Preparation method of HLA-A0201 restriction AFP antigen specific CTL

Abstract

The invention belongs to the field of biotechnology development and application and discloses a preparation method of HLA-A0201 restriction AFP antigen specific CTL. The method is characterized in that peripheral blood mononuclear cells are collected through hemapheresis, and CD8+T lymphocytes are enriched and purified; mature dendritic cells loaded with HLA-A0201 restriction AFP antigen polypeptides are used to stimulate the CD8+T lymphocytes, and rhIL-2 and rhIL-7 are combined to promote the growth of the CD8+T lymphocytes; a Tetramer marking method and a fluorescence-activated cell sorting method are used to purify the target CTL; a solid-phase-coated anti-CD3 antibody and IL-2 are used to stimulate the growth of the target CTL; autologous PBMC irradiated by gamma rays is added to enhance the activation of the target CTL; rhIL-15 is added for culture amplification, and collection and identification are performed. The target CTL prepared by the method is high in purity, high in proliferation ability, high in killing activity, high in CTL-CM proportion, and applicable to immunological treatment of 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-AFP 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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