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HER2 and MESO combined double-target CAR-T vector as well as construction method and application thereof in cancers

A carrier and binding domain technology, applied in the field of combined HER2 and MESO dual-target CAR-T carrier and its construction, which can solve the problems of homing barrier, poor CAR-T cell persistence, and lack of therapeutic targets.

Active Publication Date: 2021-11-16
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, CAR-T therapy still has many challenges in the treatment of solid tumors, including: lack of ideal therapeutic targets, homing barriers, and poor persistence of CAR-T cells caused by the immunosuppressive microenvironment

Method used

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  • HER2 and MESO combined double-target CAR-T vector as well as construction method and application thereof in cancers
  • HER2 and MESO combined double-target CAR-T vector as well as construction method and application thereof in cancers
  • HER2 and MESO combined double-target CAR-T vector as well as construction method and application thereof in cancers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0209] Example 1 Construction of HM CAR plasmid capable of simultaneously expressing HER2 single-chain antibody and MESO single-chain antibody

[0210] Connect Signal Peptide-HER2V in sequence L -(G4S) 3 -HER2V H -(G4S) 5 -MESOV H -(G4S) 3 -MESOV L - CD8α-4-1BB-CD3ζ. The two ends of the sequence were connected with BamHI and XhoI restriction sites respectively. All sequences are humanized, synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and preserved as PUC57 plasmids.

[0211] The obtained gene sequence fragment was connected to the lentiviral expression vector pLenti6.3 / V5 by restriction enzyme ligation to obtain the HM CAR plasmid. image 3 The result of agarose gel electrophoresis after the HM CAR gene constructed in the pLenti6.3 / V5 vector was identified by double digestion with BamHI and XhoI.

Embodiment 2

[0212] Example 2 lentiviral packaging

[0213] Adjust HEK293 cells to 2 × 10 6 After live cells / mL, take 25.5mL and add 1.5mL LV-MAXSupplement;

[0214] Prepare DNA / LV-MAX Transfection Reagent Complex:

[0215] Tube 1: Labeled "DNA"

[0216] (1) Add 1.5mL Opti-MEM serum-free medium;

[0217] (2) Add three helper plasmid mixtures (1.5 μg / mL) and lentiviral expression vector pLenti6.3 / V5 (1 μg / mL);

[0218] Test Tube 2: Labeled "TfxR"

[0219] (1) Add 1.5mL Opti-MEM serum-free medium;

[0220] (2) Add 180 μL of LV-MAX transfection reagent, briefly vortex and incubate at room temperature for 1 minute;

[0221] (3) After 1 minute, pour test tube 1 into test tube 2 or combine the two solutions in the opposite way, and vortex briefly;

[0222] (4) After incubating the mixed solution at room temperature for 10 minutes, directly add the DNA / LV-MAX transfection complex to HEK293 cells;

[0223] (5) 5-6 hours after transfection, add 1.2 mL of LV-MAX enhancer to the cells.

[02...

Embodiment 3

[0231] Example 3 Preparation of CAR-T cells

[0232] PBMC cell recovery and magnetic bead sorting

[0233] (1) Take the human PBMC out of the liquid nitrogen tank carefully, put it in a 37°C water bath, and put it into a safety cabinet after dissolving to a small ice cube;

[0234] (2) Pipette 5mL HBSS (containing 10% human serum albumin), then suck the cells into a 50mL centrifuge tube, rinse the frozen storage tube with 5mL HBSS (containing 10% human serum albumin);

[0235] (3) Slowly add 30 mL of HBSS (containing 10% human serum albumin) to a total volume of 40 mL, centrifuge at 400 g for 10 min, and discard the supernatant;

[0236] (4) Add 1 mL of 1640 medium (containing 10% FBS) and 8 μL of DNAse, and let stand at 37°C for 15 minutes;

[0237] (5) Add 29 mL of 1640 medium (containing 10% FBS) and let stand at 37°C for 4-6 hours;

[0238] (6) After standing for several hours, perform cell counting to determine the total cell mass;

[0239] (7) Centrifuge at 300g for ...

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Abstract

The invention provides a HER2 and MESO combined double-target CAR-T vector as well as a construction method and application thereof in cancers. Specifically, the invention provides a bispecific chimeric antigen receptor (CAR), which comprises HER2scFv, MESO scFv, a 4-1BB co-stimulatory domain and a CD3 activation domain. The bispecific CAR-T cell has a remarkable killing effect on HER2 positive target cells and MESO positive target cells, the tumor killing effect of the T cell can be improved, generated cell factors have a super addition effect, and compared with single-target CAR-T, the bispecific CAR-T cell can better remove tumor cells and relieve the antigen escape phenomenon caused by tumor heterogeneity, and the tumor killing capability of the CAR-T cells is further enhanced.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more particularly to a combined HER2 and MESO dual-target CAR-T vector and its construction method and application in cancer. Background technique [0002] Cancer is an abnormal proliferation that occurs due to the loss of normal growth regulation of normal cells in the body. It may be accompanied by distant metastasis. It has a high degree of malignancy and is a major killer that threatens human life and health in today's society. Among them, pancreatic cancer is a highly malignant gastrointestinal tumor and one of the malignant tumors with the worst prognosis, with a 5-year survival rate of only 9%. The primary tumors of the pancreas mainly include three tissue sources: exocrine, endocrine and mesenchymal tissues, among which the exocrine pancreatic tumors account for more than 90% of pancreatic tumors, and 80% are derived from the pancreatic ductal epithelium. Because of its hidden an...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K16/32C07K16/2863C07K16/303C07K14/7051C12N15/86C12N5/0636C12N5/0646C12N5/0645A61K39/001104A61K39/001102A61P35/00C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N2510/00C12N2740/15043
Inventor 姜凤婷罗剑郑眉丁亚红熊斐斐刘雪颖周旭
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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