of screening single-chain antibodies by ribosome display is as follows: image 3 shown. Because the scFv single-chain antibody library DNA prepared in Example 1 above does not contain a termination code, it is transcribed and translated in vitro to produce a complex formed by Antibody-Ribosome-mRNA ("ARM complex ").

[0105] Antibodies on the ARM complex were screened with antigen immobilized on the solid phase. After the non-specific ARM complex is eluted, RT-PCR method is used to obtain the DNA capable of binding the antigen to the antibody. Re-add T7 promoter etc. by PCR method to regenerate and obtain full-length DNA that can be used for ribosome display.

[0106] (1) Biotin-coupled PD-1 protein to avidin magnetic beads

[0107] PD-1 protein was purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.; EZ-link TM The sulfo-NHS-LC-bio...

Embodiment 3

[0141] Example 3 Single-chain antibody expression and purification

[0142] The gene encoding the single-chain antibody #hPD1-A was cloned into the expression vector pET27b(+), and pET27b-HPD1a was constructed;

[0143] Transform pET27b-HPD1a into expression bacteria E.coli BL21(DE3), and induce expression with IPTG (0.5mM) according to the method provided by Novagen; in the expressed target protein, the N-terminal of the scFv is the pelB sequence, and the pelB sequence can be The expressed scFv is secreted into the periplasmic space of BL21(DE3); the C-terminus of the scFv contains an HSV tag and a 6×His tag to facilitate the purification of the target protein;

[0144] The method provided by Qiagen was conveniently separated and purified with Ni-NTA column to obtain the single-chain antibody against human PD-1 protein.

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Abstract

The invention discloses an anti-human PD-1 protein antibody, and a coding gene and application thereof. The antibody comprises a heavy-chain variable region and a light-chain variable region; the amino acid sequences of three hypervariable regions, i.e., CDRH1, CDRH2 and CDRH3, of the heavy-chain variable region are GGSFSGYYWS, EINHSGSTNYNPSLKS and GSPDSSRARGYYMDV, respectively; and the amino acid sequences of three hypervariable regions, i.e., CDRL1, CDRL2 and CDRL3, of the light-chain variable region are RASQGIRNDLG, AASSLQS and LQHNSYPL, respectively. According to the invention, the anti-human PD-1 protein antibody prepared by constructing a human single-chain antibody library and screening single-chain antibodies by using ribosome displaying technology can specifically bind to human PD-1 protein, has high affinity, is applicable to detection of cells expressing PD-1 ad applicable as a fully human-derived anti-human PD-1 protein antibody for treating human diseases, and effective prevents and treats tumor diseases.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an anti-human PD-1 protein antibody, its coding gene and its application. Background technique [0002] Mammalian T cells play an extremely important role in the immune system. T cells rely on antigen-presenting cells (APCs) or other molecules to digest foreign or harmful antigens and re-present them in an antigenic form that T cells can recognize, so that T cells can be activated. This process is delicately regulated by many molecules, some of which promote the activation of T cells to ensure that the immune system is strong enough to fight invading pathogens, while others act as inhibitors to prevent overactivation of the immune system. [0003] There is a group of proteins involved in regulation on T cells, named CD28 receptor family (Receptor Family), including: CD28, CTLA-4, PD-1 (also known as CD279), ICOS and BTLA and other molecules (Okazaki et al.Curr .Opin...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00G01N33/569
Inventor 华绍炳
Owner 杭州贝颐药业有限公司
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