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Method for determining biology activity of PD-1 pathway inhibitor

A biologically active, PD-1 technology, applied in biochemical equipment and methods, microbial assay/inspection, cells modified by the introduction of foreign genetic material, etc., can solve the problem of long time, high cost, and large method variability. question

Active Publication Date: 2015-06-03
JIANGSU SIMCERE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These traditional methods require a long time and high cost, and the method itself is easily affected by the difference in PBMC activity between different human bodies, and the method variability is large

Method used

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  • Method for determining biology activity of PD-1 pathway inhibitor
  • Method for determining biology activity of PD-1 pathway inhibitor
  • Method for determining biology activity of PD-1 pathway inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Preparation of effector cells expressing PD-1 and NFAT-luciferase

[0087] First, pcDNA3.1(+) / PD-1 (Beijing Sino Biological Technology Co., Ltd.) and pGL4.30[Luc2P / NFAT-RE] (Promega (Beijing) Biotechnology Co., Ltd. company) plasmids were co-transfected into jurkat cells (ATCC), and then cultured in a medium containing 1 μg / ml puromycin and 1 mg / ml geneticin to obtain effector cells expressing PD-1 and NFAT-luciferase . Using jurkat cells as a control, the expression of PD-1 in the obtained cells was analyzed by flow cytometry (FACs), and the results are shown in figure 1 . Stimulate with mouse anti-human CD3 antibody (BD Biosciences, cat.555336), by Bright-Glo TM Kit (Promega, cat.E2610) was used to measure the expression of luciferase (luciferase), and the results of luciferase activity were found in figure 2. Thus, the effector cells of the present invention are obtained. The stable cell line JurKat / PD-1 / NFAT was obtained by selecting the clone wit...

Embodiment 2

[0088] Example 2: Preparation of target cells expressing αCD3TM and PD-L1

[0089] The pcDNA3.1(+) / αCD3TM and pcDNA3.1(+) / PD-L1 plasmids (Beijing Sino Biological Technology Co., Ltd.) were co-transfected into CHO-K1 cells ( ATCC), the transfected cells were then cultured in medium containing 1 μg / ml puromycin and 200 μg / ml hygromycin B. Using CHO-K1 as a control, the expression levels of αCD3TM and PD-L1 in the resulting cells were detected using donkey anti-mouse antibody and PD-L1 antibody. For the results of FACs of αCD3TM and PD-L1 expression image 3 and Figure 4 . Thus, the target cells of the present invention are obtained. The clone with higher expression level was selected to obtain a stable cell line CHO-K1 / αCD3TM / PD-L1, which was deposited in China Microorganism Culture Collection Management Committee General Microbiology on December 30, 2014 with the registration number CGMCC No.10299. center.

Embodiment 3

[0090] Example 3: Determination of biological activity of PD-1 monoclonal antibody and PD-L1 monoclonal antibody according to the method of the present invention

[0091] (1) Prepare CHO-K1 / αCD3TM / PD-L1 target cell suspension, use DMEM / F12+10% FBS medium according to 2.5×10 4 cells / well, spread evenly in 96-well plate at a density of 100 μl / well (corning), 37°C, 5% CO 2 Incubate overnight in the incubator;

[0092] (2) Use 1640+10% medium to prepare different concentrations of PD-1 monoclonal antibody (Nivolumab) or PD-L1 monoclonal antibody (MPDL3280A) solutions and dilute to 10 μg / mL, then down 3-fold ratio dilution 7 At each concentration point, absorb the DMEM / F12+10% FBS medium in the above-mentioned 96-well plate, and add the prepared monoclonal antibody solution of PD-1 monoclonal antibody or PD-L1 monoclonal antibody to 50 μl / well In 96-well plate;

[0093] (3) Use 1640+10% medium to prepare JurKat / PD-1 / NFAT effector cell suspension, and follow the 5×10 4 per well,...

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Abstract

The invention discloses a method for testing biology activity of a PD-1 pathway inhibitor. The method comprises the steps: contacting target cells expressing alpha CD3TM and PD-L1 and effector cells expressing NFAT report genes and PD-1 with the PD-1 pathway inhibitor and testing signals of the NFAT report genes to determine the biology activity of the PD-1 pathway inhibitor.

Description

technical field [0001] A method for determining the biological activity of PD-1 pathway inhibitors, said method comprising: combining target cells expressing αCD3TM and PD-L1, effector cells expressing NFAT reporter gene and PD-1 with PD-1 pathway Inhibitors were contacted, and the NFAT reporter signal was measured to determine the biological activity of PD-1 pathway inhibitors. Background technique [0002] Programmed death receptor 1 (PD-1) is an immunosuppressive receptor first expressed on activated T cells and B cells. The interaction of this receptor with its ligand has consistently shown attenuated T cell responses both in vitro and in vivo. Blocking the interaction between PD-1 and one of its ligands, PD-L1, has been shown to enhance the immunity of tumor-specific CD8+ T cells and, therefore, may help the immune system clear tumor cells. [0003] PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily related to CD28 and CTLA-4. PD-1 has bee...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02C12N5/10
CPCC12N5/10C12Q1/66C12Q1/6897
Inventor 熊新辉罗安德张弢李景荣
Owner JIANGSU SIMCERE PHARMA
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