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A kind of anti-wt1 enhanced chimeric antigen receptor modified immune cell and its application

A technology of chimeric antigen receptors and immune cells, applied in the field of biological genes

Active Publication Date: 2020-06-30
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, due to the suppressive effect of the tumor microenvironment, improving the efficacy of CAR-T cell therapy is still a difficult task

Method used

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  • A kind of anti-wt1 enhanced chimeric antigen receptor modified immune cell and its application
  • A kind of anti-wt1 enhanced chimeric antigen receptor modified immune cell and its application
  • A kind of anti-wt1 enhanced chimeric antigen receptor modified immune cell and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction of a lentiviral plasmid expressing the chimeric antigen receptor protein encoded by the nucleic acid of the present invention and virus packaging

[0025] 1. Insert the leader-scFv(WT1)-Linker-scFv(CD3)-CD8-CD137-CD3ζ into the lentiviral expression vector pLent-C-GFP.

[0026] Anti-WT1 CAR module expresses opinions figure 1 (See the appendix SEQ ID NO. 1 for the complete nucleic acid sequence).

[0027] Anti-WT1 CAR module sequence

[0028] (1) Leader nucleic acid artificial sequence (SEQ ID NO.2)

[0029] (2) Anti-WT1single chain Fv antibody (scFv) nucleic acid artificial sequence (SEQ ID NO.3)

[0030] (3) Linker nucleic acid artificial sequence (SEQ ID NO.4)

[0031] (4) Anti-CD3single chain Fv antibody (scFv) nucleic acid artificial sequence (SEQ ID NO.5)

[0032] (5) CD8 Hinge region nucleic acid artificial sequence (SEQ ID NO. 6)

[0033] (6) CD8 transmembrane region nucleic acid artificial sequence (SEQ ID NO. 7)

[0034] (7) CD137 intracellular region nu...

Embodiment 2

[0039] Example 2 Infection of CIK cells by lentivirus

[0040] 1. Preparation of CIK cells

[0041] Take 75ml of the patient's autologous peripheral blood, use TBD sample density separation solution (purchased from Tianjin Haoyang Huake Biology) to separate peripheral blood mononuclear cells. After induction culture medium (purchased from CORNING Company, 88-551-CM) containing 1000IU / ml recombinant interferon α2a (purchased from Shenyang Sansheng Pharmaceutical) for 24 hours, 1000IU / ml recombinant interleukin 2 ( (Purchased from Shenyang Sansheng Pharmaceutical), 50ng / ml OKT-3 and 5% of patients’ autologous plasma were induced to continue the culture for 24 hours. The solution was doubled every two days and cultured to the 14th day. Flow cytometry was used to detect the positive expression rate of CD3+ and CD56+ in CIK cells (CD3-FITC, CD16 / CD56-PE antibody purchased from BECKMAN, A07735). CD3+ positive rate> 80%, CD3+CD56+ double positive rate> 20%, it is deemed that CIK is succ...

Embodiment 3

[0044] Example 3 Using MS magnetic beads to sort WT1 specific CIK cells

[0045] Linked polymer reagents: including IS buffer, linked polymer magnetic beads, WT1 linked polymer MHC, etc. were purchased from German IBA company.

[0046] The preparation of WT1 / MHC linked polymer magnetic beads: 50μL of linked polymer magnetic beads, 2μg WT1 / MHC and 90μL IS buffer, incubate at 4°C for 45min in the dark, and then add 1mL IS buffer. The MS magnetic column was placed in a magnetic field, washed with 2 mL IS buffer, the liquid after the incubation was injected into the magnetic column for sorting, and the chain polymer MHC that was not bound to the WT1 magnetic beads was eluted. Move the MS magnetic column away from the magnetic field, add 250μL IS buffer to elute and collect the WT1 chain polymer magnetic bead complex.

[0047] Purification of WT1-specific CIK cells: the above-mentioned WT1-linked polymer magnetic bead complex and 2×10 7 The mononuclear cells were incubated with periphera...

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Abstract

The invention discloses an anti-WT1 reinforced chimeric antigen receptor modified immune cell and application thereof. The anti-WT1 reinforced chimeric antigen receptor modified immune cell is obtained by modifying an immune cell with an anti-WT1 reinforced chimeric antigen receptor, wherein the anti-WT1 reinforced chimeric antigen receptor comprises CD8 leader, WT1 bond zone, a CD3 bond zone, a CD8 hinge zone, a transmembrane-stimulation structural domain and a CD3 zeta stimulation signal conduction zone. In the space, the distance between tumor cells and effector T cells becomes closer by virtue of the immune cell, and the density of the effector T cells is increased in quantitative terms. Therefore, the method reinforces the killing effect of T cells for tumor cells.

Description

Technical field [0001] The present invention relates to the field of biological genes, more specifically, an immune cell modified with an anti-WT1 enhanced chimeric antigen receptor and its application. Background technique [0002] WT1 (Wilms'tumor gene 1) is the first gene related to the occurrence and development of Wilms' tumor. It is very low in normal adult tissues, but it is highly expressed in acute and chronic leukemia and some solid tumors, including lung cancer, breast cancer, and ovarian cancer. Cancer, prostate cancer, etc., which are highly expressed in 90% of breast and ovarian cancers, and are related to the poor prognosis of the disease. Antisense oligonucleotides inhibit the expression of WT1, resulting in the arrest of leukemia cells and solid tumor cells, and the ability of WT1 to induce specific cytotoxic T lymphocyte responses, suggesting that WT1 can be used as an ideal intracellular target antigen for broad-spectrum immunotherapy. [0003] Immunotherapy is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10A61K35/17A61P35/00
Inventor 刘明录韩国英王立新强邦明马洪华金海锋刘敏王亮韩庆梅卢永灿
Owner 上海兴瑞一达生物科技有限公司