A kind of preparation method and application of CTL cells

A cell and insect cell technology, applied in the field of CTL cell preparation, can solve problems such as side effects, destroy immune tolerance, etc., and achieve the effects of high stability, improved curative effect, and high-efficiency specific cytotoxicity

Active Publication Date: 2021-09-21
GUANGZHOU ANJIE BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, long-term use of immune checkpoint inhibitors may break immune tolerance, leading to serious side effects

Method used

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  • A kind of preparation method and application of CTL cells
  • A kind of preparation method and application of CTL cells
  • A kind of preparation method and application of CTL cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The preparation of embodiment 1 tumor antigen PAP-GM-CSF

[0070] 1. Construction of insect baculovirus vector

[0071] (1) Gene synthesis of tumor antigen PAP-GM-CSF

[0072] The gene sequences of prostatic acid phosphatas (Prostatic Acid Phosphatas, PAP) and granulocyte-macrophage colony stimulating factor (GM-CSF) were retrieved from Genbank, and PAP and GM-CSF were passed through two The amino acid Gly-Ser connection structure contains a signal peptide upstream of PAP, and the software is used for codon optimization to synthesize the entire DNA sequence. The corresponding DNA sequence of the PAP-GM-CSF fusion protein is shown in SEQ ID NO.: 1, and the corresponding amino acid sequence is shown in SEQ ID NO.: 2.

[0073] (2) Construction of the shuttle plasmid pFast-Bac1-PAP-GM-CSF

[0074] The vector pFast-Bac1 and the gene of the synthetic tumor antigen PAP-GM-CSF were digested, connected and transformed to construct a recombinant plasmid.

[0075] (3) Construc...

Embodiment 2

[0100] Example 2 DC cells sensitized by tumor antigen PAP-GM-CSF

[0101] Separation of peripheral blood mononuclear cells (PBMC) by a blood cell separator or Ficoll, and isolation of CD14 by magnetic bead sorting + Monocytes, add X-VIVO TM 15 (LONZA) lymphocyte serum-free medium (containing 500-1000U / ml rhGM-CSF and 500U / ml rhIL-4), 37°C, 5% CO 2 CO 2 Culture in an incubator, add tumor antigen PAP-GM-CSF on the 5th day to stimulate activated DC, add TNF-α (final concentration 20ng / ml) at the same time to induce DC maturation, continue to culture for 48h, and obtain tumor antigen PAP-GM-CSF sensitization DC cells, such as Figure 5 As shown, the proportion of CD86 of DC cells detected by flow cytometry was 54.6%.

Embodiment 3

[0102] Example 3 Sensitized DC cells induce CTL cells

[0103] Blood cell separator or Ficoll to separate PBMCs from the same source as DCs, add them to DCs stimulated and matured by PAP-GM-CSF fusion protein in Example 2 (DC:PBMCs=1:10), 37°C, 5% CO 2 co-incubated in the incubator for 3-5 days to obtain tumor antigen PAP-GM-CSF specific CTL cells, such as Image 6 shown, where Image 6 (A) Detection of CD3 in induced cells by flow cytometry + CD4 + The ratio is 19.0%, Image 6 (B) Detection of CD3 in induced cells by flow cytometry + CD8 + The proportion of 79.9%, that is, CTL cells as high as 79.9%.

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of CTL cells. The method comprises the following steps: using tumor antigen PAP-GM-CSF-sensitized DC cells to induce CTL cells; knocking out the PD-1 gene of the CTL cells to obtain PD-1 knockout CTL cells. The specific CTL cell preparation obtained by the preparation method of the present invention will not cause CTL exhaustion and disability due to PD-L1 expressed by the tumor after reinfusion into the body, thereby producing efficient and specific cytotoxicity on tumor cells, improving its curative effect and reducing side effects, can be used for the treatment of prostate cancer, especially for the treatment of PAP-positive prostate cancer patients, and has broad clinical application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of CTL cells. Background technique [0002] Prostate cancer (PC) is one of the most common malignant tumors of the male genitourinary system. It is a cancerous disease of local organs. Its course can be divided into two stages, namely hormone-dependent period and hormone-independent period. In the hormone-dependent period, prostatectomy, radiotherapy, and drug castration can be used for treatment, accompanied by serious toxic and side effects, which affect the quality of life of the patient. After the hormone-dependent period, the disease will progress to the hormone-independent period, and almost all patients develop castration-resistant prostate cancer (CRPC), while metastatic castration-resistant prostate cancer (metastatic castration-resistant prostate cancer) Prostate Cancer (mCRPC) is the leading cause of death from prostate can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N5/0784C07K19/00C12N15/866C12N15/90A61K35/17A61P35/00
CPCC12N5/0638C12N5/0639C12N9/16C12Y301/03002C07K14/535C07K14/70521C12N15/86C12N15/907A61K35/17A61P35/00C07K2319/02C12N2501/22C12N2501/2304C12N2501/25C12N2502/1121C12N2501/73C12N2710/14043C12N2800/105
Inventor 周超安鸿卢有德周玲杜永彪涂嘉琦尹海滨
Owner GUANGZHOU ANJIE BIOMEDICAL TECH CO LTD
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