Preparation method of HLA-A0201-restricted anti-HPV (human papillomavirus) antigen-specific CTL

Abstract

The invention discloses a preparation method of HLA-A0201-restricted anti-HPV (human papillomavirus) antigen-specific CTL. The preparation method comprises the following steps of: collecting peripheral blood mononuclear cells through apheresis, and enriching and purifying CD8+T lymphocytes; stimulating the CD8 + T lymphocytes by mature dendritic cells of a loaded HLA-A0201-restricted HPV antigen polypeptide; and promoting the T-cell growth through combined use of rhIL-2 and rhIL-7; purifying a target CTL by a Tetramer marking method and a flow cytometry sorting technique; stimulating the growth of the target CTL by solid phase packaged anti-human CD3 monoclonal antibody and IL-2; adding autologous PBMCs (peripheral blood mononuclear cell) after gamma-ray irradiation for enhancing activation of the target CTL; and adding rhIL-15, cultivating and amplifying, and collecting and identifying. The target CTL prepared by the prepared method has high purity, high proliferative capacity, high killing activity and a high CTL-CM ratio and can be applied to immunotherapy 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 preparation method of HLA-A0201-restricted anti-HPV antigen-specific CTL. Background technique [0002] 1. HPV infection and cervical cancer [0003] Cervical cancer is the third most common malignant tumor among women in the world. There are about 500,000 new cases of cervical cancer every year in the world, and nearly 274,000 patients die from the disease. A large number of studies have shown that the occurrence of cervical cancer is closely related to the persistent infection of high-risk human papillomavirus (human papillomavirus, HPV). More than 100 types of HPV-DNA have been isolated, more than 30 of which are related to cervical lesions. According to the size of its pathogenicity, it is divided into low-risk type and high-risk type, and the high-risk type mainly leads to the occurrence of cervical cancer, and HPV16 / 18 is more common, and ...

AI Technical Summary

Problems solved by technology

[0017] 2. Differences in CTL phenotype and function

[0035] 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;

[0036] ③TILs are mixed with CD4+CD25+Treg subsets, which can inhibit the expansion efficiency of CTLs;

[0037] ④ Due to the long time of in vitro separation and amplification, the chance of contamination is increased

[0039] ① The introduction of exogenous plasmids (retroviruses or lentiviruses, etc.) brings certain risks to clinical application;

[0040] 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

[0043] 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

[0044] 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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