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Slit2D2-chimeric antigen receptor and application thereof

A chimeric antigen receptor and antigen-binding technology, applied in the direction of antibody mimics/scaffolds, receptors/cell surface antigens/cell surface determinants, hybrid peptides, etc., can solve the problem that prostate cancer does not show therapeutic effect and other problems

Active Publication Date: 2017-10-27
ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinical trials have shown that GD2-specific CARTs have a certain effect on neuroblastoma, while aFR-specific CART cells are effective against ovarian cancer, CAIX-specific CART cells are effective against renal cell carcinoma, and PSMA-specific CART cells are effective against prostate cancer. Shown no effect of treatment

Method used

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  • Slit2D2-chimeric antigen receptor and application thereof
  • Slit2D2-chimeric antigen receptor and application thereof
  • Slit2D2-chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1 lentiviral expression vector preparation

[0085] 1. According to the known Slit2 sequence [GenBank:EAW92793.1], analyze and design the second domain Slit2D2 (Hohenester2008) of Slit2, and search for known human CD8 from the GenBank database TM Transmembrane region gene sequence, human 4-1BB intracellular region gene sequence and CD3ζ intracellular region gene sequence, the sequence list of each gene can be found in the sequence table SEQ ID NO:6-9.

[0086] 2. The above gene sequence is sequenced according to human Slit2D2 gene, CD8 TM Membrane region gene, human 4-1BB intracellular region gene and CD3ζ intracellular region gene were connected, and different enzyme cutting sites were introduced at the junction of each sequence to form a complete Slit2D2-CD8 TM - 4-1BB-CD3ζ (short for Slit2D2-CAR) gene sequence information, its sequence refer to the sequence table SEQ ID NO: 10.

[0087] 3. Insert Slit2D2-CD8 TM - The gene sequence of 4-1BB-CD3ζ was conn...

Embodiment 2

[0088] Example 2 lentivirus preparation

[0089] 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.

[0090] 2. Prepare Solution A and Solution B

[0091] Solution A: 6.25mL 2×HEPES buffer (the amount packed in 5 large dishes is the best).

[0092] Solution B: Add the mixture of the following plasmids: 112.5 μg pRRLSIN-EF-PD1 (targetplasmid); 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 A: 6.25 mL.

[0093] 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 mixture of DNA and calcium ...

Embodiment 3

[0096] Example 3 Preparation of Slit2D2-CART cells

[0097] 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.

[0098] 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 inacti...

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Abstract

The invention provides a chimeric antigen receptor (CAR) and a CAR encoding gene. The CAR comprises an extracellular structural domain capable of binding antigens, a transmembrane structural domain and intracellular immune costimulatory molecules, wherein the extracellular structural domain comprises a D2 structural domain of a Slit2 protein. In addition the invention also provides a chimeric antigen receptor expression cell, and the CAR encoding gene is introduced into the cell, so that the CAR is expressed on the surface of the cell. The CAR or the CAR expression cell provided by the invention can be used as cell medicines in the treatment of tumor diseases. The CAR provided by the invention can be used to modify cells, especially T cells, and modified T cells can specifically recognize and kill or wound tumors and have more efficient tumor killing activity.

Description

technical field [0001] The invention relates to the field of tumor treatment drugs, in particular to the application of Slit2D2-chimeric antigen receptors in drugs for preventing and / or treating tumors with high Robo1 expression. Background technique [0002] Human T lymphocytes recognize target cells through T cell receptors on their surface. This recognition is specific, that is, a certain T lymphocyte only recognizes target cells with specific antigens, and this specific antigen is present in After intracellular processing, it is presented to T lymphocytes under the action of special molecules. The molecule responsible for antigen presentation is present either on the surface of the antigen presenting cell or on the surface of the target cell. There are at least two factors that cause T lymphocytes in the body to fail to recognize cancer cells: (1) cancer cells down-regulate the expression of antigen-presenting molecules, and (2) the presented antigen has a weak affinity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K35/17A61P35/00
CPCC07K14/47C07K14/70507C07K14/7051C07K14/70514C07K14/70517C07K14/70521C07K14/70535C07K14/70575C07K14/70589C07K14/70596C12N2510/00C07K2319/33C07K2319/74A61K39/4644A61K39/4611A61K39/4631C07K14/705C07K19/00C12N5/10C12N15/09A61P35/00A61K38/00C12N2740/16043A61K48/00A61K35/17C07K14/78C12N15/85
Inventor 李华顺董宁王保垒任宝永
Owner ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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