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Compositions and methods for t cell engineering

An engineering, cell-based technology, applied in the field of compositions and methods for T cell engineering, capable of solving the problems of time and cost constraints for manufacturing patient-specific T cell products

Pending Publication Date: 2021-09-10
GRACELL BIOTECH SHANGHAI CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This approach has had early clinical success, but has been limited by the time and expense of manufacturing patient-specific T cell products

Method used

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  • Compositions and methods for t cell engineering
  • Compositions and methods for t cell engineering
  • Compositions and methods for t cell engineering

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0303] Example 1 Study of U-CAR T cells expressing CD3+CD19 dual CAR and EGFRt switch with TCR knockout

[0304] General Materials and Methods

[0305] Isolation of peripheral blood mononuclear cells (PBMC) and expansion of T cells from donor blood

[0306] Peripheral blood mononuclear cells (PBMCs) were isolated from donor blood by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich). T cells are then enriched, activated, cultured and expanded by magnetic beads coupled to anti-CD3 / anti-CD28.

[0307] Culture of cell lines and PBMCs

[0308] Raji cells (Burkitt lymphoma cells, ATCC-CCL86);

[0309] K562 cells (human erythroleukemia cell line, ATCC-CCL243);

[0310] Raji-ffluc cell line (obtained by screening Raji cells transfected with lentivirus with firefly luciferase);

[0311] 293T cells (ATCC-CRL3216).

[0312] Raji cells, K562 cells and Raji-ffluc cell line were cultured in RPMI1640 medium, and 293T cells were cultured in DMEM medium. Both RPMI1...

Embodiment 2

[0370] Example 2 Study of U-CAR-T cells expressing CAR19 and enhancer with TCR knockout

[0371] In this example, 5 x 10 5 Raji luciferase cells were transplanted into NOG mice and grown until reaching 100mm 3 . Will 1×10 6 Wild-type or TCR KO CD19 CAR-T cells were infused intravenously (IV) into Raji-transplanted mice. Tumor burden was assessed by caliper measurement of actual tumor size. The expansion of CAR-T cells in peripheral blood was measured by flow cytometry analysis. Figure 8A Tumor burden after CAR-T treatment is shown. TCR intact CD19 CAR-T cells were able to eliminate tumors, but TCR KO CD19 CAR-T could not control Raji tumor growth. Although both are based on Raji's tumor-bearing model, the subcutaneous model produces solid tumors and has higher requirements for tumor-controlled CAR-T cell proliferation than the intravenous model. Figure 8B CAR-T proliferation in peripheral blood is shown. Compared with WT CAR-T cells, TCR KOCAR-T cells showed prolif...

Embodiment 3

[0375] Example 3 has TCR and CD 7 Double-knockout U-CAR-T cells expressing CAR7 and enhancers, and TCR and CD 2 Double knockout expression of CD2 CAR and enhancer U-CAR-T cell research

[0376] General Materials and Methods

[0377] Isolation of peripheral blood mononuclear cells (PBMC) and expansion of T cells from donor blood

[0378] Peripheral blood mononuclear cells (PBMC) were isolated from donor blood by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich). T cells are then enriched, activated, cultured and expanded by magnetic beads coupled to anti-CD3 / anti-CD28.

[0379] Cell Lines and T Cell Culture

[0380] CCRF-CEM (T lymphoblastoid cells, ) TM

[0381] CCRF-CEM-luc cells (obtained by screening CCRF-CEM cells transfected with lentivirus with firefly luciferase);

[0382] K562 cells (human erythroleukemia cell line, ATCC-CCL243);

[0383] 293T cells (ATCC-CRL3216);

[0384] NK92 cells (ATCC-CRL2407).

[0385] The CCRF-CEM-luc c...

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Abstract

The disclosure relates to an engineered immune cell and use thereof. The engineered immune cell provided in the disclosure comprises a CAR or engineered TCR, which CAR or engineered TCR can comprise a first antigen binding domain and a second antigen binding domain. The engineered immune cells, when administered into a subject, can inhibit the host immune cells such as T cells and / or NK cells and enhance the survival and persistence of the engineered immune cells in vivo, thereby exhibiting more effective tumor killing activity.

Description

[0001] cross reference [0002] This application requires Chinese patent application number 201811297174.3 filed on November 1, 2018; Chinese patent application number 201811535338.1 filed on December 14, 2018; Chinese patent application number 201811549651.0 filed on December 18, 2018; June 6, 2019 Chinese Patent Application No. 201910492882.0, filed on 2019; and PCT Application No. PCT / CN2019 / 101651, filed on August 20, 2019, each of which is incorporated herein by reference in its entirety. Background technique [0003] Genetic modification to express chimeric antigen receptors (CARs) to generate tumor-specific T lymphocytes is gaining attention as a form of synthetic biology that produces potent antitumor effects. Since specificity is conferred by antibody fragments, CAR-T cells are not MHC-restricted and thus more practical than approaches based on T-cell receptors that require MHC-matching. [0004] Although early clinical data on CAR-T cells in cancer treatment have sh...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85A61K35/17A61P35/00
CPCA61P35/00C12N2510/00C12N5/0636C12N2503/00C07K14/7051C12N15/90C12N2740/16043A61K48/005C07K2319/03C07K16/2803C07K16/2878C07K2317/31A61K35/28A61K35/545C12N2310/20C12N15/1138C12N2501/515C12N2501/2307A61K2039/804A61K2239/48A61K2239/28A61K39/464412A61K39/464419A61K39/46444A61K39/464417A61K39/4631A61K39/464411A61K39/464404A61K2239/23A61K2239/26A61K2239/29A61K2239/38A61K39/464402A61K2239/31A61K39/4611C07K14/71C07K14/70596C07K16/2809C07K2319/02C07K2317/622A61K35/17A61K38/00C07K14/70507
Inventor 王欣欣金涛杨春辉施中东刘丽萍孙静邱淑怡曹卫
Owner GRACELL BIOTECH SHANGHAI CO LTD
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