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OCTS technology-based pancreatic cancer and malignant mesothelioma CAR-T therapy vector, as well as construction method and applicaitno thereof

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

Active Publication Date: 2017-10-27
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 the two antigens of MESOTHELIN and PDL1 to overcome the above shortcomings

Method used

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  • OCTS technology-based pancreatic cancer and malignant mesothelioma CAR-T therapy vector, as well as construction method and applicaitno thereof
  • OCTS technology-based pancreatic cancer and malignant mesothelioma CAR-T therapy vector, as well as construction method and applicaitno thereof
  • OCTS technology-based pancreatic cancer and malignant mesothelioma CAR-T therapy vector, as well as construction method and applicaitno thereof

Examples

Experimental program
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Embodiment approach

[0052] Below in conjunction with specific embodiment further elaborates this invention. It should be understood that the specific embodiments described herein are presented by way of example and not as limitations of the invention. The principal characteristics of this invention can be employed in various embodiments without departing from the scope of the invention. Material

[0053] 1. Lentiviral backbone plasmid pLenti-3G ​​basic, lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein plasmid pEnv-G, HEK293T / 17 cells, homologous recombination enzyme, Oligo Annealing Buffer, mycoplasma detection kit, internal Toxin Detection Kit, PDL1 + K562, MESOTHELIN + K562, PDL1 + MESOTHELIN + K562 and K562 cells were purchased from Shiao (Shanghai) Biomedical Technology Co., Ltd.; the specific preparation method of the lentiviral backbone plasmid pLenti-3G ​​basic has been published in the invention titled "A CAR-T transgene based on replication-defective recombinant le...

Embodiment 1

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

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

[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-PMs, OCTS-PMt] (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), MESOTHELIN single-chain antibody light chain VL (SEQ ID NO.18), MESOTHELIN 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 costimulator (SEQ ...

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 ...

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Abstract

The invention discloses an OCTS technology-based pancreatic cancer and malignant mesothelioma CAR-T therapy vector. The vector comprises lentivirus framework plasmid, a human EF1alpha promoter (SEQ ID NO. 14), an OCTS chimeric receptor structural domain and a PDL1 single-chain antibody; the OCTS chimeric receptor structural domain comprises CD8 leader chimeric receptor signal peptide (SEQ ID NO. 15), a PDL1 single-chain antibody light chain VL (SEQ ID NO. 16), a PDL1 single-chain antibody heavy chain VH (SEQ ID NO. 17), an MESOTHELIN single-chain antibody light chain VL (SEQ ID NO. 18), an MESOTHELIN single-chain antibody heavy chain VH (SEQ ID NO. 19), an antibody inner linker Inner-Linker (SEQ ID NO. 20), a single-chain antibody inter-linker Inter-Linker (SEQ ID NO. 21), a CD8 Hinge chimeric receptor linker (SEQ ID NO. 22), a CD8Transmembrane chimeric receptor transmembrane domain (SEQ ID NO. 23), a TCR chimeric receptor T-cell activation domain (SEQ ID NO. 26), and a chimeric receptor inducible co-stimulater area. In addition, the invention also discloses a construction method of the vector as well as application of the vector to preparation of medicines for treating pancreatic cancer and malignant mesothelioma.

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 pancreatic cancer and malignant mesothelioma 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 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 th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/62A61K35/17A61P35/00
CPCC12N15/86C07K14/70517C07K14/70521C07K14/70578C07K16/2827C12N2740/15043C07K2319/33C07K2319/02C07K2319/03C07K2319/00C07K2317/622A61K39/464411A61K39/464468A61K39/4611A61K39/4631A61K2239/54C07K14/7051C07K14/70532C07K16/30C07K2317/31C07K2317/73A61K2039/852A61K2039/86A61K38/00C07K16/2863C07K2317/24C07K2317/53C07K2319/30C12N7/00C12N2730/10144C12N2800/10
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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