A method for producing a human T-cell receptor specific for the epitope 100-108 (VLDGLDVLL) PRAME

Claims

A method for producing a human T-cell receptor specific to the epitope 100-108 (VLDGLDVLL) derived from the PRAME protein, comprising isolating peripheral blood mononuclear cells (MNCs) of HLA-A0201+ donors, then culturing the MNCs in the presence of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 4 days; on the 5th day, adding the epitope (100-108) VLDGLDVLL as a priming factor to the dendritic cell culture, followed by incubation at 37°C; on the 6th day, adding a maturation factor to complete the maturation of dendritic cells; then the obtained dendritic cells are collected, washed with a nutrient medium, the number of dendritic cells is counted, their viability is assessed and they are co-cultured with autologous CD8+ T lymphocytes in a certain ratio, and after 7 days of co-cultivation, antigen-specific CD8+ T cells are isolated,then, the TCR gene sequence is identified by sequencing the variable region of the TCRα and TCRβ chains, including CDR3, isolated from T cell clones, wherein the TCRα and TCRβ chains are connected by a peptide linker 2A (TCRb-P2A-TCRa), and the complete TCR construct is cloned into the backbone of a viral plasmid to obtain TCR complexes, characterized in that the donors are conditionally healthy individuals, from whose blood monocytes are isolated on a Ficoll gradient, on the 5th day, the epitope (100-108) VLDGLDVLL is added as a priming factor at a dose of 100 μg / ml and subsequent incubation for 24 hours, on the 6th day, TNF-α is added as a maturation factor at a concentration of 25 ng / ml for 24 hours, and co-cultivation of dendritic cells carried out with CD8+ T lymphocytes in the presence of 0.5 μg / ml anti-CD3, 1 μg / ml anti-CD28 and IL-2, IL-7, IL-15 (10 ng / ml each) in a ratio of 1:10, after 7 days of co-cultivation, antigen-specific CD8+ T cells are isolated using Flex-T reagents,then, the TCR gene sequence is identified using single-cell RNA sequencing (scRNA-seq) and a TCR clone specific to the epitope (100-108) VLDGLDVLL of the PRAME protein is selected using the ERGO-II and NetMHCpan-4.1 neural networks of the ClonoSort program, and in addition to connecting the TCRα and TCRβ chains with a peptide linker 2A (TCRb-P2A-TCRa), the untranslated region of β-globulin at the 5' and 3' ends of the insert is used, a lentivirus based on HIV-1 (pLentih_PGK) is used as a viral plasmid, one dominant TCR with one α- and one β-chain originating from the same cell is obtained.