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Preparation method and application of enhanced Slit2 CAR-T and CAR-NK cells

A technology of CD5 and extracellular domain, applied in chemical instruments and methods, biochemical equipment and methods, animal cells, etc., can solve the problem of low specificity of tumor cells

Active Publication Date: 2018-04-03
ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This chimeric receptor T cell can activate the anti-tumor effect of T cells only after 2-3 chimeric antigen receptors recognize the antigen. low specificity

Method used

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  • Preparation method and application of enhanced Slit2 CAR-T and CAR-NK cells
  • Preparation method and application of enhanced Slit2 CAR-T and CAR-NK cells
  • Preparation method and application of enhanced Slit2 CAR-T and CAR-NK cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1: Preparation of lentiviral expression vector

[0098] 1. According to the known Slit2 sequence [GenBank:EAW92793.1], the second structural domain Slit2D2 (Hohenester2008) of Slit2 was analyzed and designed. Hinge region of human CD8 and CD8 TM Transmembrane region gene sequence, human 4-1BB intracellular region gene sequence, CD3ζ intracellular region, IRES internal ribosome entry site, get Slit2D2-CAR (SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3ζ) The gene is shown in SEQ ID NO: 7, and its series diagram is shown in figure 1 Shown; synthetic HAC gene fragment, its nucleotide sequence is shown in SEQ ID NO: 2, introduces HSA gene at its C terminal simultaneously, obtains HAC-HSA fusion gene (SP2-HAC-HSA) as shown in SEQ ID NO: 8 Show.

[0099] 2. Insert the above HAC-HSA fusion gene sequence into the downstream of the Slit2D2-CAR sequence to form a complete

[0100] Slit2D2-CD8-4-1BB-CD3ζ-IRES-HAC-HSA, its construction schematic diagram is as follows figure 2 ...

Embodiment 2

[0102] Embodiment 2: lentivirus preparation

[0103] 1. 24 hours before transfection, use about 8×10 per dish 6 293T cells were seeded into 15cm culture dishes. Make sure that the cells are at about 80% confluence and evenly distributed in the culture dish during transfection.

[0104] 2. Prepare Solution A and Solution B

[0105] Solution A: 6.25mL 2×HEPES buffer (the amount packed in 5 large dishes is the best).

[0106] Solution B: Add the mixture of the following plasmids: 112.5 μg pRRSLIN-Slit2D2-CAR-IRES-HAC-HSA (target plasma); 39.5 μg pMD2.G (VSV-G envelope); 73 μg pCMVR8.74 (gag, pol, tat , rev); 625 μL 2M calcium ion solution. Total volume of solution B: 6.25 mL.

[0107] 3. Mix solution B well, and while vortexing solution A gently, add solution A drop by drop and let it stand for 5-15 minutes. Gently vortex the above mixed solution of A and B, add dropwise to the culture dish containing 293T cells, gently shake the culture dish back and forth to make the mixt...

Embodiment 3

[0110] Example 3: Preparation of CAR-T cells

[0111] 1. Take 0.5mL of blood for rapid detection of pathogenic microorganisms to exclude microbial infections such as HBV, HCV, HDV, HEV, HIV-1 / 2, Treponema pallidum and parasites; coagulation), immediately (4°C, within 24 hours) to the cell preparation laboratory to ensure that there is no pathogenic microorganism contamination in this process. After obtaining the patient's blood, in the GMP preparation room, wipe the surface of the heparin bottle with an alcohol cotton ball for disinfection and put it into a biological safety cabinet.

[0112] 2. Open two 50mL centrifuge tubes in advance, transfer the blood into two 50mL centrifuge tubes, and tighten; put the two 50mL centrifuge tubes filled with blood into the centrifuge, centrifuge at 400g (2000rpm) for 10min, and centrifuge at room temperature Afterwards, the upper layer of plasma was collected, leaving the precipitate layer; the collected autologous plasma was inactivated ...

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Abstract

The invention provides a preparation method and application of enhanced Slit2 CAR-T and CAR-NK cells. The enhanced CAR-T cell comprises a T cell of an enhanced CAR embedded antigen receptor, and the enhanced CAR-NK cell comprises a NK cell of an enhanced CAR embedded antigen receptor, wherein the enhanced CAR embedded antigen receptor includes an extracellular structural domain capable of combining with an antigen, a transmembrane structural domain, intracellular immune costimulatory molecules, an entry site IRES of internal ribosome, and HAC-HSA that are connected in sequence, wherein the extracellular structural domain includes a D2 structural domain of Slit2 protein. The enhanced CAR-T cell and CAR-NK cells can close PDL-1 inhibitory signals, improves the killing activity of CAR-immunocyte, and activates tumor-infiltrating immunocyte. The enhanced CAR-immunocyte can be taken as a cell medicine for treating tumor diseases. The transformed immunocyte can specifically recognize and kill tumor and has efficient tumor-killing activity.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a chimeric antigen receptor cell for anti-tumor stem cells, especially enhanced CAR-T cells and enhanced CAR-NK cells. Background technique [0002] The full name of PD-L1 is programmed death receptor-ligand 1, and its English name is programmed cell death-Ligand 1. It is the first type of transmembrane protein with a size of 40kDa. The full name of PD-1 is programmed death receptor 1, and its English name is programmed death 1. It is an important immunosuppressive molecule and a member of the CD28 superfamily. PD-1 (programmed death-1) is mainly expressed on the surface of T cells and primary B cells, and the two ligands of PD-1 (PD-L1 and PD-L2) are widely expressed in antigen-presenting cells (APCs). The interaction between PD1 and its receptor plays an important role in the negative regulation of immune response. The expression of PD-L1 protein can be detected in many h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00C12N15/86C12N5/10A61K35/17A61P35/00
CPCC12N5/0636C12N5/0646C12N15/86C07K14/7051C07K14/70514C07K14/70517C07K14/70521C07K14/70535C07K14/70589C07K14/70596C07K2319/02C07K2319/03C07K2319/33C12N2800/107C12N2840/203A61K39/4613A61K39/4631A61K39/4644A61K39/4611A61K48/00A61P35/00C07K19/00C12N5/10C12N15/62
Inventor 李华顺王保垒任宝永方冬冬
Owner ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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