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CD7-CAR-T cell as well as preparation and application thereof

An immune cell and molecular technology, applied in DNA/RNA fragments, recombinant DNA technology, cells modified by introducing foreign genetic material, etc., can solve the problems of successful preparation of CD7-CAR-T cells, graft-versus-host disease, CD7- CAR-T cell cannibalism and other issues

Active Publication Date: 2020-02-07
PERSONGEN BIOMEDICINESUZHOUCO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of CD7-CAR-T cells to target T-cell acute lymphoblastic leukemia still faces a big problem, because both normal effector T cells and T-cell tumors express CD7 antigens, which will lead to the development of CD7-CAR-T cells. Cannibalism, CD7-CAR-T cells are difficult to prepare successfully in vitro
Although recent reports have shown that T-ALL cells can be eliminated by CD7-CAR-T allogeneic CD7-CAR-T cells knocked out of both CD7 and T-cell receptors in mouse experiments, it is difficult to achieve practical applications. It is difficult to guarantee a 100% knockout rate, and there may still be a risk of graft-versus-host disease (GVHD) in clinical applications

Method used

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  • CD7-CAR-T cell as well as preparation and application thereof
  • CD7-CAR-T cell as well as preparation and application thereof
  • CD7-CAR-T cell as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0207] Example 1 Blocking effect of CD7 blocking molecules on CD7 molecules on the surface of T cells

[0208] Using conventional technical means in the field to construct CD7 blocking molecules, the structure is as follows Figure 17 Shown, the amino acid sequence is shown in SEQ ID NO.:2. The above-mentioned CD7 blocking molecule is inserted into the lentiviral vector, and then the CD7 blocking lentivirus is prepared by lentiviral packaging technology and transfected into T cells. The changes of CD7 molecules on the surface of T cells were detected by flow cytometry.

[0209] The result is as figure 1 As shown, 1×10 6 After the cells were transfected with 50ul of CD7-blocking virus (CD7-blocking), the expression of CD7 molecules could not be detected on the surface of T cells ( figure 1 Shown in B), while the positive rate of CD7 on the surface of T cells not transfected with CD7 blocking virus was 97% ( figure 1 Shown in A), indicating that the CD7 blocking molecule...

Embodiment 2

[0210] Example 2 Effect of CD7 blocking molecules on T cell expansion

[0211] This example detects and compares control T cells (Ctrl T) not transfected with CD7 blocking virus, CD7-CAR-T cells transfected with CD7-CAR lentivirus (CD7-CAR), and transfected with CD7 In vitro expansion of CD7-CAR-T cells (CD7-blocking&CD7-CAR) blocking virus and CD7-CAR virus.

[0212] The result is as figure 2 As shown, compared with control T cells, CD7-CAR-T cells (CD7-CAR) transfected only with CD7-CAR lentivirus are difficult to expand in vitro, because the surface of CAR-T cells expresses CD7 molecules, CD7-CAR-T cells produced a suicide phenomenon. However, CD7-CAR-T cells (CD7-blocking&CD7-CAR) transfected with CD7-blocking virus and CD7-CAR virus at the same time can expand normally, indicating that blocking CD7 molecules will basically not affect CD7-CAR-T cells. Normal amplification.

Embodiment 3

[0213] Example 3 Effect of CD7 blocking on T cell phenotype

[0214] This example analyzes control T cells (Blank), CD19-CAR-T cells (CD19-CAR), CD19-CAR-T cells that block CD7 (CD7-Blocking&CD19-CAR), T cells that block CD7 (CD7 -Blocking), the phenotype changes of CD4 and CD8 on the cell surface of CD7-CAR-T cells that block CD7 (CD7-Blocking&CD7-CAR).

[0215] Day 7 ( image 3 A) and day 14 ( image 3 B) The results of cell flow cytometry showed that the blocking of CD7 molecules basically did not affect the typing of CD4 cells and CD8 cells.

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Abstract

The invention provides a CD7-CAR-T cell as well as preparation and application thereof. Particularly, the invention provides a chimeric antigen receptor (CAR), and an antigen binding domain of the CARcomprises one or more CD7 nanobodies. The invention further provides a CAR-T cell containing the CAR, and endogenous CD7 expression of the CAR-T cell is blocked. The CAR-T cell of the invention can be applied to treatment of T cell tumor.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically relates to a CD7-CAR-T cell and its preparation and application. Background technique [0002] T-cell-derived malignancies represent a typical hematologic malignancy with high recurrence and mortality rates in both children and adults, and currently there is no very effective treatment. T-cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous hematological malignancy, accounting for about 25% of adult ALL cases and about 15% of childhood ALL cases. The clinical symptoms of T-ALL mainly include infection, anemia, fever, abnormal bleeding, etc. T-ALL progresses rapidly and can quickly infiltrate into lymph nodes, liver, spleen, central nervous system and testis and other tissues and organs in a short period of time. Without timely and effective treatment, patients will die soon after onset. Currently, the treatment strategies for T-cell acute lymphoblastic l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/86C12N5/10A61K35/17A61P35/00
CPCC07K14/7051C07K16/2896C07K2319/00A61K39/4631A61K2239/48A61K2239/31A61K39/464411A61K39/464412A61K2239/38A61K39/4611C07K16/2803C07K2317/569C07K2317/24C07K2317/76C07K2317/73C07K2317/34A61P35/00A61K39/464429A61K2239/13C07K14/70578C07K2319/33
Inventor 杨林游凤涛陈丹
Owner PERSONGEN BIOMEDICINESUZHOUCO
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