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Anti-ROR1 safe chimeric antigen receptor modified immune cell and application thereof

A chimeric antigen receptor and immune cell technology, applied in the field of biological genes, can solve problems such as HER2 attack, and achieve the effect of preventing immune response, avoiding toxic side effects, and ensuring therapeutic effect.

Active Publication Date: 2018-01-09
上海兴瑞一达生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when CAR-T cells are used to treat solid tumors, due to potential off-target toxicity, their safety has become a key issue in their application in the treatment of solid tumors (the first death case of CAR-T treatment in the world was HER2 When positive patients are treated, HER2 expressed in normal lung tissue is attacked by CAR-T cells)

Method used

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  • Anti-ROR1 safe chimeric antigen receptor modified immune cell and application thereof
  • Anti-ROR1 safe chimeric antigen receptor modified immune cell and application thereof
  • Anti-ROR1 safe chimeric antigen receptor modified immune cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction and virus packaging of lentiviral plasmid expressing chimeric antigen receptor protein encoded by nucleic acid of the present invention

[0028] 1. Insert the fusion gene fragment F36V-FKBP-linker-caspase9-T2A-Leader-scFv(ROR1)-CD8-CD137-CD3ζ into the lentiviral expression vector pLent-C-GFP.

[0029] F36V-FKBP-linker-caspase9-T2A-Leader-scFv(ROR1)-CD8-CD137-CD3ζModule opinion figure 2 (See appendix SEQ ID NO.1 for the complete nucleic acid sequence).

[0030] CAR Module Sequences of F36V-FKBP-linker-caspase9-T2A-Leader-scFv(ROR1)-CD8-CD137-CD3ζ

[0031] (1) F36V-FKBP nucleic acid artificial sequence (SEQ ID NO.2)

[0032] (2) linker nucleic acid artificial sequence (SEQ ID NO.3)

[0033] (3) Caspase9 nucleic acid artificial sequence (SEQ ID NO.4)

[0034] (4) Artificial sequence of self-cleaving polypeptide T2A nucleic acid (SEQ ID NO.5)

[0035] (5) artificial sequence of leader nucleic acid (SEQ ID NO.6)

[0036] (6) Anti-ROR1 single cha...

Embodiment 2

[0044] Example 2 lentivirus infection of CIK cells

[0045] 1. Preparation of CIK cells

[0046] Take 75ml of autologous peripheral blood from the patient, and separate peripheral blood mononuclear cells with TBD sample density separation medium (purchased from Tianjin Haoyang Huake Biology). After inducing culture for 24 hours with a culture medium (purchased from CORNING Company, 88-551-CM) containing 1000 IU / ml of recombinant interferon α2a (purchased from Shenyang Sansheng Pharmaceutical), 1000 IU / ml of recombinant interleukin 2 ( (purchased from Shenyang Sansheng Pharmaceutical), 50ng / ml of OKT-3 and 5% of the patient's autologous plasma were induced and continued to be cultured for 24 hours. Doubling liquid was added every two days, cultured until the 14th day, and the positive expression rate of CD3+ and CD56+ in CIK cells was detected by flow cytometry (CD3-FITC, CD16 / CD56-PE antibodies were purchased from BECKMAN, A07735). CD3+ positive rate > 80%, CD3 + CD56 + doub...

Embodiment 3

[0049] Example 3: Study on the killing activity of CAR-CIK cells with a safety "switch"

[0050] The ROR1-positive breast cancer cell line MDA-MB-231 was used as target cells, and the effector cells were CAR-CIK cells and CIK infected with empty lentivirus.

[0051] 1. Analysis of the effectiveness of the safety "switch" in controlling CAR-CIK cells

[0052] The CAR-CIK cells were mixed at a density of 1×10 5 cells / ml inoculated in 96-well plate, 100ul per well, placed in 5% CO 2 , Incubate in a 37°C incubator for 24 hours; add 10nM AP1903 (product of Apexbio in the United States), add 20μL of CCK-8 (product of MCE company) to each well after 12 hours, continue to incubate for 2 hours, then use a microplate reader for detection, and read the OD value at a wavelength of 450nm . A CAR-CIK cell control group without AP1903 and a blank control group with only AP1903 and no cells were set up. Mortality rate of CIK cells=[1-(OD value of group with AP1903 added-OD value of blank ...

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Abstract

The invention discloses an anti-ROR1 safe chimeric antigen receptor modified immune cell and application thereof. An immune cell is modified by an anti-ROR1 safe chimeric receptor to obtain the anti-ROR1 safe chimeric antigen receptor modified immune cell. The anti-ROR1 safe chimeric antigen receptor comprises a CD8 leader, an ROR1 binding region, a CD8 hinge region, a transmembrane-stimulus domain, a CD3zeta-stimulus signal transduction region and a safety switch element; the immune cell and application thereof, provided by the invention have the advantages that untoward effects caused by cell transduction can be effectively avoided and clinical efficacy and safety of a CAR technology are greatly improved.

Description

technical field [0001] The invention relates to the field of biological genes, more specifically, an anti-ROR1 safe chimeric antigen receptor modified immune cell and its application. Background technique [0002] Receptor tyrosine kinase-like orphan receptor 1 (receptor tyrosine kinase-like orphan receptor–1, ROR1) is a tumor-associated embryonic antigen discovered in recent years, which belongs to the ROR subfamily of cell surface receptors. Highly expressed during embryogenesis but not in normal mature tissues, except for low expression on immature B-cell precursor subsets and adipocytes. Current studies have also found that ROR1 is highly expressed in a variety of B-cell malignancies and in a variety of solid tumors such as breast cancer, colon cancer, lung cancer, ovarian cancer, and kidney cancer, and is closely related to tumor development and prognosis. Therefore, ROR1 is an ideal molecular target for targeted therapy of malignant tumors. [0003] Malignant tumor i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/62A61K35/17A61P35/00
Inventor 刘明录金海峰王立新韩国英韩庆梅强邦明万磊卢永汕
Owner 上海兴瑞一达生物科技有限公司
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