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Method for enhancing anticancer capability of cells and enhanced type cells obtained by the method

An enhanced, cellular technology, applied in the biological field, to achieve the effect of enhancing the killing effect and enhancing the ability to resist liver cancer cells

Inactive Publication Date: 2018-11-06
北京华伟康信生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for heterogeneous CIK cells, there is no literature on the function of CIK cells after knocking out PD-1.

Method used

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  • Method for enhancing anticancer capability of cells and enhanced type cells obtained by the method
  • Method for enhancing anticancer capability of cells and enhanced type cells obtained by the method
  • Method for enhancing anticancer capability of cells and enhanced type cells obtained by the method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 CIK cell culture

[0053] Human peripheral blood was obtained from patients with hepatocellular carcinoma in Beijing Shijitan Hospital, and the written consent of the patients with liver cancer was obtained and approved by the Hospital Ethics Committee.

[0054] The culture method of CIK cells was based on the protocol of previous studies (Schmidt-wolf IG et al., Use of a SCIDmouse / human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity. J. Exp. Med. 1991; 174 (1 ): 139-149). In short, 40ml of peripheral blood was obtained from a patient with hepatocellular carcinoma on day 0, and PBMC cells were separated by Ficoll-Hypaque gradient centrifugation, and the PBMC cells were suspended in GT-T551 serum-free medium (TAKARA company, Japan), and added 5% human AB plasma, 1000 U / mL IFN-γ (PeproTech). On day 1, 50 ng / mL anti-CD3 antibody (eBiosciences) and 100 U / mL recombinant human IL-2 (eBiosciences) were added to the c...

Embodiment 2

[0056] Example 2 sgRNA design and in vitro T7 transcription of sgRNA

[0057] The PD-1 exon 1 sequence was obtained from NCBI, and two CRISPR design tools (http: / / crispr.mit.edu and https: / / portals.broadinstitute.org / gpp / public / ) were used to design sgRNA, Three gRNAs were comprehensively selected:

[0058] SEQ ID NO.1 (PD-1 sgRNA1): GTCTGGGCGGTGCTACAACT

[0059] SEQ ID NO.2 (PD-1 sgRNA2): TGTAGCACCGCCCAGACGAC

[0060] SEQ ID NO.3 (PD-1 sgRNA3): ACCGCCCAGACGACTGGCCA

[0061] Use pX330 plasmid (Addgene plasmid #4223) as a template, T7 promoter + 20bp targeting sequence oligonucleotide + 20bp sgRNA scaffer as forward primer (TAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNNNNN (20bp target sequence) GTTTTAGAGCTAGAAATAGC). Specifically, the three forward primers are:

[0062] SEQ ID NO.4 (forward primer of PD-1 sgRNA1):

[0063]

[0064] SEQ ID NO.5 (forward primer of PD-1 sgRNA2):

[0065]

[0066] SEQ ID NO.6 (forward primer of PD-1 sgRNA3):

[0067]

[0068] The reverse p...

Embodiment 3

[0071] Example 3 Preparation of PD-1 knockout CIK cells and detection of knockout

[0072] Using P3 Primary Cell 4D-Nucleofector X Kit (Lonza, Germany) through 4D-Nucleofector System X (Lonza, Germany), three kinds of targeted PD- 1 Exon 1 of any sgRNA, electroporated 5×10 6 CIK cells prepared in Example 1. Preheat 50ml of serum-free GT-T551 medium before electroporation, collect CIK cells, centrifuge and add 70μl electrotransfer solution to resuspend the cells, meanwhile add Cas9 protein and sgRNA to 30μl electrotransfer solution, incubate at room temperature for 10 minutes, mix Cas9 protein and The sgRNA mixture was added to the cell suspension, and electroporation was performed with the EO-115 electroporation program using a Lonza 4D-Nucleofector X Unit electroporation instrument. After electroporation, let the cells rest for 24 hours, then resuspend the cells in 2 ml of pre-warmed GT-T551 medium containing 5% human AB plasma and 100 U / mL recombinant human IL-2, and tr...

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Abstract

A method for enhancing anticancer capability of CIK cells is provided. The method includes knockout of a PD-1 gene of the CIK cells based on a CRISPR / Cas9 technique. The invention also provides enhanced type CIK cells obtained by the method. Experiments prove that the knockout of the PD-1 gene from the CIK cells can effectively enhance anti-hepatoma capability of the CIK cells, thus providing novel effector cells for tumor cellular immunotherapy.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, the invention relates to a method for knocking out the PD-1 gene of CIK cells based on CRISPR / Cas9 technology to enhance the anti-tumor ability of CIK cells. Background technique [0002] Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related death in the world, ranking second among all malignant tumors, and its prognosis is poor. The current conventional treatment of HCC mainly involves partial hepatectomy, liver transplantation, radiofrequency ablation (RFA) and transarterial chemoembolization. Since most patients have already entered the advanced stage of liver cancer when they are clearly diagnosed, the current traditional treatment methods are difficult to provide clinically effective benefits for advanced patients. This has also led to the 5-year survival rate of liver cancer remaining at about 10-20%. [0003] In order to improve the survival rat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/113A61K35/17A61P35/00
CPCA61K35/17A61P35/00C12N5/0636C12N5/0638C12N15/113C12N2310/10C12N2510/00C12N2800/80C12N2810/10
Inventor 孙博文黄康华彭吉润
Owner 北京华伟康信生物科技有限公司
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