Recombinant CAR gene and carrier, CAR-T cell and application thereof

A gene carrier and gene technology, applied in the field of CAR-T cells and applications, recombinant CAR genes and their vectors, which can solve the problems of T cell activation and insufficient proliferation ability.

Active Publication Date: 2017-03-22
IMMUNE CELL BIOTECH CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a recombinant CAR gene and its carrier, CAR-T cell

Method used

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  • Recombinant CAR gene and carrier, CAR-T cell and application thereof
  • Recombinant CAR gene and carrier, CAR-T cell and application thereof
  • Recombinant CAR gene and carrier, CAR-T cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Construction of embodiment 1CART-19scFv plasmid

[0046] The method for obtaining the CART-19scFv plasmid is as follows: the complete sequence of the recombinant CAR gene (SEQ ID NO: 7) is synthesized, and then ligated into the lentiviral plasmid vector pLent-EF1a by AsisI / NsiI double enzyme digestion. For the plasmid map of the constructed CART-19scFv plasmid and pLent-EF1a, see Figure 5 and Figure 6 .

Embodiment 2

[0047] Embodiment 2 T lymphocyte transfection

[0048] The constructed CART-19scFv plasmid was subjected to lentiviral packaging and purification, and then the lentiviral vector technology was used (references: Tumaini B, Lee DW, Lin T, Castiello L, et al. Simplified process for the production of anti-CD19-CAR engineered T cells.Cytotherapy.2013; 15(11):1406-1415.) Express the recombinant CAR (SEQ ID NO:7) gene in the patient's T cells, and use flow cytometry to detect the positive rate of green fluorescent protein (GFP), see results Figure 4 ,Depend on Figure 4 It can be seen that the transfection efficiency of lentivirus infection of T lymphocytes is about 30%.

Embodiment 3

[0049] Example 3 In vitro proliferative ability detection of T lymphocytes after transfection

[0050] The transfected T lymphocytes (CAR-T cells) were expanded in vitro for 2 weeks, and the results are shown in figure 1 . And use flow cytometry to measure the ratio of CD3 positive T cells and CD8 positive T cells before and after expansion, the results are shown in figure 2 and image 3 . Depend on figure 2 It can be seen that the proportion of CD3 positive T cells before expansion is 55.8%+16.8%, about 72.7% in total; the proportion of CD3 positive T cells after expansion is 30.4%+56.2%, about 86.9% in total, compared with before expansion, After 2 weeks of in vitro expansion, CD3 positive T cells increased by 14.2%. Depend on image 3 It can be seen that the proportion of CD8-positive T cells before expansion was 13.8%; the proportion of CD8-positive T cells after expansion was 42.9%. Compared with before expansion, after 2 weeks of in vitro expansion, CD8-positive...

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Abstract

The invention belongs to the field of tumor cell immunotherapy, and particularly relates to a recombinant CAR gene and carrier, CAR-T cell and application thereof. The recombinant CAR gene includes a nucleotide sequence coded the following parts of an antigen-binding part of a CD19 antibody, a transmembrane part and a CD28 endochylema functional domain, a 41BB endochylema functional domain and a CD3zeta endochylema functional domain connected in a random sequence. The efficiency of using the recombination CAR gene and the carrier containing the recombinant CAR gene to transfect T cell is high, and the positive T cell through transfection has good multiplication capacity. Besides, the recombination CAR gene is high in specificity to B cell tumor antigen, and prompts CAR-T to kill and wound B cell tumor specifically.

Description

technical field [0001] The invention belongs to the field of tumor cell immunotherapy, and specifically relates to a recombinant CAR gene and its carrier, CAR-T cells and applications. Background technique [0002] B lymphocytic neoplasms include a variety of malignancies, such as most non-Hodgkin's lymphomas, acute lymphoblastic leukemia, and chronic lymphocytic leukemia. Current treatments for B lymphocyte malignancies include chemotherapy, radiation therapy, bone marrow transplantation, and peripheral blood stem cell transplantation. Despite a variety of treatments, there are still many patients who cannot be cured. For example, B-lineage acute leukemia is sensitive to chemotherapy but cannot be eradicated, with relapse occurring in about 65% of adults and 20% of children. So far, people can only improve the sensitivity of chemotherapeutic drugs by enhancing their cytotoxicity, but it also increases the mortality rate. Although adoptive immunotherapy using leukocytes f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/85C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/2803C07K2319/03C07K2319/30C12N5/0636C12N2510/00
Inventor 孙秀莲
Owner IMMUNE CELL BIOTECH CO LTD
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