A kind of prostate cancer car-t treatment vector based on octs technology and its construction method and application

A prostate cancer and carrier technology, applied in the field of medical biology, can solve problems that have not yet been overcome, and achieve the effects of saving economic expenditure, expanding the scope of identification, and eliminating thoroughly

Active Publication Date: 2019-09-03
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 relevant reports on CAR-T therapy targeting PSMA and PD-L1 antigens to overcome the above shortcomings

Method used

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  • A kind of prostate cancer car-t treatment vector based on octs technology and its construction method and application
  • A kind of prostate cancer car-t treatment vector based on octs technology and its construction method and application
  • A kind of prostate cancer car-t treatment vector 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-PDL1PSMAs and lvOCTS-PDL1PSMAt.

[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-PDL1PSMAs, OCTS-PDL1PSMAt] (CD8 leader chimeric receptor signal peptide (SEQ ID NO.15), PSMA single-chain antibody light chain VL (SEQ ID NO.16), PSMA single-chain antibody heavy chain VH (SEQ ID NO.17), PDL1 single-chain antibody light chain VL (SEQ ID NO.18), PDL1 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

[0206] OCTS-CAR-T cell pathogen detection and expression detection.

[0207] 1. Endotoxin detection;

[0208] (1), endotoxin working standard is 15EU / branch;

[0209] (2), Limulus reagent sensitivity λ=0.25EU / ml, 0.5ml / tube

[0210] (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;

[0211] (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;

[0212] Two are positive control tubes, add 0.1ml of endotoxin working standard solution with 2λ concentration;

[0213] 2 tubes are sample positive control tubes, add 0.1ml sample solution containing 2λ endotoxin ...

Embodiment 3

[0243] Example 3 Functional testing of OCTS-CAR-T cells.

[0244] 1. Evaluation of target cell killing effect.

[0245] (1) Culture target cells separately [PSMA + K562, PDL1 + K562, PDL1 + PSMA + K562, K562 cells] and effector cells [OCTS-CAR-T cells];

[0246] (2) Collect target cells 4x10 5 cells and OCTS-CAR-T cells 2.8x10 6 cells, 800g, centrifuge for 6min, discard the supernatant;

[0247] (3) Resuspend the target cells and effector cells in 1ml D-PBS(-) solution, centrifuge at 800g for 6min, discard the supernatant;

[0248] (4) Repeat step 3 once;

[0249] (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);

[0250] (6) Experimental wells with effect-to-target ratios of 1:1, 5:1, and 10:1 were set, and the grouping of effector cells co-incubated with single-target cells and double-target cells was shown in Table 6, and a control group ( K562 cells), 3 replicate wells...

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Abstract

Provided is a prostate cancer CAR-T therapeutic vector targeting both PSMA and PDL1, comprising a lentiviral backbone plasmid, a human EF1α promoter, a double targeting chimeric receptor domain, and a PDL1 single-chain antibody. The double targeting chimeric receptor domain comprises a CD8 leader chimeric receptor signal peptide, a PSMA single-chain antibody light chain variable region, a PSMA single-chain antibody heavy chain variable region, a PDL1 single-chain antibody light chain variable region, a PDL1 single-chain antibody heavy chain variable region, an inner-antibody hinge, an inter-single-chain antibody hinge, a CD8 chimeric receptor hinge, a CD8 chimeric receptor transmembrane region, a TCR chimeric receptor T cell activation domain, and a chimeric receptor costimulatory factor region. Also provided are a method for constructing said vector and use thereof in the preparation of a medicament for treating prostate cancer.

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 prostate cancer 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 ant...

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
CPCC12N5/0636C12N15/86C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/2827C07K16/3069C07K2317/622C07K2319/03C07K2319/02C07K2319/33C12N2740/15043A61K39/4611A61K39/4631A61K39/464411A61K2239/29A61K39/464495
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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