Preparation method for HLA-A0201 restrictive anti-PSA antigen-specificity CTL

Abstract

The invention belongs to the field of biotechnology development and application research and discloses a preparation method for HLA-A0201 restrictive anti-PSA antigen-specificity CTL. The method includes the steps that peripheral mononuclear cells are collected through a single sampling instrument, and CD8+T lymphocyte is enriched and purified; mature dendritic cells loaded with HLA-A0201 restrictive PSA polypeptide are used for stimulating the CD8+T lymphocyte, and growth of T cells is promoted through combination of rhIL-2 and rhIL-7; target CTL is purified through a Tetramer labeling method and fluorescence-activated cell sorting; growth of the target CTL is stimulated through an anti-CD3 monoclonal antibody coated with a solid phase and IL-2; gamma-ray irradiated autologous PBMCs are added to enhance activation of the target CTL; rhIL-5 is added for cultivation and amplification, and collection and identification are conducted. The target CTL prepared through the method has high purity, high multiplication capacity, high killing activity and high proportion CTL-CM, and is used for tumor therapy and other immunological therapy.

Description

technical field [0001] The invention belongs to the field of biotechnology development and application research, and relates to a method for preparing HLA-A0201-restricted anti-PSA antigen-specific CTL. Background technique [0002] 1. Difficulties and opportunities in cancer treatment [0003] Malignant tumors have become the number one "killer" of human beings. Surgery, radiotherapy and chemotherapy are the three conventional methods for treating tumors, which can effectively reduce the tumor burden in a short period of time, but they still cannot effectively remove tumor cells. Minimal residual lesions, partial sensitivity and drug resistance to radiotherapy and chemotherapy are the root causes of tumor recurrence and metastasis, and recurrence and metastasis are the main causes of death in cancer patients. Conventional treatment methods have been unable to do what they want, and the development of new tumor treatment technologies and products is imminent. [0004] 2. T...

AI Technical Summary

Problems solved by technology

[0020] 2. Differences in CTL phenotype and function

[0038] ② TIL isolation, CTL clone acquisition and identification methods are complex, various cell components in tumor tissue are more complicated, and the time for cell isolation is long, usually more than 1 month;

[0039] ③TILs are mixed with CD4+CD25+Treg subsets, which can inhibit the expansion efficiency of CTLs;

[0040] ④ Due to the long time of in vitro separation and amplification, the chance of contamination is increased

[0042] ① The introduction of exogenous plasmids (retroviruses or lentiviruses, etc.) brings certain risks to clinical application;

[0043] ②Endogenous TCR interference, the imported TCR gene may not be imported into the expected site, but misaligned with the endogenous TCR, its ability to recognize the antigen is not the expected design, and is unknown, there is a great risk

[0046] ② The cycle of artificial APC in vitro sensitization of CTL is long and takes 3-4 weeks. The acquisition of CTL needs to be expanded and cultured to meet the needs of clinical treatment. Therefore, the cycle of in vitro culture is long and the probability of contamination is high

[0047] At present, no literature has reported the preparation method of HLA-A201-restricted antigen-specific CTL with short preparation time, high amplification factor, high CTL purity and good killing effect

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