Chimeric antigen receptor (CAR) gene for expressing soluble PD-1, and application of CAR gene

A chimeric antigen receptor, PD-1 technology, applied in the direction of DNA / RNA fragments, applications, genetic engineering, etc.

Inactive Publication Date: 2019-11-08
EAST CHINA NORMAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no product that co-expresses soluble PD-1 and chimeric antigen receptor CAR to improve the lethality of CAR-T cells against tumor cells

Method used

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  • Chimeric antigen receptor (CAR) gene for expressing soluble PD-1, and application of CAR gene
  • Chimeric antigen receptor (CAR) gene for expressing soluble PD-1, and application of CAR gene
  • Chimeric antigen receptor (CAR) gene for expressing soluble PD-1, and application of CAR gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Obtaining the extracellular sequence of human PD-1 gene

[0048] Find the CDS region of the extracellular segment sequence of the PD-1 gene through the website https: / / www.ncbi.nlm.nih.gov / pubmed / , and find the PD-1 protein at https: / / www.uniprot.org / The extracellular segment region. Use SnapGene software to design and synthesize primers in the company (the primers contain the signal peptide sequence of CD8a), use human T cell cDNA as a template to amplify the extracellular segment of PD-1 gene by PCR, and sequence the amplified products The signal peptide gene sequence containing CD8a shown in positions 1201-1263 of SEQ ID No. 2 is connected to the gene sequence of PD-1 extracellular segment shown in positions 1264-1713 of SEQ ID No. 2. The amino acid sequences are respectively As shown in SEQ ID No.3 and SEQ ID No.1.

Embodiment 2

[0049] Example 2. Construction of pCDH-NKG2D-sPD-1-CAR vector

[0050] The constructed vector PCDH-NKG2D-CAR was double digested with the quick digestion enzymes BamHI and SalⅠ (for the connection between the EcoRI and BamHI restriction sites of the vector pCDH figure 1 The CAR element shown in the upper figure), the double digested vector PCDH-NKG2D-CAR with ligase and the PCR amplification product (containing sPD-1 sequence) and T2A gene fragment (SEQ ID No. 1) of Example 1 2) to obtain a recombinant vector, transform the recombinant vector into Escherichia coli, and confirm the pCDH-NKG2D-sPD-1-CAR vector (containing figure 1 CAR element shown in the middle and lower pictures).

[0051] The above recombinant vector pCDH-NKG2D-sPD-1-CAR is connected between the SalI and EcoRI restriction sites of the lentiviral expression vector pCDH figure 1 The CAR element encoding gene shown in the middle and lower figure (ie the CAR gene shown in SEQ ID No. 2),

[0052] In the CAR gene shown in...

Embodiment 3

[0054] Example 3. Virus packaging

[0055] 1. Plasmid transfection

[0056] The recombinant vector (the vector pCDH-NKG2D-sPD-1-CAR of Example 2 or the vector PCDH-NKG2D-CAR) as the destination vector was sent into the 293T cell through the three-plasmid transfection system, according to the helper plasmid psPAX2: helper plasmid pMD2. G: Target vector = 5:3:3 mass ratio is added into the Opti-MEM medium, 50ul PEI solution is added, and after repeated pipetting 20 times to mix, the mixture is allowed to stand at room temperature for 20 minutes to obtain a DNA / PEI mixture. Slowly drop 1ml DNA / PEI mixture into the 293T culture dish laid the day before, mix gently, and incubate at 37°C incubator. After 6-8 hours, replace with fresh medium and put it in 37°C incubator to continue incubating.

[0057] 2. Virus collection and concentration

[0058] 48h after plasmid transfection, after collecting the supernatant, add 10ml of fresh Opti-MEM medium to continue culturing to 72h, collect the su...

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Abstract

The invention discloses a chimeric antigen receptor (CAR) gene for expressing a soluble PD-1, and application of the CAR gene. The CAR gene comprises an antigen binding domain gene, a transmembrane domain gene, an intracellular domain gene and a soluble PD-1 gene which are sequentially connected, the soluble PD-1 is an extracellular domain area of the PD-1, and preferably, the soluble PD-1 includes an amino acid sequence shown in SEQ ID No.1. CAR-T cells can secrete sPD-1 in a tumor microenvironment, through combination of sPD-1 and PD-L1 on the surfaces of tumor cells, combination with transmembrane PD-1 on the surfaces of the CAR-T cells is inhibited competitively, thus inhibition of the CAR-T cells is avoided, the killing and treating effects of the CAR-T cells on the tumor cells are improved, and the purpose of treating tumors, especially solid tumors, is achieved. A new way is provided for improving the effect of the CAR-T cells on killing the tumor cells.

Description

Technical field [0001] The invention relates to the field of biomedicine, in particular to a chimeric antigen receptor CAR gene expressing soluble PD-1 and its application. Background technique [0002] Tumor is a serious disease threatening human life, and its treatment methods have always been highly concerned by the medical and biological circles. In recent years, as people's understanding of the tumor immune microenvironment continues to deepen, a new approach to tumor treatment has gradually entered people's field of vision, namely "tumor immunotherapy". In 2013, the US FDA called it "breakthrough therapy" , Enough to see its importance, and chimeric antigen receptor T cell therapy (CAR-T) is currently one of the most promising tumor immunotherapy. [0003] The chimeric antigen receptor CAR generally consists of an antigen binding region / extracellular region / transmembrane region and an intracellular signal region that can activate T cells after binding to the antigen. CAR-T c...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCA61P35/00A61K39/001102C07K14/70521C07K2319/02C07K2319/03C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 江文正王熙何聪刘明耀席在喜
Owner EAST CHINA NORMAL UNIVERSITY
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