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T cell synergist for CAR T cell therapy of leukemia and method for obtaining synergistic T cells

A synergist and cell technology, which is used to target specific cell fusion, receptor/cell surface antigen/cell surface determinant, blood cell cancer vaccine, etc., and can solve problems such as poor treatment effect

Pending Publication Date: 2022-05-03
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Another key factor determining the efficacy of CAR T cells is whether CAR T cells can persist in the body. Short-term survival of CAR T cells in vivo often leads to poor therapeutic effect.

Method used

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  • T cell synergist for CAR T cell therapy of leukemia and method for obtaining synergistic T cells
  • T cell synergist for CAR T cell therapy of leukemia and method for obtaining synergistic T cells
  • T cell synergist for CAR T cell therapy of leukemia and method for obtaining synergistic T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction and preparation of EpCAM CAR T cells and CD33 CAR T cells

[0040] In this example, EpCAM CAR T cells and CD33 CAR T cells were prepared. Except for the difference in CAR chimeric sequence, other operations and reagents were the same for the two cells.

[0041] CAR chimeric sequence for EpCAM CAR T cells: The murine anti-human EpCAM scFv was connected to the CD8 transmembrane region, 41bb co-stimulatory domain and CD3ζ to obtain the murine anti-human EpCAM chimeric sequence (AE4 scFv-CD8-CD28- CD3ζ, whose sequence is shown in SEQ ID No: 1), was inserted into PCDH-MSCV-MCS-EF1-copGFP (purchased from Addgene).

[0042] CAR chimeric sequence of CD33 CAR T cells: The humanized anti-human CD33 scFv is connected to the CD8 transmembrane region, 41bb co-stimulatory domain and CD3ζ to obtain a humanized anti-human CD33 chimeric sequence (such as SEQ ID No: 3), inserted into PCDH-MSCV-MCS-EF1-copGFP (purchased from Addgene)

[0043] Then, the three plasm...

Embodiment 2

[0048] Example 2. Down-regulation of chemokines and activation of mTOR in CAR T cells cultured in vitro

[0049] Determination groups: T cells initially isolated in Example 1 (indicated by T), and EpCAM CAR T cells cultured in vitro for 5 days in Example 1 (indicated by CAR T).

[0050] Flow Cytometry

[0051] The cells to be determined were detected by BD LSRII flow cytometer and analyzed by Flowjo V10.

[0052] In the determination of this example, the anti-human antibody EpCAM was purchased from biolengend, (324208), the anti-human CXCR4 antibody was purchased from eBioscience (12-9999-42), the anti-human mTOR antibody was purchased from BD (583489), and the anti-human S6 The antibody was purchased from CST (14733S), the anti-human CD62L antibody was purchased from BD (555544), and the anti-human CD45RO antibody was purchased from BD (560607).

[0053] Wash the EpCAM T cell line harvested in vitro once in phosphate buffered saline supplemented with 2% fetal bovine serum, ...

Embodiment 3

[0057] Example 3. Effect of rapamycin treatment on chemokines of EpCAM CAR T cells

[0058] Determination groups: EpCAM CAR T cells cultured in vitro and treated with rapamycin at 24 hours (indicated by +Rapa), EpCAM CAR T cells cultured in vitro but not treated with rapamycin (indicated by -RaPa). Sample collection time points: Days 3, 6, 9, and 12.

[0059] Concentration screening: T cells were isolated and transfected with lentivirus as in Example 1, and CAR T cells were treated with 0nM, 5nM, 10nM, 20nM, and 40nM rapamycin respectively, and the expression of CXCR4 was detected by flow cytometry after 6 days of culture. The results are shown in image 3 .

[0060] The results showed that 5nM, 10nM, 20nM, and 40nM can all be used to treat CAR T cells, and 20nM is the optimal concentration, which can most effectively up-regulate the expression of CXCR4.

[0061] T cells were isolated as in Example 1 and subjected to lentiviral transfection and rapamycin (20nM) treatment to...

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Abstract

The present invention relates to T cell potentiators for use in CAR T cell therapy against hematologic tumors, such as acute myelogenous leukemia, and methods of using the same to enhance the effect of CAR T cell therapy. The method comprises treating T cells used in CAR T therapy in an in vitro culture process using a T cell potentiator. The T cell synergist provided by the invention can enhance migration of CAR T / NK cells to bone marrow, and improve the effect of CAR T cell therapy in removing tumors.

Description

technical field [0001] The present invention relates to chimeric antigen receptor (CAR) T cell therapy for tumors, in particular to T cell potentiators used in CAR T cell therapy for hematological tumors such as acute myeloid leukemia, and the use thereof to enhance CAR T cells method of therapeutic effect. Background technique [0002] Acute myeloid leukemia (AML) is a tumor derived from the bone marrow. Although chemotherapy can induce a remission rate of up to 70%, most patients will relapse. ((Bishop,1997)), when the leukemia cells in the bone marrow exceed 5%, it is an intramedullary relapse, and if it occurs in the extramedullary part, it becomes an extramedullary relapse (usually the central nervous system and testis). Intramedullary and extramedullary leukemia in children Recurrence is about half and half, and 95% of adults have intramedullary recurrence. Whether the leukemia stem cells in the bone marrow can be eliminated is a key factor in the success or failure ...

Claims

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Application Information

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IPC IPC(8): C12N5/10A61K35/17A61K39/00A61P35/00A61P35/02
CPCC12N5/0636C07K16/30C07K16/2803C07K14/7051A61P35/00A61P35/02C07K2319/03C07K2319/33C07K2317/622C12N2501/04C12N2501/2302C12N2501/2307C12N2501/2315A61K2039/804A61K39/464411A61K39/464466A61K2239/31A61K2239/48A61K2239/38A61K39/4611C12N5/10A61K31/436C12N5/06
Inventor 魏海明粘志刚郑小虎孙汭田志刚
Owner UNIV OF SCI & TECH OF CHINA
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