CD19-CAR-T cell carrying iCasp9 suicide gene and use thereof

A gene and lymphocyte technology, applied in the fields of genetic engineering and cell biology, can solve problems such as side effects and patient death, and achieve the effect of increasing safety

Active Publication Date: 2017-11-21
山东省齐鲁细胞治疗工程技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In early July 2016, Seattle-based Juno Therapeutics reported that in the company's clinical trial JCAR015, three patients (later confirmed to be four) died after receiving CAR-T cell therapy, and the FDA immediately stopped In the clinical trial of JCAR015, Juno later explained that the use of the chemotherapy drug fludarabine caused neurotoxins, but many people speculated that it was due to the strong cytokine storm (CRS) generated by the input T cells that caused the death of the patient
Regardless of the cause, most of the patients receiving CAR-T cell therapy will develop CRS, which can lead to high fever and severe side effects

Method used

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  • CD19-CAR-T cell carrying iCasp9 suicide gene and use thereof
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  • CD19-CAR-T cell carrying iCasp9 suicide gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Design of CD19CAR containing iCasp9 suicide gene and construction of expression vector

[0056]According to the different construction strategies of iCasp9 and CD19CAR dual expression vectors, two CD19CARs containing iCasp9 suicide gene were designed and constructed, named pHR-iCasp9-2A-CD19CAR-4-1BB-CD3ζ and pHR-iCasp9-IRES-CD19CAR-4 respectively -1BB-CD3ζ, wherein the amino acid residue sequence expressed by pHR-iCasp9-2A-CD19CAR-4-1BB-CD3ζ is as shown in SEQID NO:17, expressed by pHR-iCasp9-IRES-CD19CAR-4-1BB-CD3ζ The amino acid residue sequence is shown in SEQID NO: 19, and the pattern is shown in figure 1 As shown, the structure diagram is as follows figure 2 , image 3 shown.

Embodiment 2

[0057] Example 2: Packaging and concentration of lentivirus

[0058] The lentiviral expression vector carrying the gene of interest (pHR-iCasp9-2A-CD19CAR-4-1BB-CD3ζ, pHR-iCasp9-IRES-CD19CAR-4-1BB-CD3ζ constructed in Example 1), pCMV vector and pMD. 2G vectors were mixed and transfected into 293FT cells, replaced with complete medium for culture 6-8 hours after transfection, collected the culture medium after 48 hours, retained the supernatant after centrifugation and filtered the supernatant with a 0.45 μm filter, and retained the filtrate. The filtrate is the solution of the recombinant lentivirus.

[0059] Lentivirus concentration was carried out according to the instructions of Lenti-XTM Concentrator (takara, cat: 631231).

Embodiment 3

[0060] Embodiment 3: Verification of iCasp9 / CID suicide gene system

[0061] 1. Preparation of K562 cells expressing lentiviral vector

[0062] Resuspend the cells in 1640 medium to 1x106 / mL. Add lentivirus. 37°C, 5% CO 2 Cultivate in an incubator for 6-8 hours, and replace the culture medium by centrifugation with fresh K562 cell proliferation culture medium. Add fresh K562 cell proliferation medium every 2-3 days to maintain the cell density at 0.5x10 6 / mL or so. After 48 hours of virus infection, the proportion of CAR-positive cells was detected by flow cytometry (Fluorescein (FITC) AffiniPure Goat Anti-Mouse IgG, F(ab')2 fragment specific, jackson immunoresearch, cat: 115-095-006).

[0063] The result is as Figure 4 As shown, the proportion of CD19CAR-K562 cells containing the iCasp9 suicide gene is 34%, indicating that the CD19CAR-K562 cells containing the iCasp9 suicide gene have been successfully obtained and named as iCasp9-CD19CAR-K562 cells. As effector cell...

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Abstract

The invention discloses a lentivirus expression vector. The lentivirus expression vector comprises a gene encoding a chimeric antigen receptor and an iCasp9 apoptotic gene. The invention also discloses a CD19-CAR-T cell carrying an iCasp9 suicide gene. The CD19-CAR-T cell is a T lymphocyte comprising a lentivirus expression vector or a T lymphocyte of which the chromosome is integrated with a gene encoding a chimeric antigen receptor and an iCasp9 apoptotic gene. The invention also discloses a use of the CD19-CAR-T cell carrying an iCasp9 suicide gene in preparation of drugs for preventing and / or treating and / or assistantly treating cancers. Compared with the existing CD19-CAR-T cell, the CD19-CAR-T cell carrying an iCasp9 suicide gene utilizes a suicide mechanism and can express CAR and co-express a suicide gene iCasp9. When serious side effects occur, through inducing T cell apoptosis, CRS side effects are controlled and safety is improved.

Description

technical field [0001] The present invention relates to a chimeric antigen receptor T cell (Chimeric Antigen Receptor T cell, CAR-T) carrying iCasp9 (induciblecaspase9) suicide gene, and its preparation for preventing and / or treating and / or adjuvantly treating malignant tumors The application in belongs to the fields of genetic engineering and cell biology. Background technique [0002] CD19 chimeric antigen receptor modified T cells (anti-CD19 chimeric antigen receptor T cells, referred to as CD19CAR-T cells) have achieved great success in the treatment of refractory B cell malignancies; while targeting other tumor targets CAR-T technology has also shown good application prospects in the treatment of other solid tumors. Coexisting with the effectiveness of CD19CAR-T therapy is its side effects and cytotoxicity. In clinical application, it may cause false attack on normal tissue cells (on target / off-tumor effect), or cause toxic side effects such as cytokine storm due to t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N7/01C12N5/10C12N15/12A61K35/17A61P35/02
CPCA61K35/17C07K14/47C07K14/7051C12N5/0636C12N7/00C12N15/86C12N2510/00C12N2740/15043
Inventor 谭毅张慧慧
Owner 山东省齐鲁细胞治疗工程技术有限公司
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