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Combined application of oncolytic virus and CAR-T to treatment of solid tumor

A CAR-T, oncolytic virus technology, applied in the direction of virus/phage, application, virus, etc., can solve the problem of unsatisfactory tumor effect, low effect of lysis and killing ability of tissue and cell functional activity, lack of targeted in vivo immune response, etc. , to achieve the effect of overcoming the unsatisfactory killing effect

Active Publication Date: 2019-04-05
WUHAN BIO RAID BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the unsatisfactory effect of simple CAR-T cell therapy on solid tumors in the prior art, and the lack of targeting and easy immune response in vivo when administered with simple oncolytic virus, in view of the genetically modified oncolytic virus The ability to lyse and kill tumor cells and have little or no effect on the functional activity of normal tissue cells, we propose a combination of oncolytic virus and CAR-T cells

Method used

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  • Combined application of oncolytic virus and CAR-T to treatment of solid tumor
  • Combined application of oncolytic virus and CAR-T to treatment of solid tumor
  • Combined application of oncolytic virus and CAR-T to treatment of solid tumor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1 Lentiviral construction:

[0036] 1.1 Using OE PCR technology according to figure 1 The structure shown constructs the anti-EGFR vⅢ CAR gene into the lentiviral vector pLVX-EF1α-IRES-Puro.

[0037] 1.2 Transfect HEK293T cells with the lentiviral vector constructed in step 1, lentiviral vector psPAX2 and lentiviral vector pMD2.G at a ratio of 6:3:1. The total amount of plasmid per 10 cm dish is 10 μg, and the PEI cell transfection solution is 30 μg. For the specific transfection protocol, refer to the instruction manual of PEI cell transfection solution.

[0038] 1.3 On the second day after plasmid transfection, add 5ml of liquid to each dish, and put it into the cell incubator to continue culturing.

[0039] 1.4 On the third day of transfection, collect the supernatant into a 50ml tube, and centrifuge at 4000g at 4°C for half an hour.

[0040] 1.5 After centrifugation, filter with a 0.22 μm filter membrane, separate 3ml of the filtrate and store it in a 4°C refrige...

Embodiment 2

[0085] Example 2 In vitro tumor killing experiment

[0086] 1. Digest U251-MG cells and U373-MG cells with Versene, resuspend the cells and count. Follow 1 x 10 per well 4 Cells were seeded in a 94-well plate, with 100 μl of medium per well. Place in a cell culture incubator for 6-8 hours.

[0087] 2. According to the effect-to-target ratio of 1 / 4, 1 / 2, 1, 2, 4, the above-mentioned OCAR-T cells loaded with oncolytic virus, OT cells loaded with oncolytic virus, and CAR- T cells, T cells not loaded with oncolytic virus, oncolytic virus. Add 100 μl of the system to the target cells per well. Incubate in the cell incubator for 16 to 24 hours (the specific time is determined by microscopic observation of the tumor killing effect, and the specific time of the oncolytic virus group is consistent with that of the experimental group).

[0088] 3. After the tumor-killing incubation, all the supernatant of each well was discarded, and the T cells at the bottom of the plate were wash...

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Abstract

The invention discloses combined application of an oncolytic virus and CAR-T, mainly directing at treatment of solid tumor. As a carrier of the oncolytic virus for treating the solid tumor, the CAR-Tcarries the oncolytic virus to a solid tumor location, so that the defect of undesirable killing effect on the solid tumor of the CAR-T is overcome, and the oncolytic virus is not separately exposed to a humoral immunity environment. The oncolytic virus is specifically carried and delivered to the solid tumor location according to a solid tumor target of the CAR-T, so that the cancer cell lysis ability of the oncolytic virus and the specific target cell killing ability of a CAR-T cell are brought into full play.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and targeted immunization, in particular to the technology for the combined application of oncolytic virus and CAR-T for the treatment of solid tumors Background technique [0002] CAR-T cells are called Chimeric Antigen Receptor T-Cells. It is to express T cells from the peripheral blood of patients through lentiviral transfection and other means to express single-chain antibodies targeting specific targets, and specifically target target cells. However, it does not have the ability to recognize and kill cells expressing non-target proteins. At present, chimeric antigen receptor T-cell immunotherapy (Chimeric Antigen Receptor T-Cell Immunotherapy, CAR-TImmunotherapy) has made great achievements in cancer treatment, especially in the treatment of hematological tumors. The ability to target tumor cells, so as to quickly find and bind target cells, and kill target cells[6 ,7] . At p...

Claims

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Application Information

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IPC IPC(8): A61K35/768A61P35/00C12N5/10C12N15/867A61K35/17
CPCA61K35/17A61K35/768A61P35/00C12N5/0636C12N15/86C12N2510/00C12N2740/15043A61K2300/00
Inventor 张同存顾潮江张子健徐瑶
Owner WUHAN BIO RAID BIOTECH CO LTD
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