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Chimeric antigen receptor taking TCR[gamma][delta] as target, and application of chimeric antigen receptor

An antigen and receptor technology, applied in the field of medicine and biology, can solve the problems of poor therapeutic effect, high recurrence rate, and high mortality, and achieve the effect of improving tumor killing efficiency and remarkable effect

Active Publication Date: 2019-12-27
WUHAN BIO RAID BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

γδT-cell lymphoma is clinically faced with the phenomenon of no specific drug target, which leads to poor treatment effect, high recurrence rate and high mortality
However, there is no effective treatment of tumors targeting TCRγδ

Method used

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  • Chimeric antigen receptor taking TCR[gamma][delta] as target, and application of chimeric antigen receptor
  • Chimeric antigen receptor taking TCR[gamma][delta] as target, and application of chimeric antigen receptor
  • Chimeric antigen receptor taking TCR[gamma][delta] as target, and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of PTK881-EF1α-C3-1, PTK881-EF1α-C3-2, PTK881-EF1α-C4 plasmids

[0028] 1. Artificially synthesize the nucleotide fragments shown in SEQ ID NO.3, the connecting polypeptide Linker nucleotide sequence, and SEQ ID NO.4 in sequence to form SP-C3-1. Artificial sequential synthesis of SEQ ID NO.7, connecting polypeptide linker nucleotide sequence, and the nucleotide fragment shown in SEQ ID NO.8 constitute SP-C3-2. The connecting polypeptide Linker has an amino acid sequence of GGGGSGGGGSGGGGS and a nucleotide sequence of ggcggtggcggtagcggtggcggtggctctggtggtggtggcagc.

[0029] 2. Using the human cDNA library as a template, design primers for PCR to amplify fragments CD8 hinge, CD28 transmembrane region, CD28 intracellular domain, 4-1BB, CD3ζ, IL-7, and CCL19 respectively, and obtain Strep tag by primer complementation Ⅱ, F2A short fragment. Using Overlap PCR technology, SP-C3-1 and fragments Strep tag Ⅱ, CD8 hinge, CD28 transmembrane region, CD28 in...

Embodiment 2

[0035] Example 2, preparation and sequencing of plasmids

[0036] 1. Preparation of plasmid

[0037] Inoculate DH5α strains containing plasmids PTK881-EF1α-C3-1, PTK881-EF1α-C3-2, and PTK881-EF1α-C4 into 250 mL of LB culture medium containing 100 μg / mL ampicillin, and culture at 37°C and 220 rpm overnight. The culture solution was centrifuged at 6000g for 20min at 4°C, and the supernatant was discarded.

[0038] Take out Buffers P1 from the EndoFree plasma mega kit (Qiagen), add 120mL pre-cooled Buffers P1 to the centrifuged E. coli pellet, cover the cap of the centrifuge bottle, shake the centrifuge bottle vigorously to completely disperse the E. coli pellet in Buffers P1 .

[0039] Add 120mL Buffers P2 to the centrifuge bottle, put the cap on the roller mixer, slowly increase the speed to 50rpm, mix thoroughly and place at room temperature for 5min.

[0040] Add 120mL Buffers P3 to the centrifuge bottle, put the cap on the roller mixer, slowly increase the speed to the m...

Embodiment 3

[0053] Example 3, Preparation of Lenti3-C3-1-CAR, Lenti3-C3-2-CAR, Lenti3-C4-CAR Lentiviral Vectors and Live Droplet Detection

[0054]1. Preparation of lentiviral vector

[0055] Insert 130.0~140.0×10 in the multilayer cell culture flask (Hyperflask) 6 Number of 293T cells (Takara), a total of 560 mL DMEM complete medium (50 mL fetal bovine serum, 5 mL Antibiotic-Antimycotic (100×)), at 37 °C with 5% CO 2 Incubate for 24 hours in the incubator. Mix 320 μg plasmid (PTK881-EF1α-C3-1: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid=12:10:5:6), mix 320 μg plasmid (PTK881-EF1α-C3-2: BZ1 plasmid: BZ2 Plasmid: BZ3 plasmid=12:10:5:6), 960 μg PEI was added to the DMEM basal medium mixed with 320 μg plasmid (PTK881-EF1α-C4: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid=12:10:5:6) In the tube, vortex, and equilibrate at room temperature for 10 minutes. Mix the above-mentioned 35mL PEI-plasmid mixture with 525mL DMEM complete medium, and replace it into the above-mentioned multi-layer cell culture fla...

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PUM

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Abstract

The invention discloses a chimeric antigen receptor taking TCR[gamma][delta] as a target, and application of the chimeric antigen receptor. A signal peptide, a single-chain antibody ScFv, a Strep tagII, a CD8 hinge, a CD28 transmembrane domain, a CD28 intracellular structural domain, 4-1BB and a CD3[xi] chain are spliced in sequence form a N terminal to a C terminal, and the single-chain antibodyScFv can identify TCR[gamma][delta] on the surfaces of tumor cells. The chimeric antigen receptor taking TCR[gamma][delta] as the target is used for modifying immune cells, and the modified immune cells can effectively kill any tumor cells of which the surfaces express TCR[gamma][delta].

Description

technical field [0001] The invention relates to the field of medicine and biology, and specifically refers to a chimeric antigen receptor (CAR) for treating γδT cell lymphoma / leukemia with TCRγδ as a target and its application. Background technique [0002] CAR-T cell therapy has become one of the most popular and effective tumor treatments in recent years. The full name of CAR-T cells is Chimeric Antigen Receptor T-Cell (Chimeric Antigen Receptor T-Cell). The single-chain variable region (Scfv) of an antibody that can recognize a certain tumor antigen and the intracellular region of the CD3ζ chain are coupled into a chimeric protein in vitro, and transfected into patient T cells cultured in vitro by gene transduction to make it Expression of Chimeric Antibody Receptor (CAR). After the patient's T cells are "reprogrammed", a large number of lethal CAR-T cells are generated, which can specifically target tumor cells. Compared with traditional immunotherapy, CAR-T has signif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/66C12N15/867C12N5/10A61K39/00A61P35/00A61P35/02
CPCA61K2039/5158A61P35/00A61P35/02A61K39/001102C07K14/521C07K14/5418C07K14/7051C07K16/2809C07K2317/56C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N15/66C12N15/86C12N2740/15043Y02A50/30
Inventor 张同存祝海川张子健
Owner WUHAN BIO RAID BIOTECH CO LTD
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