Bispecific chimeric antigen receptor (CAR) targeting ROR1 and CD33 and application thereof

A chimeric antigen receptor and bispecific technology, applied in the bispecific chimeric antigen receptor and the application field of preparing anti-tumor drugs, can solve the problem of lack of CART cells, etc., to inhibit tumor growth and prevent off-target effects , the effect of promoting cytokine secretion

Active Publication Date: 2022-03-04
郑州源创基因科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] It can be seen that there is still a lack of CART cells that can fully exert anti-tumor activity based on ROR1 targets, especially how to develop bispecific CART cells that include ROR1 targets, and how to select a second antigen that can produce synergistic benefits, so as to improve tumor therapy targetin

Method used

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  • Bispecific chimeric antigen receptor (CAR) targeting ROR1 and CD33 and application thereof
  • Bispecific chimeric antigen receptor (CAR) targeting ROR1 and CD33 and application thereof
  • Bispecific chimeric antigen receptor (CAR) targeting ROR1 and CD33 and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: CART cell preparation

[0025] 1.1 Chimeric antigen receptor design

[0026] The bispecific chimeric antigen receptor provided in the present invention has the following structure: ROR1 scFv-X scFv-H-TM-C-CD3ζ where "-" is a connecting peptide or a peptide bond; H is a hinge region; TM is Transmembrane domain; C is a co-stimulatory signal molecule; CD3ζ is an intracellular signal transduction sequence; X scFv is selected from scFv targeting CD19, CD20, CD33, CD7 or CD3. The amino acid sequence and nucleotide sequence of each scFv were prepared and preserved by the inventors in earlier experiments.

[0027] 1.2 Viral vector transfection of T cells

[0028] In the present invention, multiple chimeric antigen receptor structures with bispecificity were constructed in order to verify the combined effects of ROR1 targets and other anti-tumor targets such as CD19, CD20, CD33, CD7 or CD3, for the convenience of distinction and follow-up Detection, respectively...

Embodiment 2

[0030] Example 2 In vitro tumor cell killing effect of CART cells

[0031] In order to verify the tumor killing effect of CART cells, K562 (chronic myeloid leukemia cell line) and Raji (lymphoma cell line) were selected as research objects in the present invention.

[0032] 2.1 Tumor cell culture

[0033] Remove and revive K562 cells and Raji cells from liquid nitrogen at 1 × 10 6 cells / mL inoculated in RPMI 1640 complete medium (containing 10% FBS), 37°C, 5% CO 2 Culture in a sterile cell culture incubator. Count and passage once every 48h, observe the state of the cells, when the cell survival rate reaches more than 80%, carry out subsequent experiments.

[0034] 2.2 CART cell co-culture

[0035] Harvest the CART cells and the above tumor cells separately, wash 3 times with sterile PBS, then mix the CART cells and tumor cells at a ratio of 2:1, and adjust the cell density to 1×10 6 cells / mL, inoculate in 96-well plate, carry out co-cultivation of CART cells and tumor ce...

Embodiment 3

[0040] Embodiment 3CART promotes cytokine release

[0041] The CART cells provided in the present invention are co-cultured with Raji cells, the specific method is the same as section 2.2. After co-cultivation for 48 hours, the cell supernatant was collected, and the contents of IFN-γ and TNF-α in the supernatant were detected using an ELISA kit (purchased from solarbio).

[0042] Such as image 3 As shown, after co-culture of CART cells and Raji cells, the expression level of IFN-γ can be greatly increased, and the bispecific CART targeting ROR1 and CD33 has the strongest ability to promote the secretion of IFN-γ, while the bispecific CART targeting ROR1 and CD7 CARTs and ROR1 and CD20 bispecific CARTs followed, and ROR1 and CD19 bispecific CARTs and ROR1 and CD3 bispecific CARTs were relatively weak in inducing IFN-γ secretion.

[0043] A similar situation occurs in the regulation of TNF-α expression, such as Figure 4 As shown, CART can increase the expression level of T...

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Abstract

The invention provides a bispecific chimeric antigen receptor (CAR) targeting ROR1 and CD33 and application thereof, the bispecific chimeric antigen receptor (CAR) has the following structure: ROR1 scFv-CD33 scFv-H-TM-C-CD3 zeta, in which '-' is a connecting peptide or a peptide bond; h is a hinge area; tM is a transmembrane domain; c is a costimulatory signal molecule; cD3zeta is an intracellular signal transduction sequence. The chimeric antigen receptor can inhibit tumor growth in vivo and in vitro, prolong the life cycle, promote cytokine secretion and play a synergistic anti-tumor role.

Description

Technical field: [0001] The invention belongs to the field of tumor immunotherapy, and specifically provides a bispecific chimeric antigen receptor (CAR) targeting ROR1 and CD33 and its application in the preparation of antitumor drugs. Background technique: [0002] At present, cancer has become one of the diseases with the highest fatality rate in the world. However, the existing methods such as surgical resection, radiotherapy and chemotherapy are effective in the treatment of early tumors, but it is difficult to treat patients in the middle and advanced stages, and it will also cause severe disease in patients. Adverse reactions affect the quality of life of patients and increase the burden on patients and their families. In recent years, with the development of tumor immunotherapy, Chimeric Antigen Receptor T Cells (CART) therapy has achieved great success in anticancer. This therapy was first proposed by Gross and colleagues in 1989, who fused the antigen-binding doma...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K39/00A61P35/00
CPCC07K16/2803C07K14/7051C12N5/0636A61K39/001111A61P35/00C07K2317/622C07K2317/31C07K2317/565C07K2317/56C07K2319/03C07K2319/33C07K2319/74C12N2510/00A61K2039/5156
Inventor 张克礼雷林均
Owner 郑州源创基因科技有限公司
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