Induction of expression of T-cell receptors against epitopes of melanoma-associated antigens, cloning and production of genetically modified TCR-T cells

• Yulia A. Shevchenko
• Julia A. Lopatnikova
• Marina S. Fisher
• Vasily V. Kurilin
• Julia G. Philippova
• Alaa Alsalloum
• Saleh Alrhmoun
• Olga Yu. Perik-Zavodskaia
• Roman Yu. Perik-Zavodskiy
• Kirill V. Nazarov
• Aleksey S. Bulygin
• Elena A. Golikova
• Polina A. Shangina
• Anastasia A. Gizbrecht
• Olga P. Vorobyeva
• Alexander N. Silkov
• Sergey V. Sennikov

Abstract

Introduction. The ability of immune cells to destroy tumor cells due to the recognition of tumor antigens by the T-cell receptor (TCR) and the transmission of activating signals inside the cell underlies modern immunotherapy of oncological diseases. The development of technology for isolating and cloning T-cell receptors and genetic engineering has made it possible to create patient T-cells encoding TCR for tumor antigens, capable of effectively destroying tumor cells.

Aim – production of genetically modified TCR-T cells aimed at recognizing epitopes of antigens associated with melanoma by induction of antigen-specific T cells and sequencing of single cell RNA.

Material and methods. Peripheral blood mononuclear cells from healthy donors with the HLA-A02 genotype were used to generate dendritic cells loaded with peptides to target antigens and subsequent clonal expansion of antigen-specific T cells. TCR sequence analysis was performed using single cell RNA sequencing. Bioinformatic analysis using different approaches allowed us to obtain TCR sequences for subsequent cloning and expression in a lentiviral vector. The obtained vectors were used for T cell transduction and evaluation of cytotoxic activity against melanoma cell lines with the expression of PRAME and MART-1 [SK-Mel-5 and SK-Mel-37, provided by Professor H. Shiku from the collection of the Graduate School of Medicine of the University of Mie (Japan)]. The transduced T cells were cultured with tumor cells in a 10 : 1 ratio, and the cytotoxic effect was assessed by the level of the enzyme lactate dehydrogenase, which is released from the lysed cells.

Results. As a result of clonal expansion, an almost 1000-fold enrichment of the initial population of antigen-specific cells was obtained. Cells with stable expansion of specific TCR and proliferative activity were used for transcriptome analysis on the BD Rhapsody platform. To determine the optimal strategy for selecting antigen-specific TCR, we used the ERGO-II neural network, which predicts the affinity of TCR to the target peptide of the PRAME antigen and determines the dominance of the clonotype for TCR recognizing MART-1. The transduction of T-lymphocytes by lentiviral constructs encoding the obtained TCR resulted in the lysis of at least 30 % of target cells on the first day of interaction with tumor cells.

Conclusion. We have achieved significant enrichment of the target antigen-specific cell population in quantities sufficient for single cell transcriptome analysis and T cell clonotypes. Using single cell RNA sequencing technology, we obtained detailed information about the sequence of each TCR and the immune transcriptome of a single T cell, which made it possible to accurately design the resulting TCR clones, assess the functional state of the cells, and improve the selection of TCR candidate clones. The obtained sequences for TCR were used to develop lentiviral constructs that have shown their effectiveness in the interaction of T cells transduced by these constructs with target cells.

Keywords: T-cell receptor (TCR); melanoma; transciptome; sequencing; affinity; clonotype

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