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Car-t therapy carrier for malignant glioma based on octs technology and its construction method and application

A malignant glioma, carrier technology, applied in the field of medical biology, can solve the problems that have not been overcome, and achieve the effects of improving curative effect, inhibiting immune escape, and ensuring reliable protection

Active Publication Date: 2020-03-20
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, there are no reports on CAR-T therapy targeting EGFRvIII and PDL1 antigens to overcome the above shortcomings

Method used

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  • Car-t therapy carrier for malignant glioma based on octs technology and its construction method and application
  • Car-t therapy carrier for malignant glioma based on octs technology and its construction method and application
  • Car-t therapy carrier for malignant glioma based on octs technology and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1 Construction of OCTS-CAR-T cells

[0095] 1. Construction, purification and detection methods of recombinant lentiviral vectors lvOCTS-PEvIIIs and lvOCTS-PEvIIIt.

[0096] see image 3 , the construction method of the recombinant lentiviral vector of the present invention is as follows:

[0097] 1. Human EF1α promoter (SEQ ID NO.14), OCTS structure [OCTS-PEvIIIs, OCTS-PEvIIIt] (CD8leader chimeric receptor signal peptide (SEQ ID NO.15), PDL1 single-chain antibody light chain VL ( SEQ ID NO.16), PDL1 single-chain antibody heavy chain VH (SEQ ID NO.17), EGFRvIII single-chain antibody light chain VL (SEQ ID NO.18), EGFRvIII single-chain antibody heavy chain VH (SEQ ID NO.19 ), the hinge Inner-Linker (SEQ ID NO.20) in the antibody, the hinge Inter-Linker (SEQ ID NO.21) between single-chain antibodies, the CD8Hinge chimeric receptor hinge (SEQ ID NO.22), the CD8Transmembrane chimeric receptor body transmembrane region (SEQ ID NO.23), CD28 chimeric receptor costim...

Embodiment 2

[0204] OCTS-CAR-T cell pathogen detection and expression detection.

[0205] 1. Endotoxin detection;

[0206] (1), endotoxin working standard is 15EU / branch;

[0207] (2), Limulus reagent sensitivity λ=0.25EU / ml, 0.5ml / tube

[0208] (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;

[0209] (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;

[0210] Two are positive control tubes, add 0.1ml of endotoxin working standard solution with 2λ concentration;

[0211] 2 tubes are sample positive control tubes, add 0.1ml sample solution containing 2λ endotoxin ...

Embodiment 3

[0241] Example 3 Functional detection of OCTS-CAR-T cells.

[0242] 1. Evaluation of target cell killing effect.

[0243] (1) Culture target cells separately [PDL1 + K562, EGFRvIII + K562, PDL1 + EGFRvIII + K562, K562 cells] and effector cells [OCTS-CAR-T cells];

[0244] (2) Collect target cells 4x10 5 cells and OCTS-CAR-T cells 2.8x10 6 cells, 800g, centrifuge for 6min, discard the supernatant;

[0245] (3) Resuspend the target cells and effector cells in 1ml D-PBS(-) solution, centrifuge at 800g for 6min, discard the supernatant;

[0246] (4) Repeat step 3 once;

[0247] (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);

[0248] (6) Set up experimental wells with effector-target ratios of 1:1, 5:1, and 10:1. The grouping of effector cells co-incubated with single-target cells and double-target cells is shown in Table 6, and a control group ( K562 cells), 3 replicate wel...

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Abstract

The present invention discloses a malignant glioma CAR-T therapeutic vector based on OCTS technology, including lentiviral backbone plasmid, human EF1α promoter (SEQ ID NO.14), OCTS chimeric receptor domain and PDL1 single-chain antibody ; OCTS chimeric receptor domain includes: CD8 leader chimeric receptor signal peptide (SEQ ID NO.15), PDL1 single-chain antibody light chain VL (SEQ ID NO.16), PDL1 single-chain antibody heavy chain VH (SEQ ID NO.16) ID NO.17), EGFRvIII single-chain antibody light chain VL (SEQ ID NO.18), EGFRvIII single-chain antibody heavy chain VH (SEQ ID NO.19), antibody internal hinge Inner-Linker (SEQ ID NO.20), Single-chain antibody Inter-Linker (SEQ ID NO.21), CD8 Hinge chimeric receptor hinge (SEQ ID NO.22), CD8 Transmembrane chimeric receptor transmembrane region (SEQ ID NO.23), TCR chimeric receptor Synthetic receptor T cell activation domain (SEQ ID NO.26) and chimeric receptor co-stimulatory factor region. In addition, the invention also discloses the construction method of the carrier and its application in the preparation of medicine for treating malignant glioma.

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 treatment carrier for malignant glioma based on OCTS technology. 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 an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/62C12N5/10A61K35/17A61P35/00
CPCA61K39/464411A61K39/464404A61K39/4631A61K39/4611C12N5/0636C12N15/86C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/2827C07K2319/03C07K2319/02C07K2317/622C07K2319/33C12N2740/15043A61K2239/11A61K2239/29C07K16/2863C07K16/30C07K2317/24C12N2510/00C12N2501/51C12N2501/515A61P35/00C12N2740/16043A61K38/00C07K2317/53C07K2319/30C12N7/00C12N2730/10144C12N2800/10
Inventor 祁伟俞磊康立清林高武余宙
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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