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Preparation method and kit of bispecific chimeric antigen receptor gene modified natural killer cells

A technology of chimeric antigen receptors and natural killer cells, applied to cells modified by introducing foreign genetic material, receptors/cell surface antigens/cell surface determinants, chemical instruments and methods, etc. Poor antitumor activity, low antitumor activity, insufficient immune function against tumors, etc.

Inactive Publication Date: 2016-06-08
ZICHENG RUISHENGHUI BEIJING BIOTECH DEV CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention aims at the deficiencies of the existing clinical application of NK cell technology, especially because NK cells target tumor cells with poor killing specificity; NK cells express inhibitory receptors, so that tumor cells can generate immune escape by binding to them, and activate patients in vivo Insufficient autoimmunity against tumors, resulting in low tumoricidal activity and limited treatment for most malignancies

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  • Preparation method and kit of bispecific chimeric antigen receptor gene modified natural killer cells
  • Preparation method and kit of bispecific chimeric antigen receptor gene modified natural killer cells
  • Preparation method and kit of bispecific chimeric antigen receptor gene modified natural killer cells

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preparation example Construction

[0040] The invention relates to a method for preparing natural killer cells genetically modified with bispecific chimeric antigen receptors. Human natural killer cells are transfected, and the bispecific chimeric antigen receptor gene is highly expressed, which can specifically bind to tumor cells expressing signaling lymphocyte activation molecule family member 7 and fibronectin variants, and inhibit the inhibitory effect of natural killer cells. The body expression avoids the immune evasion of tumor cells; simultaneously activates the first signal and co-stimulatory signal to trigger anti-tumor cytotoxic activity, and activates a strong anti-tumor response in vivo and in vitro.

[0041] The SLAMF7 and FNv single-chain antibodies in the bispecific chimeric antigen receptor gene of the present invention specifically bind to the SLAMF7 and FNv antigens expressed by malignant tumor cells, so that NK cells can produce anti-tumor targeting specificity, preferably targeting melanin ...

Embodiment 1

[0075] Construction of recombinant Di-CAR gene and preparation of lentivirus

[0076] Human IFNγ-signal peptide-scFv(SLAMF7)-CD8a hinge region-CD28-CD137-CD3ζ intracellular region, and scFv(FNv)-IgD hinge region-c-Myc mutant gene through Furin cleavage site and linker sequence Fragment composition, the 362nd leucine of the c-Myc mutant is mutated to valine, namely c-Myc-L / V, see figure 1 , which is the schematic diagram of Di-CAR fusion protein.

[0077] The human IFNγ-signal peptide-scFv (SLAMF7)-CD8a hinge region-CD28-CD137-CD3ζ intracellular region, and the scFv (FNv)-IgD hinge region-c-Myc mutant were passed through the Furin cleavage site and linking sequence (Linker ) are linked together to form a recombinant gene sequence, which is obtained by chemical synthesis to form a complete cDNA of the bispecific chimeric antigen receptor gene, that is, Di-CAR;

[0078] After the target DNA fragment of Di-CAR and the pUC229 vector HindIII / BamHI were double-digested, under the a...

Embodiment 2

[0083] Preparation of Natural Killer Cells Genetically Modified with Bispecific Chimeric Antigen Receptor

[0084] Collect 30ml of patients with advanced melanoma and uveal melanoma (with informed consent), add an equal amount of 1×PBS to dilute, gently superimpose on the lymphocyte separation medium along the wall of the centrifuge tube to form a clear interface, and centrifuge at 2000rpm at room temperature After 20 minutes, the PBMCs in the interface layer were sucked and washed twice with 1×PBS (pH 7.4). After counting orchids, resuspend with RPMI1640 medium and dilute to 3×10 6 Cells / ml, resuspended with RPMI1640 medium and diluted to 3×10 6 Cells / ml, supplemented with 50ng / mlIL-15, 20ng / mlIL-12, 20ng / mlIL-18, 100IU / mlIL-2 and 10% autologous serum, 1.2ml per well was added to mouse anti-human CD16-coated 24 In an orifice plate, at 37°C, containing 5% CO 2 Continue culturing in the incubator until day 5. NK cells were harvested by centrifugation on day 5 and counted by...

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Abstract

The invention provides a preparation method of bispecific chimeric antigen receptor gene modified natural killer cells, comprising the features: constructing a bispecific chimeric antigen receptor gene containing specific-binding signaling lymphocyte activation molecule family member 7 and fibronectin mutants, recombining the bispecific chimeric antigen receptor gene to a viral vector, transfecting with human natural killer cells, and highly expressing the bispecific chimeric antigen receptor gene, thus specifically binding tumor cells that express the signaling lymphocyte activation molecule family member 7 and fibronectin mutants, inhibiting expression of natural killer cell inhibitory receptors, and preventing immune escape of tumor cells; meanwhile, activating a first signal and a co-stimulatory signal to trigger toxic activity of the tumor cells, and great anti-tumor killing toxicity is shown in in-vivo and in-vitro experiments; the invention also provides a kit for the preparation of autologous natural killer cells through the above method, and with the kit, it is possible to highly express the bispecific chimeric antigen receptor gene and trigger great anti-tumor effect.

Description

technical field [0001] The present invention relates to a method for preparing natural killer cells genetically modified with bispecific chimeric antigen receptors and a kit thereof, which includes the following features: a cell containing specific binding signal Lymphocyte Activation Molecule Family Member 7 (SignalingLymphocytic ActivationMoleculeFamily, Member7, SLAMF7) and fibronectin variant (fibronectin variant, FNv) dual-specific chimeric antigen receptor (double chimeric antigen receptor, Di-CAR) genes recombined into viral vectors and transfected into human natural killer cells (Natural killer cells, NK), high expression of the The bispecific chimeric antigen receptor gene can specifically bind to tumor cells expressing signaling lymphocyte activation molecule family member 7 and fibronectin variants, and inhibit the expression of natural killer cell inhibitory receptors, avoiding immune evasion of tumor cells; Simultaneous activation of the first signal and co-stimul...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/86C12N15/62A61K35/17A61P35/00
CPCA61K35/17C07K14/705
Inventor 陈建勋黄启明
Owner ZICHENG RUISHENGHUI BEIJING BIOTECH DEV CO LTD
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