CAR-T (chimeric antigen receptor T-cell immunotherapy) therapeutic vector for T lymphocytic leukemia as well as construction method and application of CAR-T therapeutic vector

A technology of lymphocytes and leukemia, applied in the field of medical biology, can solve the problems of CAR-T cell therapy and achieve flexible anti-tumor activity

Active Publication Date: 2018-05-11
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, there is no relevant

Method used

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  • CAR-T (chimeric antigen receptor T-cell immunotherapy) therapeutic vector for T lymphocytic leukemia as well as construction method and application of CAR-T therapeutic vector
  • CAR-T (chimeric antigen receptor T-cell immunotherapy) therapeutic vector for T lymphocytic leukemia as well as construction method and application of CAR-T therapeutic vector
  • CAR-T (chimeric antigen receptor T-cell immunotherapy) therapeutic vector for T lymphocytic leukemia as well as construction method and application of CAR-T therapeutic vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 Construction of CAR-T cells

[0093] 1. Construction, purification and detection methods of recombinant lentiviral vectors lvCAR7-1-lvCAR7-7.

[0094] see image 3 , the construction method of the recombinant lentiviral vector of the present invention is as follows:

[0095] 1. The human EF1α promoter and CAR structure [CAR7-1~CAR7-7] were cloned into the lentiviral backbone plasmid pLenti-3G ​​basic to obtain recombinant lentiviral plasmids pCAR7-1~pCAR7-7 respectively. The sequence and number of components are shown in Table 1;

[0096] Table 1

[0097]

[0098] (1) The lentiviral backbone plasmid pLenti-3G ​​basic was double-digested with Cla I and EcoR I restriction endonucleases, and the product was subjected to 1.5% agarose gel electrophoresis to confirm the 5823bp fragment V1, which was recovered by tapping and placed in In the Eppendorf tube, recover the corresponding fragments (see Table 2) with the agarose gel recovery kit of MN Company, and m...

Embodiment 2

[0210] CAR-T cell pathogen detection and expression detection.

[0211] 1. Endotoxin detection;

[0212] (1), endotoxin working standard is 15EU / branch;

[0213] (2), Limulus reagent sensitivity λ=0.25EU / ml, 0.5ml / tube

[0214] (3) Dilution of endotoxin standard substance: Take one endotoxin standard substance, dilute it with BET water in proportion to dissolve into 4λ and 2λ respectively, seal with parafilm, shake and dissolve for 15min; each step of dilution should be mixed in the vortex Mix on the mixer for 30s;

[0215] (4) Adding samples: Take several LAL reagents, add 0.5 ml of BET water to each tube to dissolve, and distribute to several endotoxin-free test tubes, each tube has 0.1 ml. Two of them are negative control tubes, add 0.1ml of BET water;

[0216] Two are positive control tubes, add 0.1ml of endotoxin working standard solution with 2λ concentration;

[0217] 2 tubes are sample positive control tubes, add 0.1ml sample solution containing 2λ endotoxin stand...

Embodiment 3

[0247] Example 3 Functional testing of CAR-T cells.

[0248] 1. Evaluation of target cell killing effect.

[0249] (1) Culture target cells separately [CD7 + K562, K562 cells] and effector cells [CAR-T cells];

[0250] (2) Collect target cells 4x10 5 cells and CAR-T cells 2.8x10 6 cells, 800g, centrifuge for 6min, discard the supernatant;

[0251] (3) Resuspend the target cells and effector cells in 1ml D-PBS(-) solution, centrifuge at 800g for 6min, discard the supernatant;

[0252] (4) Repeat step 3 once;

[0253] (5) Resuspend effector cells with 700ul medium (AIM-V medium + 1-10% FBS), and resuspend target cells with 2ml medium (AIM-V medium + 1-10% FBS);

[0254] (6) Set up experimental wells with effect-to-target ratios of 1:1, 5:1, and 10:1, and the grouping conditions are as follows Figure 11 As shown, and set up the control group (K562 cells), each group has 3 duplicate wells;

[0255] (7) 250g, 5min plate centrifugation;

[0256] (8) Cultivate for 4 hours i...

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Abstract

The invention discloses a CAR-T (Chimeric antigen receptor T-cell immunotherapy) therapeutic vector for T lymphocytic leukemia. The therapeutic vector comprises a lentivirus skeleton plasmid, a humanEF1 alpha promoter and a CAR chimeric receptor domain, wherein the CAR chimeric receptor domain contains CD8 leader chimeric acceptor signal peptide, a CD7 single-chain antibody light chain VL, a CD7single-chain antibody heavy chain VH, an antibody inner hinge UCLinker, a CD8 Hinge chimeric receptor hinge, a CD8 Transmembrane chimeric receptor transmembrane region, a TCR chimeric receptor T cellactivation domain and a chimeric receptor co-stimulatory factor region. The invention further discloses a construction method of the vector and an application of the vector in preparation of a drug for treating T lymphocytic leukemia.

Description

technical field [0001] The invention belongs to the field of medical biology, and in particular relates to a carrier, in particular to a CAR-T therapeutic carrier for T lymphocytic leukemia. In addition, the present invention also relates to the construction method and application of the carrier. Background technique [0002] The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body's ability to attack tumor cells (highly specific cytolysis). In the 1950s, Burnet and Thomas put forward the theory of "immune surveillance", thinking that the mutated tumor cells that often appear in the body can be recognized and eliminated by the immune system, which laid the theoretical foundation for tumor immunotherapy [Burnet FM. Immunological aspects of malignant disease. Lancet, 1967; 1:1171-4]. Subsequently, various tumor immunotherapies, including cytokine therapy, monoclonal antibody therapy, a...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/65C12N15/66A61K35/17A61P35/02
Inventor 祁伟俞磊康立清林高武余宙晏祥孺
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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