CAR-NK cell as well as preparation method and application thereof

A kind of NK cell and cell technology, applied in the biological field, can solve the problems that the application precedent of CAR-NK cell tumor immunotherapy has not been established, and achieve the effect of wide application range and low preparation cost

Active Publication Date: 2017-08-11
国健亦诺生物科技(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, an effective method for the preparation of CAR-NK cells and its application precedent in tumor immunotherapy have not been established

Method used

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  • CAR-NK cell as well as preparation method and application thereof
  • CAR-NK cell as well as preparation method and application thereof
  • CAR-NK cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Preparation of recombinant vector CAR-CD244-antiCD19 and recombinant vector CAR-CD244-antiPSMA

[0055] 1. The specific antibody-encoding gene is amplified by PCR using primers with BfuAI restriction endonuclease recognition sites at the end;

[0056] The specific antibody coding gene sequences used in this example are the partial sequence of CD19 antibody (antiCD19) (sequence listing sequence 2) and the partial sequence of PSMA antibody (antiPSMA) (sequence listing sequence 3);

[0057] 2. Purify the PCR products of step 1 respectively;

[0058] 3. Carry out single enzyme digestion (BfuAI) to the PCR product purified in step 2, and simultaneously carry out single enzyme digestion (BfuAI) to the cloning vector containing the transformed CD244 backbone;

[0059] 4. Purify the digested product;

[0060] 5. After the purified product is prepared according to the molar ratio of the antibody fragment and the cloning carrier containing the modified CD244 backbone...

Embodiment 2

[0063] Embodiment 2, preparation of recombinant lentiviral vector

[0064] 1. Using the recombinant vector CAR-CD244-antiCD19 prepared in Example 1 and the recombinant vector CAR-CD244-antiPSMA as templates respectively, PCR amplification was performed using primers with BamHI and XbaI restriction endonuclease site sequences;

[0065] 2. Purify, digest (BamHI and XbaI) and purify the PCR products respectively;

[0066] 3. Use T4 ligase to connect the products of step 2 into the Plenti lentiviral vector (named pLentiCMV / TO eGFP Puro (w159-1), purchased from addgene, website: http: / / www.addgene.org / 17481 / ) between BamHI and XbaI sites;

[0067] 4. Transformation, plating, picking single clones to extract plasmids for sequencing, and named the recombinant lentiviral vectors with correct sequencing as recombinant lentiviral vectors Plenti-CAR-CD244-antiCD19 and Plenti-CAR-CD244-antiPSMA respectively.

[0068] The schematic diagram of the recombinant lentiviral vector Plenti-CAR...

Embodiment 3

[0069] Example 3, preparation and concentration of lentivirus

[0070] 1. Preparation of lentivirus

[0071] Use the recombinant lentiviral vector Plenti-CAR-CD244-antiCD19 prepared in Example 2 and psPAX2 (purchased from addgene, website at https: / / www.addgene.org / 12260 / ), pMD2.G (purchased from addgene, website 293T cells (about 3X10 6 293T cells were plated in a 10cm dish), and replaced with fresh medium (DMEM medium, supplemented with 10% fetal bovine serum) after 8 hours, and then the supernatant was collected every 24 hours and added with fresh medium (DMEM medium, supplemented with 10% Fetal bovine serum), collected 3 times in total to obtain lentiviral supernatant A.

[0072] Using the prepared recombinant lentiviral vector Plenti-CAR-CD244-antiPSMA and psPAX2, pMD2.G vectors prepared in Example 2, the mass ratio was 5ug:3.2ug:1.8ug to transfect 293T cells (about 3×10 6 293T cells were plated in a 10cm dish), and replaced with fresh medium (DMEM medium, supplemented...

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Abstract

The invention relates to a CAR-NK cell as well as a preparation method and an application thereof. The method comprises the following steps: S1, linking a specific antibody coding gene into a cloning site region of a modified CAR skeleton so as to obtain a CAR gene, wherein the modified CAR skeleton comprises a CD244 episporium signal region, the cloning site region, a CD244 extracellular hinge region, a CD244 transmembrane part region and a CD244 intracellular part region which are sequentially connected; and S2, infecting the CAR gene with NK cell through lentivirus, so that the NK cell expressing the CAR protein is obtained, namely the CAR-NK cell. The prepared tumor immnuotherapy cell, namely the CAR-NK cell, of the invention, besides a patient with cancer, is also applicable to allosome infusion; therefore, the prepared tumor immnuotherapy cell, in comparison with existing CAR-T, is wider in application scope and is lower in preparation cost.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a method for preparing CAR-NK (or other immune cells) for clinical medical treatment, etc., for cellular immunotherapy of tumors. Background technique [0002] Cell biological therapy is a new type of cancer treatment method using the autoimmune system, and it is a treatment mode with significant curative effect. It uses molecular biology technology and cell engineering technology to culture and expand the immune cells collected from the patient's body with biological agents, and then reinfuse them back into the patient's body, so as to stimulate and enhance the body's own immune function and achieve surveillance. And the purpose of killing diseased cells or repairing damaged cells. [0003] Natural killer cells (NK) belong to granular lymphocytes and are important immune cells in the body. It is generally believed that NK cells are derived from bone marrow lymphoid stem ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C12N15/62A61K35/17A61P35/00
CPCA61K35/17C07K14/70503C07K16/2803C07K16/3069C07K2319/02C07K2319/03C07K2319/33C12N5/0646C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 顾春雨尹乐齐浩刘建强
Owner 国健亦诺生物科技(北京)有限公司
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