Preparation method for HLA-A0201 limited antigen specificity CTL (cytotoxic T lymphocyte)

Abstract

The invention belongs to the field of biotechnology development and application research, and discloses a preparation method for an HLA-A0201 limited antigen specificity CTL (cytotoxic T lymphocyte). The method comprises the following steps: collecting eripheral mononuclear cells one by one, so as to enrich and purify a CD8+T lymphocyte; stimulating the CD8+T lymphocyte with a mature dendritic cell bearing an HLA-A0201 limited target antigen polypeptide, and promoting the growth of the T lymphocyte through the combination of rhIL-2 and rhIL-7; purifying the target CTL according to a Tetramer marking method and a flow cell sorting method; stimulating the growth of the target CTL according to a solid phase coated anti-human-CD3mAb and IL-2; adding an autologous PBMC (peripheral blood mononuclear cell) to enhance the actification of the target CTL; and adding rhIL-15 for enlarging cultivation, collecting and identifying. The CTL prepared according to the method has the advantage that the purity, the proliferation capability, the cytotoxicity and the CTL-CM proportion are high, so as to be used for immunological therapy 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 antigen-specific cytotoxic T lymphocytes (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 imminen...

AI Technical Summary

Problems solved by technology

[0016] 2. Differences in CTL phenotype and function

[0034] 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;

[0035] ③TILs are mixed with CD4+CD25+Treg subsets, which can inhibit the expansion efficiency of CTLs;

[0036] ④ Due to the long time of in vitro separation and amplification, the chance of contamination is increased

[0038] ① The introduction of exogenous plasmids (retroviruses or lentiviruses, etc.) brings certain risks to clinical application;

[0039] 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

[0042] 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

[0043] 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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