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Modified T cell and preparation method and application thereof

A cell and cell surface technology, applied in the fields of gene editing and tumor immunotherapy, can solve the problems of large tumor heterogeneity, complex tumor microenvironment, and difficult infiltration of CAR-T cells

Inactive Publication Date: 2020-03-24
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although CAR-T cell therapy has been successful in early clinical studies for the treatment of CD19-positive hematological malignancies (Daviala et al, 2014; Lee et al, 2015; Maude et al, 2014), however, the use of CAR-T cells to target entities The clinical response to tumor antigens is very limited, due to the large heterogeneity of solid tumors, the complex tumor microenvironment, and the difficulty of CAR-T cell infiltration, etc.

Method used

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  • Modified T cell and preparation method and application thereof
  • Modified T cell and preparation method and application thereof
  • Modified T cell and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0212] Example 1. Effects of CAR-T cells with different CAR structures

[0213] Four structures of CAR (P4-z, P4-BBz, P4-28z and P4-28BBz were designed, the structures are as follows figure 1 Shown, wherein P4 is anti-mesothelin scFv), and prepare CAR-T cells.

[0214] The resulting CAR-T cells were co-cultured with H226 cells for 20 h at an effector cell:target cell ratio of 1:1, and the release of IFN-γ and IL-2 was measured.

[0215] The resulting CAR-T cells were co-cultured with luciferase-expressing H226 cells (H226-luci) at a ratio of 2:1 effector cells:target cells for 3 days, and the target cells were calculated by measuring the luciferase activity of the remaining tumor cells. Percentage of cell lysis.

[0216] Such as figure 2 As shown, compared with P4-z, P4-BBz and P4-28BBz, P4-28z showed higher release of IFN-γ and IL-2 ( figure 2 A), and higher specific cell lysis ( figure 2 B, * means P<0.05, **** means P<0.0001).

[0217] The following experiments all...

Embodiment 2

[0218] Example 2. The effect of gene editing of inhibitory receptors in CAR-T cells on tumor killing

[0219] This example studies the effect of inhibitory receptors TIGIT, BTLA, CD160, 2B4 and CD200R in CAR-T cells being knocked out through gene editing on tumor killing.

[0220] The TIGIT knockout group showed a killing effect not lower than that of the control CAR-T cells in the short-term (24h, 48h) killing of target cells with a high-efficiency target ratio (1:1) of CRL5826-luci and CRL5826-PDL1-luci, It shows a stronger killing advantage than control CAR-T cells in the killing of low-efficiency targets for a long time. ( image 3 )

[0221] The TIGIT knockout group showed a killing effect not lower than that of the control CAR-T cells in the short-term (24h, 48h) killing of the target cells OVCAR3-luci and HCT116-luci with a high-efficiency target ratio (1"1). The effect target ratio showed a stronger killing advantage than the control CAR-T cells in the long-term kil...

Embodiment 3

[0230] Example 3. The effect of gene editing of T cell exhaustion-related transcription factors in CAR-T cells on tumor killing

[0231] This example studies the effect of T cell exhaustion-related transcription factors BATF, GATA3, IRF4, and RARA in CAR-T cells being knocked out through gene editing on tumor killing.

[0232] The BATF knockout group showed a killing effect not lower than that of the control CAR-T cells in the short-term (24h, 48h) killing of target cells with high-efficiency target ratio (1:1) of CRL5826-luci and CRL5826-PDL1-luci, It has a very obvious killing advantage over control CAR-T cells in the killing of low-efficiency targets and long-term killing. ( Figure 13 )

[0233] The BATF knockout group showed a killing effect not lower than that of the control CAR-T cells in the short-term (24h, 48h) killing of the target cells OVCAR3-luci and HCT116-luci with a high-efficiency target ratio (1:1). Compared with the control CAR-T cells, the effect target...

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Abstract

The invention relates to the field of gene editing and tumor immunotherapy. Specifically, the present invention relates to a method for preparing a modified T cell, such as a CAR-T cell, by gene editing, and the modified T cell prepared by the method and application thereof.

Description

technical field [0001] The invention relates to the fields of gene editing and tumor immunotherapy. In particular, the present invention relates to a method for preparing modified T cells such as CAR-T cells by gene editing, as well as the modified T cells prepared by the method and uses thereof. Background technique [0002] T cells play an important role in anti-tumor immunity. However, in tumor patients, the content of specific cytotoxic T lymphocytes (CTL) is very small, its acquisition and in vitro expansion are difficult, and the low affinity of CTL limits its application in clinical treatment of tumors. [0003] Adoptive transfer of T cells is a specific and low-toxic anti-tumor method that has gained more attention in recent years. For example, genetic modification of T cells with T cell receptor (TCR) or chimeric antigen receptor (CAR) is the most commonly used method to generate tumor-specific T cells. [0004] TCR gene transfer technology is to clone the α and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/90C12N9/22A61K35/17A61P35/00A61P35/02
CPCC12N5/0636C12N15/907C12N9/22A61K35/17A61P35/00A61P35/02C12N2510/00C12N15/90C07K14/705C07K14/7155C07K14/71C07K14/70596C07K14/70578C07K14/70575A61K39/001168A61K2039/5156A61K2039/5158C07K16/30
Inventor 王皓毅张兴颖程晨唐娜李娜
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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