Preparation method of HLA-A0201 restrictive anti-carcinoembryonic antigen (CEA) antigenic specificity cytotoxic T lymphocyte (CTL)

Abstract

The invention discloses a preparation method of an HLA-A0201 restrictive anti-CEA antigenic specificity CTL. The preparation method includes collecting peripheral blood mononuclear cells (PBMCs) through hemapheresis, and enriching and purifying CD8+T lymphocytes; stimulating the CD8+T lymphocytes by utilizing mature dendritic cells loaded with the HLA-A0201 restrictive CEA antigen polypeptides and promoting T cells to grow by using rhIL-2 and rhIL-7; purifying a target CTL by utilizing a tetramer marking method and a fluorescene-activated cell sorting; stimulating the target CTL to grow with an anti-human CD3 monoclonal antibody coated by solid phase and IL-2; adding autologous PBMCs after radiation of gamma rays to strengthen activation of the target CTL; and adding rhIL-15 for breeding and amplification, collecting and identifying. The prepared target CTL has high purity, multiplication capacity, killing activity and proportion of CTL-CM, and can be applied to immunotherapy such as tumors.

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-CEA 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...

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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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