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Double-chimeric antigen receptor, T cell and construction method and application thereof

A chimeric antigen receptor and cell technology, applied in the direction of receptor/cell surface antigen/cell surface determinant, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, animal cells, etc., can solve normal Tissue damage, safety issues, cytokine release syndrome and other issues, to achieve the effect of improving safety

Pending Publication Date: 2020-01-10
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The third-generation CAR added two co-stimulatory molecules, and it has been clinically shown that this CAR-T cell has a strong ability to eliminate tumor cells, but it faces serious safety issues, mainly cytokine release syndrome
Relatively speaking, the probability of expressing two TAAs at the same time in normal tissues or even tumor paracancerous tissues is relatively low, and they often only overexpress one TAA. s reason

Method used

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  • Double-chimeric antigen receptor, T cell and construction method and application thereof
  • Double-chimeric antigen receptor, T cell and construction method and application thereof
  • Double-chimeric antigen receptor, T cell and construction method and application thereof

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Embodiment 1

[0074] Determination of the whole gene sequence of double CAR

[0075] 1.1 Obtain the gene sequence of hMSLNscFv and hCEAscFv, analyze and optimize their gene sequences to ensure that they are more suitable for high-efficiency expression in human T lymphocytes under the condition that the encoded amino acid sequence remains unchanged. For the sequence information of the hMSLNscFv gene, see SEQUENCE LISTING (SEQ ID NO.3); for the sequence information of the hCEAscFv gene, see SEQUENCE LISTING (SEQ ID NO.5).

[0076] 1.2 Obtain the gene sequences of human CD8α signal peptide gene, human CD8α hinge region gene, human CD8 transmembrane region and intracellular region gene, and human CD3ζ and 4 / 1BB signal domain.

[0077] 1.3 The above gene sequence was sequenced according to human CD8α signal peptide, hMSLNscFv, human CD8α hinge region, human CD8 transmembrane region and intracellular region, human 4 / 1BB co-stimulatory signal domain, IRES sequence, human CD8α signal peptide, hCEAs...

Embodiment 2

[0079] Construction of expression plasmids

[0080] 2.1 The optimized and complete double chimeric receptor gene sequence was subjected to whole gene synthesis, and cloned into The overexpression vector pFC-MCS of the site-specific recombinase system, and finally the recombinant plasmid pFC-dCAR was constructed, and the GFP fluorescent tag was introduced to facilitate the detection and tracking of the constructed dCAR-T cells.

[0081] 2.2 Sequence the recombinant plasmid, and compare the sequencing result with the designed CAR gene sequence. The results confirm that the synthetic sequence obtained is correct and that the target gene fragment has been connected to the corresponding overexpression vector.

[0082] See attached for some sequencing results figure 2 .

Embodiment 3

[0084] control group genetic design

[0085] In order to fully verify the effectiveness and safety of the designed dual-receptor CAR-T cells, multiple control groups were designed in the experiment. That is, two negative control structures: CAR1, Signal peptide-hCEAscFv-CD8α-CD8TM-CD3ζ-IRES-GFP and CAR2, Signal peptide-hMSLNscFv-CD8α-CD8TM-4 / 1BB-IRES-GFP; one positive control structure: CAR3, Signal peptide-hCEAscFv-CD8α-CD8TM-4 / 1BB-CD3ζ-IRES-GFP. The sequence information of each element is detailed in Example 1, and the schematic diagrams of the gene sequence elements of the three control groups are detailed in image 3 , the schematic diagram of various types of CAR-T cells constructed is detailed in Figure 4 .

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Abstract

The invention discloses a double-chimeric antigen receptor, a T cell and a construction method and an application thereof, which belong to the field of cellular immunotherapy of tumors. The inventionspecifically relates to a specific structure and a construction method of the double chimeric antigen receptor T cell (dCAR-T cell), and preliminarily discusses the in-vivo and in-vitro activity of the dCAR-T cell. The selected tumor-associated antigens are mesothelin and carcino-embryonic antigens, and researches show that the two tumor antigens can be simultaneously expressed on the surface of asolid tumor, such as pancreatic cancer. The invention discloses an antigen receptor. The in-vitro and in-vivo tests prove that the constructed dCAR-T cell can be permanently and effectively activatedonly under the condition that two antigens are simultaneously recognized, and has efficient anti-tumor activity, so that a specific tumor killing function can be exerted, and the application of CAR-Tcell immunotherapy is improved.

Description

technical field [0001] The present invention belongs to the field of tumor cell immunotherapy, more specifically, relates to the field of tumor cell immunotherapy. Construction of a dual chimeric antigen receptor (dual-receptor CAR, dCAR), T cell and its construction method and application. Background technique [0002] With the continuous development of cellular immunology, the role of immune T cells in alleviating the development of tumors and clearing tumors has been paid more and more attention by scientists. Studies have found that when endogenous T cells are used for tumor immunotherapy, the target antigens of tumor cells need to be processed and presented by the main histocompatibility complex (MHC) on the surface before being presented on the surface of T cells in vivo. Recognized by the receptors, so that tumor cells are eliminated, that is, the function of T cells has the characteristics of "MHC restriction". However, the process of tumor immunoediting will signi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N5/10A61K39/00A61P35/00
CPCC07K16/3007C07K16/30C07K14/7051C12N5/0636A61K39/0011A61P35/00C07K2319/02C07K2319/33C07K2319/60C07K2317/622C12N2510/00A61K2039/5156A61K2039/852A61K2039/86A61K2039/828A61K2039/82A61K2039/812A61K2039/844A61K2039/892A61K2039/876A61K2039/884
Inventor 徐寒梅杨培伟迟骁玮
Owner CHINA PHARM UNIV
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