PSCA (prostate stem cell antigen) and PD-L1 targeted CAR based on OCTS (one CAR with two ScFvs)-CAR (chimeric antigen receptor), encoding gene and expression vector

A technology of OCTS-CAR and chimeric antigen receptor, applied in DNA/RNA fragments, genetic engineering, plant gene improvement, etc., can solve the problems of occupying precious capacity of lentiviral transgene vectors, decreased killing function, and low overall efficiency. Achieve the effect of eliminating self-replication, improving expression efficiency, and eliminating possibility

Active Publication Date: 2017-11-07
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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  • PSCA (prostate stem cell antigen) and PD-L1 targeted CAR based on OCTS (one CAR with two ScFvs)-CAR (chimeric antigen receptor), encoding gene and expression vector
  • PSCA (prostate stem cell antigen) and PD-L1 targeted CAR based on OCTS (one CAR with two ScFvs)-CAR (chimeric antigen receptor), encoding gene and expression vector
  • PSCA (prostate stem cell antigen) and PD-L1 targeted CAR based on OCTS (one CAR with two ScFvs)-CAR (chimeric antigen receptor), encoding gene and expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Construction of OCTS-CAR-T cells

[0083] 1. Construction, purification and detection of recombinant lentiviral vectors lvOCTS-PDL1PSCAs and lvOCTS-PDL1PSCAt

[0084] Such as image 3 As shown, the construction method of the recombinant lentiviral vector of the present invention is as follows:

[0085] 1. The human EF1α promoter, OCTS structure [OCTS-PDL1PSCAs, OCTS-PDL1PSCAt], and PDL1 single-chain antibody were cloned into the lentiviral backbone plasmid pLenti-3G ​​basic to obtain recombinant lentiviral plasmids pOCTS-PDL1PSCAs and pOCTS-PDL1PSCAt respectively.

[0086] (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, a...

Embodiment 2

[0197] Example 2 OCTS-CAR-T cell pathogen detection and expression detection

[0198] 1. Endotoxin detection;

[0199] (1) Endotoxin working standard is 15EU / cartridge;

[0200] (2) Limulus reagent sensitivity λ=0.25EU / ml, 0.5ml / tube

[0201] (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;

[0202] (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. Two of them are negative control tubes, add 0.1ml of BET water; two are positive control tubes, add 0.1ml of endotoxin working standard solution with 2λ concentration; two are sample positive control tubes, add 0.1ml of endotoxin standard containing 2λ sample solution (1ml of 20-fold...

Embodiment 3

[0232] Example 3 Functional detection of OCTS-CAR-T cells

[0233] 1. Evaluation of target cell killing effect.

[0234] (1) Culture target cells [PSCA+K562, PDL1+K562, PDL1+PSCA+K562, K562 cells] and effector cells [OCTS-CAR-T cells] respectively, and the effector cells are co-incubated with single target cells and double target cells respectively The groupings are shown in Table 9.

[0235] Table 9 Grouping list of effector cells co-incubated with single target cells and double target cells

[0236] effector cells

target cell 1

target cell 2

target cell 3

OCTS-PDL1 PSCAs-CAR-T

PDL1 + K562

PSCA + K562

PDL1 + PSCA + K562

OCTS-PDL1PSCAt-CAR-T

PDL1 + K562

PSCA + K562

PDL1 + PSCA + K562

[0237] (2) Collect target cells 4x10 5 cells and OCTS-CAR-T cells 2.8x10 6 cells, 800g, centrifuge for 6min, discard the supernatant;

[0238] (3) Resuspend the target cells and effector cells in 1ml D-PBS(-) sol...

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Abstract

The invention provides a PSCA (prostate stem cell antigen) and PD-L1 targeted CAR based on OCTS (one CAR with two ScFvs)-CAR (chimeric antigen receptor), an encoding gene, an OCTS-CAR-T recombinant expression vector as well as a construction method and an application of the OCTS-CAR-T recombinant expression vector. The PSCA and PD-L1 targeted CAR comprises a CD8 leader membrane receptor signal peptide, a double-antigen binding region, a CD8 Hinge chimeric receptor linker, a CD8 Transmembrane chimeric receptor transmembrane region, a CD28 chimeric receptor co-stimulator, an OX40 chimeric receptor co-stimulator and a TCR chimeric receptor T-cell activation domain which are connected in series sequentially, wherein the double-antigen binding region comprises heavy chains VH and light chains VL of PSCA and PDL1 single-chain antibodies, Inner-Linker in antibodies and Inter-Linker among the single-chain antibodies, which are in series connection or turn connection. Besides, the invention provides the encoding gene of the PSCA and PD-L1 targeted CAR, the recombinant expression vector as well as the construction method and the application of the recombinant expression vector.

Description

technical field [0001] The invention belongs to the technical field of tumor immunotherapy, and specifically relates to an OCTS-CAR-based anti-PSCA and PDL1 dual-targeting chimeric antigen receptor, encoding gene, recombinant expression vector and its construction method for malignant tumor immunotherapy and application. 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, including cytokine therapy, m...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
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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