Production of CD87 Antigen-Specific CAR-T Lymphocytes and Assessment of Their In Vitro Functional Activity

Abstract

Aim. To generate anti-CD87 CAR-T lymphocytes and to assess their in vitro functional activity. Materials & Methods. Т-lymphocytes isolated from healthy donor peripheral blood were transduced with the anti-CD87-CAR, T2A, and FusionRed gene coding lentiviral vector. Transduction efficacy assessed by reporter protein FusionRed signal, subpopulation structure, and functional status of CAR-T lymphocytes were determined by flow cytometry. Interferon-y (IFN-y) expression by CAR-T lymphocytes was analyzed using immunoassay. Cytotoxic activity of CAR-T lymphocytes was evaluated during their co-cultivation with HeLa target cells by means of xCELLigence real-time assay. Results. The efficacy of T-lymphocyte transduction was 8.4 %. The obtained CAR-T cells contained the markers of both CD27 and/or CD28 activation (92.91 % cases) and PD1 exhaustion (20.66 % cases). The population of CAR-T lymphocytes showed 98.51 % central memory T-cell phenotype and CD4/CD8 ratio of 1:7. IFN-y concentration in the medium after co-cultivation of CAR-T lymphocytes with target cells appeared to be significantly higher than in control samples. The study demonstrates that generated CAR-T lymphocytes manifest specific cytotoxicity towards target cells with both unmodified expression and overexpression of CD87 antigen in HeLa cell lines. Cytotoxicity proved to be more pronounced with respect to the cell line with CD87 antigen overexpression. Conclusion. Despite overexpression of PD1 exhaustion marker, CAR-T lymphocytes showed specific IFN-y secretion and pronounced cytotoxic activity in interaction with CD87 antigen on target cell membranes. Therefore, anti-CD87 CAR-T lymphocytes can be applied in the treatment of hematologic as well as solid tumors. Since the observed difference in cytotoxicity does not linearly correlate with CD87 antigen density on the surface of attacked cells, the in vivo administration of a CAR-T cell drug should be designed to prevent cytotoxic risk for CD87-expressing healthy cells.