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Lymphoblastic leukemia CAR-T (Chimeric Antigen Receptor-T Cell Immunotherapy) therapy carrier based on OCTS (One CAR with Two SeFvs) technology as well as constructing method and application of lymphoblastic leukemia CAR-T therapy carrier

A leukemia, carrier technology, applied in the field of medical biology

Active Publication Date: 2017-10-13
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] At present, there is no report on CAR-T therapy targeting CD19 and any one of the antigens selected from CD22, CD20, CD30, and CD123 to overcome the above shortcomings.

Method used

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  • Lymphoblastic leukemia CAR-T (Chimeric Antigen Receptor-T Cell Immunotherapy) therapy carrier based on OCTS (One CAR with Two SeFvs) technology as well as constructing method and application of lymphoblastic leukemia CAR-T therapy carrier
  • Lymphoblastic leukemia CAR-T (Chimeric Antigen Receptor-T Cell Immunotherapy) therapy carrier based on OCTS (One CAR with Two SeFvs) technology as well as constructing method and application of lymphoblastic leukemia CAR-T therapy carrier
  • Lymphoblastic leukemia CAR-T (Chimeric Antigen Receptor-T Cell Immunotherapy) therapy carrier based on OCTS (One CAR with Two SeFvs) technology as well as constructing method and application of lymphoblastic leukemia CAR-T therapy carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0060] 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

[0061] 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, CD19 + K562, CD20 + K562, CD22 + K562, CD30 + K562, CD123 + K562, CD19 + CD123 + K562, CD19 + CD20 + K562, CD19 + CD22 + K562, CD19 + CD30 + 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 ...

Embodiment 1

[0103] Example 1 Construction of OCTS-CAR-T cells

[0104] 1. Construction, purification and detection methods of recombinant lentiviral vectors lvOCTS12319s, lvOCTS2219s, lvOCTS2019s, lvOCTS3019s, lvOCTS12319t, lvOCTS2219t, lvOCTS2019t, lvOCTS3019t.

[0105] see image 3 , the construction method of the recombinant lentiviral vector of the present invention is as follows:

[0106] 1. Human EF1α promoter (SEQ ID NO.14), OCTS structure [CD8leader chimeric receptor signal peptide as shown in SEQ ID NO.15, two groups of single-chain antibodies: the first group is selected from the following four groups of single-chain antibodies Any group of chain antibodies: CD20 single-chain antibody light chain VL as shown in SEQ ID NO.18, CD20 single-chain antibody heavy chain VH as shown in SEQ ID NO.19; CD22 as shown in SEQ ID NO.20 Single-chain antibody light chain VL, CD22 single-chain antibody heavy chain VH as shown in SEQ ID NO.21; CD30 single-chain antibody light chain VL as shown i...

Embodiment 2

[0221] OCTS-CAR-T cell pathogen detection and expression detection.

[0222] 1. Endotoxin detection;

[0223] (1), endotoxin working standard is 15EU / branch;

[0224] (2), Limulus reagent sensitivity λ=0.25EU / ml, 0.5ml / tube

[0225] (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;

[0226] (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;

[0227] Two are positive control tubes, add 0.1ml of endotoxin working standard solution with 2λ concentration;

[0228] 2 tubes are sample positive control tubes, add 0.1ml sample solution containing 2λ endotoxin ...

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Abstract

The invention discloses a lymphoblastic leukemia CAR-T (Chimeric Antigen Receptor-T Cell Immunotherapy) therapy carrier based on an OCTS (One CAR with Two SeFvs) technology. The lymphoblastic leukemia CAR-T therapy carrier comprises a lentivirus skeleton plasma, a human EF1alpha promoter, an OCTS chimeric receptor structural domain and an IL6R single-chain antibody, wherein the OCTS chimeric receptor structural domain comprises a CD8 leader chimeric receptor signal peptide and two groups of single-chain antibodies; the first group of single-chain antibodies is selected from any one of the following four groups of single-chain antibodies: a CD20 single-chain antibody light chain VL and a CD20 single-chain antibody heavy chain VH, a CD22 single-chain antibody light chain VL and a CD22 single-chain antibody heavy chain VH, a CD30 single-chain antibody light chain VL and a CD30 single-chain antibody heavy chain VH, and a CD123 single-chain antibody light chain VL and a CD123 single-chain antibody heavy chain VH; and the second group of the single-chain antibodies is a CD19 single-chain antibody light chain VL and a CD19 single-chain antibody heavy chain VH, an antibody Inner-Linker, a single-chain antibody Inter-Linker, a CD8-Hinge chimeric receptor linker, a CD8 Transmembrane chimeric receptor transmembrane zone, a TCR (T Cell Receptor) chimeric receptor T cell activation domain and a chimeric receptor co-stimulator zone. Besides, the invention discloses a constructing method for the carrier and application of the carrier to preparation of a drug for treating lymphoblastic leukemia.

Description

technical field [0001] The invention belongs to the field of medical biology, in particular to a carrier, in particular to a CAR-T treatment carrier for lymphoid leukemia 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 therapy, monoclonal antibody therapy, adopt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/62C12N5/10A61K35/17A61P35/02
CPCC12N5/0636C12N15/86C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/2827C07K16/3069C12N2740/15043C07K2319/33C07K2319/03C07K2319/02C07K2317/622A61K39/4611A61K39/464419A61K39/464424A61K39/464412A61K39/4631A61K39/464417A61K2239/29A61K39/464413
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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