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Construction method and application of lentiviral vector for expressing TGF-beta antibody as well as construction method and application of CAR-T cell

A lentiviral vector, TGF- technology, applied in the field of tumor immunotherapy, can solve the problems of poor treatment effect and achieve the effect of reducing inhibitory molecules

Inactive Publication Date: 2018-12-07
安徽古一生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, CAR-T immunotherapy has achieved outstanding results in hematological tumors, but in the immunotherapy of solid tumors, the therapeutic effect is not good. biggest obstacle

Method used

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  • Construction method and application of lentiviral vector for expressing TGF-beta antibody as well as construction method and application of CAR-T cell
  • Construction method and application of lentiviral vector for expressing TGF-beta antibody as well as construction method and application of CAR-T cell
  • Construction method and application of lentiviral vector for expressing TGF-beta antibody as well as construction method and application of CAR-T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] figure 1 A technical roadmap for the construction of TGF-β-expressing CAR-T cells carrying GPC3 targets

[0075] (1) Construction of TGF-β-expressing CAR-T cells carrying GPC3 targets

[0076] ① Whole gene synthesis of anti-GPC3 scFV-CD8α hinge / TM-CD137-CD3ζ and anti-GPC3 scFV-CD8α hinge / TM-CD137-CD3ζ-T2A-antiTGF-β gene fragments:

[0077] The anti-GPC3 scFV-CD8α hinge / TM-CD137-CD3ζ and anti-GPC3 scFV-CD8αhinge / TM-CD137-CD3ζ-T2A-antiTGF-β synthetic gene sequence map is as follows figure 2 shown.

[0078] CD8α signal peptide, CD8α signal peptide sequence: the CD8α signal peptide sequence NM_001768 (890..952) was found in the Genebank database, and the sequence is shown in SEQ ID NO.1;

[0079] Anti-GPC3 molecular light chain variable region (anti-GPC3 VL fragment), as a part of the scFv fragment, refer to the corresponding VL fragment nucleic acid sequence of the corresponding GC33 antibody in the patent document US7919086B2, the sequence is shown in SEQ ID NO.2 Sho...

Embodiment 2

[0122] Figure 11 A technical roadmap for the construction of TGF-β-expressing CAR-T cells carrying EpCAM targets

[0123] (1) Construction of CAR-T cells expressing TGF-β carrying the EpCAM target

[0124] ① Whole gene synthesis of anti-EpCAM scFV-CD8α hinge / TM-CD137-CD3ζ and anti-EpCAM scFV-CD8α hinge / TM-CD137-CD3ζ-T2A-antiTGF-β gene fragments

[0125] The sequences of CD8α signal peptide, CD8α hinge region and transmembrane region, co-stimulatory factors CD137 and CD3ζ in the sequence anti-EpCAM scFV-CD8α hinge / TM-CD137-CD3ζ are the same as in Example 1;

[0126] CD8α signal peptide, CD8α hinge region and transmembrane region, co-stimulatory factor CD137, CD3ζ, GM-CSF signal peptide signal peptide, anti -The heavy chain and light chain sequences of the TGF antibody are the same as in Example 1;

[0127] Vector construction method, lentivirus preparation and titer determination method and final EpCAM.CAR-T cell preparation method are the same

[0128] Embodiment 1;

[0...

Embodiment 3

[0140] Figure 21 A technical roadmap for the construction of CAR-T cells expressing TGF-β antibodies carrying Her2 targets

[0141] (1) Construction of CAR-T cells expressing TGF-β antibody carrying Her2 target

[0142] ① Whole gene synthesis of anti-Her2 scFV-CD8α hinge / TM-CD137-CD3ζ and anti-Her2 scFV-CD8α hinge / TM-CD137-CD3ζ-T2A-antiTGF-β gene fragments

[0143] The sequences of CD8α signal peptide, CD8α hinge region and transmembrane region, co-stimulatory factors CD137 and CD3ζ in the sequence anti-Her2 scFV-CD8α hinge / TM-CD137-CD3ζ are the same as in Example 1;

[0144] CD8α signal peptide, CD8α hinge region and transmembrane region, co-stimulatory factors CD137, CD3ζ, GM-CSF signal peptide signal peptide, anti -The heavy chain and light chain sequences of the TGF antibody are the same as in Example 1;

[0145] The vector construction method, lentivirus preparation and titer determination method, and the final Her2.CAR-T cell preparation method are the same as in Exa...

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Abstract

The invention discloses a construction method and an application of a lentiviral vector for expressing a TGF-beta antibody as well as a construction method and an application of a CAR-T cell based onthe problem that immunosuppression molecules and suppressor cells are the biggest barriers in the treatment of solid tumors in a tumor immunosuppression microenvironment, relating to the technical field of tumor immunotherapy. The invention discloses the lentiviral vector which carries a CAR gene of a solid tumor specific antigen target spot of the TGF-beta antibody as well as the CAR-T cell for expressing the solid tumor specific antigen target spot of the TGF-beta antibody. The construction methods and the application have the beneficial effects that the targeting effect on the tumor cells is realized, the suppressor cells in the tumor microenvironment are reduced, the solid tumors are killed to an extreme extend, and the new breakthrough on the immunotherapy of the solid tumors is realized.

Description

technical field [0001] The invention relates to the technical field of tumor immunotherapy, in particular to a lentiviral vector expressing TGF-β antibody, a construction method and application of CAR-T cells. Background technique [0002] More and more research findings and clinical data show that the occurrence, development and growth of tumor cells are significantly related to the strength of the body's own immune function. Tumor patients with low immune function have significantly higher tumor incidence and recurrence rates. Aiming at the relationship between tumors and the immune system, Schreiber et al. proposed the "tumor immunoediting theory" in the early 20th century. Tumor immunoediting includes three stages: immune clearance, immune stalemate, and immune escape. The body's immune system recognizes and eliminates most tumor cells through innate immunity and adaptive immunity; however, in order to avoid the attack of the immune system, there may be a small number o...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 李杰韩江萌刘振云王维程丰伟
Owner 安徽古一生物科技有限公司
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