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CAR-iNKT with high amplification, survival capacity and tumour killing effect, and application

A technology of chimeric antigen receptors and cells, applied in DNA/RNA fragments, antineoplastic drugs, genetically modified cells, etc., can solve the problems affecting the efficacy of CAR-T cells, adverse long-term survival of CAR-T cells, hypoxia, etc. problem, to achieve the effect of improving tumor killing efficiency, enhancing tumor killing efficiency, and reducing recurrence

Active Publication Date: 2021-01-15
BEIJING GENE KEY LIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ordinary T cells cannot effectively infiltrate into solid tumors; the tumor microenvironment of solid tumors is hypoxic and acidic, which is very unfavorable to the expansion and long-term survival of CAR-T cells, thus seriously affecting the efficacy of CAR-T cells

Method used

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  • CAR-iNKT with high amplification, survival capacity and tumour killing effect, and application
  • CAR-iNKT with high amplification, survival capacity and tumour killing effect, and application
  • CAR-iNKT with high amplification, survival capacity and tumour killing effect, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Preparation of Lentivirus Containing Chimeric Antigen Receptor Molecules

[0069]Passage HEK-293T cells. After the cells grow to 60%-70% confluent, transfect the expression vector containing the CAR molecule together with the packaging plasmid into HEK-293T cells with pEI reagent, and replace with fresh culture 4 hours after transfection. base. Cell culture supernatants were harvested 48-72 hours after transfection. The supernatant was subjected to ultracentrifugation to concentrate the packaged lentivirus containing the CAR molecule. The concentrated lentivirus was tested for viral titer and stored at -80°C for later use.

[0070] For the specific nucleotide and amino acid sequences of each part in the chimeric antigen receptor, refer to the sequence listing, where the names of each part have been marked.

Embodiment 2

[0071] Example 2 The effects of construction of different intracellular signal stimulation domains and expression of IL-15-IL15Rα fusion protein in anti-GPC3 CAR on the proliferation of total iNKT cells

[0072] After isolating iNKT cells from human peripheral blood mononuclear cells with anti-iNKT microbeads, in a 24-well plate according to 2x10 per well 5 For cell seeding, add X-VIVO complete medium (containing 100 IU / ml and 100 ng / ml α-Galcer) to each well and culture for 48 hours, collect the cells in each well, centrifuge at 400xg for 5 minutes, discard the supernatant, and add fresh X-VIVO complete medium was resuspended and reseeded in a 24-well culture plate. Cells in each well were added with 4-1BB, ICD1, ICD2, ICD3, 4-1BB-IL-15, ICD1-IL-15, ICD2- Different lentiviruses of IL-15 and ICD3-IL-15 CAR were used for infection. After 24 hours, collect the cells in each well in a centrifuge tube, centrifuge at 400xg for 5 minutes, count, and count according to 5x10 5 Add...

Embodiment 3

[0075] Example 3 Effects of different intracellular signaling domains and the expression of fusion protein IL-15-IL-15Rα on the ability of CAR-iNKT to kill tumors

[0076] When CAR-iNKT cells in different groups were cultured to the 21st day, 0.33x10 5 CAR-iNKT cells, 1x10 5 CAR-iNKT cells, 3x10 5 CAR-iNKT cells, with 1x10 5 After Huh-7 cells (mixed and incubated with fluorescent dye CFSE for 10 minutes) were co-cultured for 6 hours, the culture supernatants of each group were taken to detect the fluorescence intensity in the supernatants with a microplate reader (the stronger the killing ability of CAR-iNKT cells, the stronger the killing ability of Huh-7 cells). The more CFSE released in the cells, the greater the fluorescence intensity), and the killing efficiency of CAR-iNKT cells in each group was calculated (Figure 3). The CAR-iNKT cells whose intracellular signaling domains are ICD1 and ICD3 have significantly improved killing efficiency against liver cancer cell...

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Abstract

The invention provides a chimeric antigen receptor. The chimeric antigen receptor comprises a GPC3 antigen binding structural domain, an ICD1, ICD2 or ICD3 intracellular signal stimulation domain withamino acid sequences of SEQ ID NO.29, 31 and 33 and IL-15-IL-15alpha fusion protein with an amino acid sequence of SEQ ID NO.7. After the chimeric antigen receptor is transferred into immune cells, especially iNKT cells, the cell proliferation speed, the survival time and the tumour killing effect can be effectively improved. The invention further provides a corresponding expression vector, a transduction system, pharmaceutical application, independent ICD1, ICD2 and ICD3 intracellular signal stimulation domains and IL-15-IL-15alpha fusion protein.

Description

technical field [0001] This application is in the field of cancer immunotherapy. Specifically, the application provides a chimeric antigen receptor, which includes a GPC3 antigen-binding domain, an intracellular signal stimulation domain, and an IL-15-IL-15α fusion protein; and provides corresponding expression vectors, transduction systems, pharmaceutical Use etc. Background technique [0002] According to the 2018 Global Cancer Report, there were 841,080 new cases of liver cancer worldwide, and 781,631 people died of liver cancer [1]. Among them, there were 392,868 new cases of liver cancer in China, and 368,960 people died of liver cancer [2], accounting for almost half of the new cases and deaths of liver cancer in the world. [0003] Existing treatments for liver cancer include surgical resection, liver transplantation, local therapy (RFA, TACE, TAE, HIFA, etc.), systemic therapy (sorafenib, lenvatinib, etc.). Surgical resection can be cured, but most liver cancers h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/12C12N15/867C12N5/10A61K39/00A61P35/00A61P1/16
CPCC07K16/303C07K14/5443C07K14/7051C07K14/70539C07K14/70507C12N15/86C12N5/0636C12N5/0646A61K39/001102A61K39/00114A61P35/00A61P1/16C07K2317/622C07K2319/33C07K2319/74C07K2319/00C07K2319/03C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156A61K2039/844
Inventor 闾军熊福银冯纪开
Owner BEIJING GENE KEY LIFE TECH CO LTD
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