CD7 chimeric antigen receptor modified NK-92MI cell and application thereof

A technology of chimeric antigen receptors and NK cells, applied in genetically modified cells, cells modified by introducing foreign genetic material, receptors/cell surface antigens/cell surface determinants, etc., can solve CAR-T cell killing And other issues

Active Publication Date: 2019-04-19
PERSONGEN BIOMEDICINESUZHOUCO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, designing CARs against T-cell tumors still faces great challenges, because the same antigens are expressed on normal T-cells and T-cell malignancies, causing CAR-T cells to kill each other

Method used

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  • CD7 chimeric antigen receptor modified NK-92MI cell and application thereof
  • CD7 chimeric antigen receptor modified NK-92MI cell and application thereof
  • CD7 chimeric antigen receptor modified NK-92MI cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0225] Example 1 Construction of CD7-CAR / dCD7-CAR vector and CAR-NK-92MI cells

[0226] Two CD7-CARs were constructed using the CD7 Nanobody sequence VHH6 monovalent (SEQ ID NO.: 1) and bivalent sequence (SEQ ID NO.: 2). The monovalent CD7-CAR is composed of signal peptide, anti-CD7 Nanobody sequence (VHH6), human Fc hinge region, CD28 transmembrane domain and CD28 and 4-1BB intracellular signaling domains in series with CD3ζ signaling domain. The bivalent dCD7-CAR contains signal peptide, anti-CD7 Nanobody repeat sequence (VHH6-VHH6), human Fc hinge region, CD28 transmembrane domain, and CD28 and 4-1BB intracellular signal transduction in series with CD3ζ signaling domain Structural domain. The structure diagram of CAR is as figure 1 Shown in A. The CD7-CAR and dCD7-CAR sequences were cloned into the pHULK PiggyBac electrotransformation expression vector and named as the CD7-CAR plasmid and dCD7-CAR plasmid, respectively.

[0227] After electroporation, CD7-CAR-NK-92MI and dCD...

Embodiment 2

[0229] Example 2 Expression of CD7 in NK-92MI and T-ALL cells

[0230] The expression of CD7 in NK-92MI, CD7-CAR-NK-92MI and dCD7-CAR-NK-92MI cells was detected by flow cytometry. The results showed that the positive rate of CD7 in NK-92MI cells was 8.42%, while the positive rate of CD7 in NK-92MI cells transfected with CD7-CAR (dCD7-CAR) was less than 1% ( figure 2 A). It shows that CD7-CAR-NK-92MI or dCD7-CAR-NK-92MI cells can specifically kill CD7-positive NK-92MI cells. This example also detected the expression of CD7 on the surface of leukemia cell lines (Jurkat and CCRF-CEM), lymphoblastic cell lines (Raji) and primary tumor cells from T-ALL. The results showed that the CD7 positive rate in CCRF-CEM and Jurkat cells was almost 100% ( figure 2 B), the CD7 positive rate of T-ALL primary tumor cells is 93% ( figure 2 C, the control group is T-ALL cells that have not been incubated with CD7 antibody), while Raji cells are CD7-negative cells ( figure 2 B).

Embodiment 3

[0231] Example 3 In vitro killing activity of CD7-CAR-NK-92MI and dCD7-CAR-NK-92MI cells on T-ALL cell line

[0232] CD7-positive T-ALL cell lines (CCRF-CEM and Jurkat cells) were used to evaluate the in vitro anti-tumor activity of CD7-CAR-NK92-MI and dCD7-CAR-NK92-MI cells. Raji serves as a negative cell line. After co-cultured with CCRF-CEM cells in vitro for 4 hours or 24 hours (effective target ratio 1:1), the cytotoxicity of CD7-CAR-NK92-MI and dCD7-CAR-NK92-MI cells to target cells was detected by flow cytometry. The results showed that compared with control NK92-MI cells, CD7-CAR-NK-92MI and dCD7-CAR-NK-92MI cells showed significant specific cytotoxicity to CCRF-CEM cells under conditions of different effective target ratios. ( image 3 A). This example also evaluated the cytotoxicity of CD7-CAR-NK-92MI and dCD7-CAR-NK-92MI cells to Jurkat cells under the conditions of the effective target ratio of 1:1 and 5:1. The results showed that after 24 hours of incubation with ...

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Abstract

The invention provides a CD7 chimeric antigen receptor modified NK-92MI cell and an application thereof. Particularly, the invention provides an engineered NK cell that expresses a chimeric antigen receptor CAR, the antigen binding domain of which contains a nanoantibody VHH sequence targeting CD7. The NK cell can effectively kill tumor cells, especially T cell tumors, and has very good treatmenteffect on T cell leukemia (such as T-ALL).

Description

Technical field [0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to CD7 chimeric antigen receptor modified NK-92MI cells and applications thereof. Background technique [0002] T-cell malignant tumors represent a type of blood system cancer, with high recurrence and mortality rates in children and adults. There is currently no effective or targeted therapy. T-cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous hematological malignancy, accounting for 25% of adult acute lymphoblastic leukemia cases and 15% of pediatric acute lymphoblastic leukemia cases. Currently, T-ALL treatment strategies include intensive chemotherapy, allogeneic hematopoietic stem cell transplantation (allo-HSCT), antiviral therapy and molecular targeted therapy. However, intensive chemotherapy and allo-HSCT usually cannot prevent and treat refractory relapses. For those patients who relapse after initial treatment, the remission r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/62A61K35/17A61P35/00
CPCA61P35/00A61K35/17C12N5/0646C07K14/7051C07K16/28C07K2319/00C12N2510/00C07K14/70578A61K38/00A61P35/02C07K14/70521C07K14/70517C07K14/705A61K39/4631C07K16/2803C07K2317/569C07K2317/22A61K39/4613C07K2317/73A61K2039/505A61K2039/5156C07K2317/35C07K2317/53C07K2319/02C07K2319/03C07K2319/30C07K2319/33
Inventor 杨林游凤涛
Owner PERSONGEN BIOMEDICINESUZHOUCO
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