Anti-human PD-1 protein antibody as well as encoding gene and application thereof

A PD-1, encoding gene technology, 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: 2017-05-24
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 cells often leads to poor prognosis of patients with these tumors

Method used

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  • Anti-human PD-1 protein antibody as well as encoding gene and application thereof
  • Anti-human PD-1 protein antibody as well as encoding gene and application thereof
  • Anti-human PD-1 protein antibody as well as encoding 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] Human PD-1 protein (catalog number 10377-H03H) was purchased from Beijing Sino Biological Technology Co., Ltd.; E...

Embodiment 3

[0142] Example 3 Single-chain antibody expression and purification

[0143] The gene encoding the single-chain antibody #hPD1-B was cloned into the expression vector pET27b(+), and pET27b-HPD1b was constructed;

[0144] Transform pET27b-HPD1b 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;

[0145] 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 as well as an encoding gene and an application thereof. The antibody comprises a heavy chain variable region and a light chain variable region, wherein amino acid sequences of three hypervariable regions CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are shown as GFTFSSYAMS, AISGSGGTTYYADSVKG and AWAPFVNSIVVVTANDL respectively; amino acid sequences of three hypervariable regions CDRL1, CDRL2 and CDRL3 of the light chain variable region are shown as RASQGISTWLA, AASSLQS and QQANSFPI respectively. A human single-chain antibody library is established, single-chain antibodies are screened with a ribosome display technology, and the obtained anti-human PD-1 protein antibody can be specially bonded with human PD-1 protein, has high affinity, can be used for detecting cells expressing PD-1, can be used as a fully-human anti-human PD-1 antibody for treating human diseases and effectively 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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Patent Type & Authority Applications(China)
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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