Preparation method of HLA-A0201 (Human Leukocyte Antigen-A0201) restrictive anti-MAGE (MicroArray and Gene Expression) antigenic specificity CTL (Cytotoxic T Lymphocyte)

Abstract

The invention discloses a preparation method of an HLA-A0201 (Human Leukocyte Antigen-A0201) restrictive anti-MAGE (Micro Array and Gene Expression) antigenic specificity CTL (Cytotoxic T Lymphocyte). The method comprises the following steps of: carrying out hemapheresis to collect peripheral blood mononuclear cells; enriching and purifying CD8+T lymphocytes; utilizing a mature dendritic cell loaded with an HLA-A0201 restrictive anti-MAGE antigen polypeptide to stimulate the CD8+T lymphocytes; utilizing rhIL-2 and rhIL-7 to accelerate the T cells to grow; utilizing a Tetramer marking method and a flow type cell separation technology to purify the target CTL; utilizing an anti-human CD3 monoclonal antibody covered by a solid phase and an IL-2 to stimulate the target CTL to grow; adding an autologous PBMC (Peripheral Blood Mononuclear Cell) radiated by gamma rays to enhance the activation of the target CTL; adding rhIL-15 to cultivate and reproduce; and collecting and identifying. The prepared target CTL has high purity, high reproduction capability, high killing activity and high ratio CTL-CM, and can be used for immune 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 preparation method of HLA-A0201-restricted anti-MAGE 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....

AI Technical Summary

Problems solved by technology

[0019] 2. Differences in CTL phenotype and function

[0037] 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;

[0038] ③TILs are mixed with CD4+CD25+Treg subsets, which can inhibit the expansion efficiency of CTLs;

[0039] ④ Due to the long time of in vitro separation and amplification, the chance of contamination is increased

[0041] ① The introduction of exogenous plasmids (retroviruses or lentiviruses, etc.) brings certain risks to clinical application;

[0042] 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

[0045] 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

[0046] 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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