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Plasmid carrier pair and immune cells modified by same, and application thereof

A plasmid carrier and immune cell technology, applied to genetically modified cells, cells modified by introducing foreign genetic material, blood/immune system cells, etc., can solve the problems that do not mention the necessity and beneficial effects of IL-12 , to avoid toxic side effects, reduce toxicity and enhance the effect

Inactive Publication Date: 2019-07-26
CRAGE MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, this US patent is silent on the need for controlled expression and local secretion of IL-12 in immune cells and its beneficial effects

Method used

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  • Plasmid carrier pair and immune cells modified by same, and application thereof
  • Plasmid carrier pair and immune cells modified by same, and application thereof
  • Plasmid carrier pair and immune cells modified by same, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1: Construction of lentiviral plasmid and viral packaging of chimeric antigen receptor protein encoded by nucleic acid

[0108] Table 1 below illustrates the linking sequence of the various parts of the chimeric antigen receptor of the example of the present invention.

[0109] Table 1 Connection sequence of each part of chimeric antigen receptor

[0110]

[0111] 1. AntiGPC3-synNotch-GAL4VP64 nucleic acid fragment amplification

[0112] 1) Amplification of GPC3 scFv sequence

[0113] Obtain the nucleic acid sequence (SEQ ID NO: 2) of the scFv of antiGPC3 by conventional PCR method

[0114] Other nucleic acid sequences were obtained by PCR using PHR-PGK-antiCD19-synNotch-GAL4VP64 (purchased from addgene) as a template. Wherein the signal sequence (SEQ ID NO:1) is represented by a primer pair [upstream primer: 5'-tctcacgcgtcaagtggagc-3'(SEQ ID NO:14), downstream primer: 5'-tctgcaccagctgcacctcgaggtcctcttcagag-3'(SEQ ID NO:15)] PCR amplification was carried ...

Embodiment 2

[0148] Example 2: Infection of NK92 cells with recombinant lentivirus

[0149] Infect NK92 cells with the lentivirus 1 prepared in Example 1 to obtain GPC3-SYN-IL12-NK92 cells, the specific operations are as follows:

[0150] 1) The day before infection, coat a 24-well plate with recombinant human fibronectin (Retronectin), add 380 μl of 5 μg / ml recombinant human fibronectin solution (PBS) to each well, and incubate overnight at 4°C;

[0151] 2) Discard the recombinant human fibronectin solution (PBS) in the 24-well plate during infection, and wash twice with 1 ml of PBS. Infect with the above-mentioned recombinant lentivirus at MOI=30, and at the same time add polybrene at a final concentration of 10 μg / ml to improve the infection efficiency, and the number of cells per well is 5×10 5 , culture medium 500μl, 32°C, 1800g, after centrifugation for 90min, transfer to the cell culture incubator;

[0152] 3) Cells after infection were treated with 5×10 cells every other day 5 / ...

Embodiment 3

[0153] Example 3: Identification of GPC3-SYN-IL12-NK92 cells

[0154] On the 7th day of culture, the lentivirus-infected NK92 cells were detected by flow cytometry to detect the expression of different receptors. Since the N-terminus of antiGPC3 has a Myc tag, the detection of Myc expression is a positive cell successfully infected by pHRLSIN-antiGPC3-synNotch-GAL4VP64, Detecting the expression of mcherry means the positive cells successfully infected by pHRLSIN-GAL4UAS-IL12-PGK-mcherry, and the simultaneous expression of Myc and mcherry means the positive cells successfully double infected by GPC3-SYN-IL12-NK92.

[0155] 1) 1×10 NK92 cells infected with different 6 Divide the cells into 2ml centrifuge tubes, centrifuge at 5000rpm at 4°C for 5min, discard the supernatant, and wash twice with PBS;

[0156] 2) The cells in the control group for detection of myc expression were washed twice with direct PBS (2% NBS) and resuspended as a control; the cells in the detection group w...

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Abstract

The invention discloses a plasmid carrier pair and immune cells modified by the same, and an application thereof in preparation of drugs for local secretion of IL12 or regulating expression of IL12 and preparation of drugs for enhancing the killing effect of CAR-T cells, and also discloses a composition and combined drug box of the immune cells and the CAR-T cells. The plasmid vector pair includesa first carrier and a second carrier, the first carrier contains a Notch core, and other coding nucleic acid molecules of extracellular and intracellular segments, wherein the intracellular segment preferably contains transcription activating factors; the second carrier contains nucleic acid molecules specifically recognizing and binding with the intracellular segment in the first carrier and nucleic acid molecules encoding IL12. The combined drug box comprises a drug box A containing the immune cells and a drug box B containing the CAR-T cells. The immune cells can control the expression andsecretion of IL12, and have the effect of synergistically treating tumors with the CAR-T cells.

Description

technical field [0001] The invention belongs to the field of immunotherapy, and specifically relates to a pair of plasmid vectors, immune cells modified by the same and application thereof, and the immune cells are immune cells that regulate the expression of IL12 dependently on target antigens. Background technique [0002] IL12 is an immune cell growth stimulator with multiple biological activities and a heterodimeric cytokine that promotes the proliferation of T helper 1 (Th1); induces NK cells and T cells to produce interferon-γ ; Improve the cytotoxic effect of NK cells; Promote the formation of cytotoxic T cells. At present, IL12 is directly loaded to enhance the anti-tumor efficacy of chimeric antigen receptor (CAR), but this loading method will cause IL12 to be secreted systemically along with CAR-T, and it is easy to cause the expression of IL12 to increase with T cells. The expansion of the uncontrolled, which will bring serious toxic side effects to the body. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10A61K35/17A61P35/00
CPCC12N15/85C07K16/30C07K14/5434A61K35/17A61P35/00C12N2510/00C12N2740/16043C12N2830/48C12N15/86C12N2830/002C07K16/303C07K2317/622A61K2039/5156A61K39/001174C07K14/7051C07K2319/00C12N5/0636C12N5/0646C12N5/16C12N15/90C12N2501/606C12N2800/107
Inventor 李宗海骆红王华茂
Owner CRAGE MEDICAL CO LTD
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