OCTS-CAR double-targeting chimeric antigen receptor, coding gene, recombinant expression vector and establishment and application of OCTS-CAR double-targeting chimeric antigen receptor, coding gene and recombinant expression vector

A technology of OCTS-CAR and chimeric antigen receptor, which can be used to target specific cell fusion, polypeptides containing positioning/targeting motifs, receptors/cell surface antigens/cell surface determinants, etc. It is difficult to transduce into primary T lymphocytes, the killing function is reduced, and the transduction cycle time is long, so as to eliminate the possibility of self-replication, expand the recognition range, and improve the transduction efficiency.

Active Publication Date: 2017-11-10
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The disadvantage of option 1 is that it occupies the precious capacity of the lentiviral transgene vector, which is not conducive to loading other functional elements; the packaging efficiency of the transgene vector is low; the gene transduction efficiency is very low, and it is difficult to transduce into primary T lymphocytes
[

Method used

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  • OCTS-CAR double-targeting chimeric antigen receptor, coding gene, recombinant expression vector and establishment and application of OCTS-CAR double-targeting chimeric antigen receptor, coding gene and recombinant expression vector
  • OCTS-CAR double-targeting chimeric antigen receptor, coding gene, recombinant expression vector and establishment and application of OCTS-CAR double-targeting chimeric antigen receptor, coding gene and recombinant expression vector
  • OCTS-CAR double-targeting chimeric antigen receptor, coding gene, recombinant expression vector and establishment and application of OCTS-CAR double-targeting chimeric antigen receptor, coding gene and recombinant expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1 Construction of OCTS-CAR-T cells

[0090] 1. Construction, purification and detection of recombinant lentiviral vectors lvOCTS123BCMAs, lvOCTS319BCMAs, lvOCTS38BCMAs, lvOCTS-PDL1BCMAs, lvOCTS123BCMAt, lvOCTS319BCMAt, lvOCTS38BCMAt, lvOCTS-PDL1BCMAt

[0091] Such as image 3 As shown, the construction method of the recombinant lentiviral vector of the present invention is as follows:

[0092]1. Cloning human EF1α promoter, OCTS-based CAR dual-targeting chimeric antigen receptors (OCTS123BCMAs, OCTS319BCMAs, OCTS38BCMAs, OCTS-PDL1BCMAs, OCTS123BCMAt, OCTS319BCMAt, OCTS38BCMAt, OCTS-PDL1BCMAt), and PDL1 single-chain antibody into lentivirus The backbone plasmid pLenti-3G ​​basic was used to obtain recombinant lentiviral plasmids pOCTS123BCMAs, pOCTS319BCMAs, pOCTS38BCMAs, pOCTS-PDL1BCMAs, pOCTS123BCMAt, pOCTS319BCMAt, pOCTS38BCMAt, pOCTS-PDL1BCMAt.

[0093] (1) The lentiviral backbone plasmid pLenti-3G ​​basic was double-digested with Cla I and EcoR I restricti...

Embodiment 2

[0217] Example 2 OCTS-CAR-T cell pathogen detection and expression detection

[0218] 1. Endotoxin detection;

[0219] (1) Endotoxin working standard is 15EU / cartridge;

[0220] (2) Limulus reagent sensitivity λ=0.25EU / ml, 0.5ml / tube

[0221] (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 15 minutes; each dilution step should be diluted in a vortex mixer Mix well for 30s;

[0222] (4) Adding samples: Take several LAL reagents, add 0.5ml of BET water to dissolve each, and distribute to several endotoxin-free test tubes, each with 0.1ml. 2 of them are negative control tubes, add 0.1ml of BET water; 2 are positive control tubes, add 0.1ml of endotoxin working standard solution with 2λ concentration; 2 are sample positive control tubes, add 0.1ml of endotoxin standard containing 2λ sample solution (1ml of 20-fold dilut...

Embodiment 3

[0253] Example 3 Functional detection of OCTS-CAR-T cells

[0254] 1. Evaluation of target cell killing effect.

[0255] (1) Culture target cells separately [BCMA + K562, CD319 + K562, CD38 + K562, PDL1 + K562, CD123 + K562, BCMA + CD123 + K562, BCMA + CD319 + K562, BCMA + CD38 + K562, BCMA + PDL1 + K562, K562 cells] and effector cells [OCTS-CAR-T cells], the groups of effector cells co-incubated with single target cells and double target cells are shown in Table 9.

[0256] Table 9 Grouping list of effector cells co-incubated with single target cells and double target cells

[0257] effector cells

target cell 1

target cell 2

target cell 3

OCTS123BCMAs-CAR-T

CD123 + K562

BCMA + K562

BCMA + CD123 + K562

OCTS319BCMAs-CAR-T

CD319 + K562

BCMA + K562

BMCA + CD319 + K562

OCTS38BCMAs-CAR-T

CD38 + K562

BCMA + K562

BCMA + CD38 + K562

OCTS-PDL1BCMAs-CAR-T

PDL1 +...

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Abstract

The invention provides an OCTS-CAR technique-based OCTS-CAR double-targeting chimeric antigen receptor, a coding gene and a recombinant expression vector and establishment and application of the OCTS-CAR double-targeting chimeric antigen receptor, the coding gene and the recombinant expression vector. The OCTS-CAR double-targeting chimeric antigen receptor includes a CD8 leader membrane receptor signal peptide, a double-antigen binding region, a CD8 Hinge chimeric receptor gemel, a CD8 transmembrane chimeric receptor transmembrane region, a CD28 chimeric receptor co-stimulatory factor, a CD134 chimeric receptor co-stimulatory factor and a TCR chimeric receptor T cell activating domain which are connected sequentially and in series, wherein the double-antigen binding region comprises heavy-chain VH and light-chain VL, connected in a certain mode, of two single-chain antibodies, an antibody Inner-Linker and an Inter-Linker between single-chain antibodies, and the two single-chain antibodies are formed by combining any two of a BCMA single-chain antibody, a CD319 single-chain antibody, a CD38 single-chain antibody, a PDL1 single-chain antibody and a CD123 single-chain antibody; in addition, the invention further provides a gene encoding the OCTS-CAR double-targeting chimeric antigen receptor, the recombinant expression vector and an establishment method and application of the gene encoding the OCTS-CAR double-targeting chimeric antigen receptor and the recombinant expression vector.

Description

technical field [0001] The invention belongs to the technical field of tumor immunotherapy, and specifically relates to a CAR dual-targeting chimeric antigen receptor based on OCTS technology, an encoding gene, an OCTS-CAR recombinant expression vector, and a construction method and application thereof for immunotherapy of malignant tumors . 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 (eg, highly specific cytolysis). In the 1950s, Burnet and Thomas proposed the theory of "immune surveillance", believing 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, includin...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K16/2866C07K16/2896C07K16/30C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043
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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