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Chimeric antigen receptor T cell expressing IL-6R blocking antibody and targeting CD19, preparation method and application of T cell

A technology of chimeric antigen receptors and expression vectors, which is applied in the fields of genetic engineering and cell biology, can solve the problems of patient death, patient economic burden, and the high price of Tocilizumab, and achieve the goal of reducing the impact of CRS and improving safety Effect

Inactive Publication Date: 2020-02-28
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, Juno Therapeutics released a report that in the company’s clinical trial JCAR015, four patients died after receiving CAR-T cell therapy. The FDA immediately stopped the clinical trial of JCAR015, and Juno later explained that it was because 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 infused T cells that caused the death of the patient
However, Tocilizumab is expensive, which is not a small economic burden for patients, and the timing of drug administration needs to be monitored

Method used

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  • Chimeric antigen receptor T cell expressing IL-6R blocking antibody and targeting CD19, preparation method and application of T cell
  • Chimeric antigen receptor T cell expressing IL-6R blocking antibody and targeting CD19, preparation method and application of T cell
  • Chimeric antigen receptor T cell expressing IL-6R blocking antibody and targeting CD19, preparation method and application of T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of dual expression vectors for CD19 CAR and Tocilizumab

[0052] Such as figure 1 As shown, the CD8 transmembrane signal peptide, anti-CD19 Scfv, CD8 transmembrane region, 4-1BB co-stimulatory signal region, CD3Zeta TCR activation region, IRES, and Tocilizumab were sequentially cloned into the lentiviral backbone plasmid pHR to obtain pHR- antiCD19CAR-Tocilizumab plasmid.

[0053] The nucleotide sequence of antiCD19CAR-Tocilizumab is shown in SEQ ID NO:9, and the amino acid sequence of antiCD19CAR-Tocilizumab is shown in SEQ ID NO:10. Transfect 1ug of the lentiviral expression vector carrying the target gene into 293FT cells, collect 293FT cells 5×105 after 24h, add 10ul of FITC-LabeledHumanCD19 (20-291) Protein to each sample, and incubate for 1h at 4°C in the dark , washed 3 times, centrifuged, and the pellet was resuspended in 200 μL PBS and tested on a machine (Millipore guava easyCyte HT). The transfection efficiency of pHR-antiCD19CAR-To...

Embodiment 2

[0054] Example 2: Packaging and concentration of lentivirus

[0055] The lentiviral expression vector carrying the target gene, pCMV vector and pMD.2G vector were mixed and transfected into 293FT cells. After transfection, 6h-8h was replaced with complete medium for culture. After 48h, the culture medium was collected and centrifuged. Keep the supernatant and filter the supernatant with a 0.45 μm filter, keep the filtrate, and the filtrate is the solution of the recombinant lentivirus.

[0056] Lentivirus concentration was carried out according to the instructions of Lenti-XTM Concentrator (takara, cat: 631231).

Embodiment 3

[0057] Example 3: Preparation of T cells with dual expression vectors of CD19 CAR and Tocilizumab

[0058] Take 50mL of fresh blood and conduct density gradient centrifugation with lymphocyte separation medium (Tianjin Haoyang) to separate mononuclear cells. Resuspend mononuclear cells into CTS TM AIM V TM In SFM medium (GIBCO, product number A3021002), the medium should be supplemented with 5% ICS (GIBCO, A2596101). At the same time, CD3 monoclonal antibody and CD28 monoclonal antibody were added to activate T lymphocytes, and cultured at 37°C with 5% CO2 for 48 hours.

[0059] Take 2×106 cells, add the concentrated lentivirus in Example 2 according to MOI=5, add IL-2 and polybrene at the same time, mix well, incubate at 37°C 5% CO2 for 6-8 hours, centrifuge at 300g for 5 minutes to replace the fresh medium CTS TM AIM V TM SFM medium (containing IL-2).

[0060] Add fresh CTS every 2-3 daysTM AIM V TM SFM medium (containing IL-2), maintain the cell density at abou...

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Abstract

The invention belongs to the field of genetic engineering and cell biology, and relates to a chimeric antigen receptor T cell expressing an IL-6R blocking antibody and targeting CD19, a preparation method and an application of the T cell. Specifically, a dual expression carrier targeting a CD19 chimeric antigen receptor and an IL-6R blocker tocilizumab is constructed. The dual expression carrier targeting the CD19 chimeric antigen receptor and the IL-6R blocker tocilizumab is transduced to the T cell by a lentivirus mediated method. The pHR-antiCD19CAR-Tocilizumab-T cell enable the cells to autocrine the IL-6R blocker tocilizumab through a genetic engineering technology under the condition of not affecting the characteristics of phenotype, killing, cytokine release and the like of the pHR-antiCD19CAR-Tocilizumab-T cell, and thus the generation of CRS side effects is controlled, and the safety is improved.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and cell biology, and in particular relates to a CD19-targeting chimeric antigen receptor T cell (Chimeric Antigen Receptor T cell, CAR-T) expressing an IL-6R blocking antibody and a preparation method thereof, application. Background technique [0002] In 1989, the concept of chimeric antigen receptor-modified T cells (CAR-T) was first proposed to establish adoptive cell therapy with tumor-specific recognition capabilities. The design of the first-generation CAR is to simply replace the extracellular region of the TCR (T cell receptor) with scFv (single-chain antibody variable region fragment) segments derived from specific monoclonal antibodies, retaining the transmembrane structure and intracellular signal transmission In the CD3ζ region, some research groups also use FcR for the intracellular signal transmission part. However, the results of the first-generation CAR clinical tri...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61K39/395A61K39/00A61P35/00A61P35/02
CPCA61K39/395A61P35/00A61P35/02A61K39/001102C07K14/7051C07K16/2803C07K16/2866C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043A61K2300/00
Inventor 谭毅张慧慧陈倩韩镇
Owner 山东省齐鲁细胞治疗工程技术有限公司
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