Preparation method of HLA-A0201 restriction anti-Her2-neu antigen-specific CTL (cytotoxic lymphocyte)

Abstract

The invention belongs to the field of biotechnology development and application and discloses a preparation method of HLA-A0201 restriction anti-Her2-neu antigen-specific CTL (cytotoxic lymphocyte). The method includes: collecting peripheral mononuclear cells by single collecting, and enriching and purifying CD8+T lymphocyte; stimulating the CD8+T lymphocyte with mature dendritic cells that support HLA-A0201 restriction Her2-neu antigen polypeptides, and promoting T cell growth with rhIL-2 and rhIL-7; purifying a target CTL by Tetramer labeling and flow activated cell sorting; stimulating the target CTL to grow with antihuman CD3 monoclonal antibodies and IL-2; enhancing activation of the target CTL through autologous PBMC (peripheral blood mononuclear cells) after irradiation of gamma ray; adding rhIL-15 for culturing and amplification, and collecting and identifying. The target CTL prepared by the method is high in purity, multiplication capacity, killing activity and CTL-CM ratio and is used in immunotherapy for tumors and the like.

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-Her2-neu 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]...

AI Technical Summary

Problems solved by technology

[0022] 2. Differences in CTL phenotype and function

[0040] ②TIL separation, the acquisition and identification methods of CTL clones are complicated, and there are many and complicated cell components in the tumor tissue, and the time to separate cells is long, usually more than 1 month;

[0041] ③TILs are mixed with CD4+CD25+Treg subsets, which can inhibit the expansion efficiency of CTLs;

[0042] ④ Due to the long time of in vitro separation and amplification, the chance of contamination is increased

[0044] ① The introduction of exogenous plasmids (retroviruses or lentiviruses, etc.) brings certain risks to clinical application;

[0045] 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

[0048] 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

[0049] At present, no literature has reported the preparation method of HLA-A0201-restricted antigen-specific CTL with short preparation time, high amplification factor, high CTL purity and good killing effect

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