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CD19-targeting CAR-T cell of over-expression ATP5IF1 gene and application of CD19-targeting CAR-T cell

A cell-targeted technology, applied to CD19-targeting CAR-T cells and its application fields, to achieve the effect of increasing ratio, increasing TCM and TSCM, and significant tumor killing activity

Active Publication Date: 2022-07-15
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the existing problems of CAR-T cell drugs targeting CD19 in the prior art, and to provide a CD19-targeting CAR-T cell overexpressing ATP5IF1 and its application

Method used

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  • CD19-targeting CAR-T cell of over-expression ATP5IF1 gene and application of CD19-targeting CAR-T cell
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  • CD19-targeting CAR-T cell of over-expression ATP5IF1 gene and application of CD19-targeting CAR-T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Preparation of CD19-IF1-CAR-T cells

[0028] 1. Construction of lentiviral expression vector

[0029] (1) Construction of lentiviral vector expressing chimeric antigen receptor gene for CD19 and ATP5IF1 gene

[0030] The fusion gene in this example includes the ATP5IF1 gene and the gene encoding the CD19-targeting chimeric antigen receptor. The nucleotide sequence of the fusion gene is shown in positions 2491-4338 of SEQ ID NO. 1, wherein SEQ ID NO. Positions 3949-4020 of NO.1 are P2A restriction sites, and positions 4021-4338 are ATP5IF1 gene.

[0031] Chimeric Antigen Receptor ( figure 1 B) includes sequentially concatenated:

[0032] a. CD8 leader peptide, position 2491-2553 of SEQ ID NO.1;

[0033] b. Anti-CD19 scFv, position 2554-3279 of SEQ ID NO.1;

[0034] c. CD8 hinge region (position 3280-3414 of SEQ ID NO. 1), CD8 transmembrane region (position 3415-3477 of SEQ ID NO. 1) and CD8 intracellular region (position 3478 of SEQ ID NO. 1) -3489 bits); ...

Embodiment 2

[0055] Example 2: Determination of anti-tumor effect of CD19-IF1-CAR-T cells in vitro

[0056] 1. CD19-IF1-CAR-T cells against CD19 + NALM-6 luc+ Killing effect:

[0057] The CD19-CAR-T cells, CD19-IF1-CAR-T cells and UT cells prepared in Example 1 were mixed with 1 × 10 4 CD19 + NALM-6 Luc+ Cells were co-cultured in 96-well plates according to different ratios (E:T=5:1, 3:1, 1:1, 1:2), and after 24 hours, the firefly luciferase reporter gene (Biyuntian, One-LumiTM II) was detected. Firefly luciferase reporter gene detection kit) represents the killing activity of the corresponding CAR-T cells by the tumor cell lysis rate.

[0058] The result is as Figure 4 As shown, the CD19-IF1-CAR-T cells had a higher tumor lysis rate by measuring Luci in the cell culture supernatant ( Figure 4 ), that is, ATP5IF1-overexpressing CAR-T cells indeed enhanced their tumor-killing effect and performed better when targeting CD19.

[0059] 2. Determination of the expression level of IFN-...

Embodiment 3

[0077] Example 3: Evaluation of anti-tumor effect of CD19-IF1-CAR-T cells in vivo

[0078] 18 6-week-old NCG female mice were taken, and each mouse was injected with 1.2 × 10 6 NALM-6 Luc+ The cells were then randomly divided into 3 groups: UT cell group, CD19-CAR-T cell group and CD19-IF1-CAR-T group. One week later, 1.2 × 10 was injected into the tail vein 6 UT cells, 1.2×10 6 CD19-CAR-T cells and 1.2 × 10 6 Three groups of mice were treated with CD19-IF1-CAR-T cells, respectively. period, on day 22 ( Figure 8 A) and 28 days ( Figure 8 B) Venous blood was obtained from mouse tail and analyzed by flow cytometry for CD19 of tumor cells in mice + NALM-6 Luc+ The absolute number of cancer cells. Observe the state of the mice within 50 days and record the survival period of the mice, draw the survival curve, Figure 9 Schematic diagram of mouse survival time.

[0079] Figure 8-9 The results showed that CD19 in tumor-bearing mice injected with CD19-IF1-CAR-T cells ...

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Abstract

The invention belongs to the technical field of cellular immunotherapy, and particularly relates to a CD19-targeting CAR-T cell of an overexpressed ATP5IF1 gene and application of the CD19-targeting CAR-T cell. Specifically, on one hand, the invention relates to a fusion gene which comprises an ATP5IF1 gene and a coding gene of a CD19-targeted chimeric antigen receptor, and the nucleotide sequence of the fusion gene is as shown in the 2491th site to the 4338th site in SEQ ID NO.1. The invention also relates to a lentiviral expression vector, a recombinant lentivirus and a CD19-targeting CAR-T cell for overexpressing the ATP5IF1 gene, and in-vivo and in-vitro experiments show that the CD19-targeting CAR-T cell for overexpressing the ATP5IF1 gene (i.e., the CD19-IF1-CAR-T cell) can specifically kill tumor cells, enhance the curative effect of resisting B cell malignant lymphoma and obviously prolong the lifetime of tumor-bearing mice.

Description

technical field [0001] The invention belongs to the category of cellular immunotherapy technology, and specifically relates to a CAR-T cell targeting CD19 that overexpresses the ATP5IF1 gene and its application. Background technique [0002] Adoptive immunotherapy based on the infusion of genetically redirected autologous T cells has been demonstrated for the treatment of hematological malignancies and solid tumors. Therefore, a variety of functional acquisition strategies based on T cell receptors (TCRs) or chimeric antigen receptors (CARs) have emerged to confer desired antigen receptors on T cells. Among them, the use of CAR has been shown to be effective in enhancing the immune response against cancer. Although CAR-T cell therapy can have a significant impact on disease clearance, it still faces challenges such as strong toxic side effects and difficulty in maintaining long-term remission. [0003] CD8 + T cell subsets play a key role in protective immunity against tu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/867C12N7/01C12N5/10C12N5/0783A61K39/00A61P35/00
CPCC07K16/2803C07K14/7051C07K14/4703C12N15/86C12N7/00C12N5/0636A61K39/001112A61P35/00C07K2319/00C12N2510/00C12N2740/15043C12N2740/15021
Inventor 钟根深王崎吴敏娜许芝山杨如张忠新
Owner XINXIANG MEDICAL UNIV
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