Классические и активирующие химерные антигенные рецепторы PD-1 как элемент мультитаргетного подхода в лечении гематологических и солидных новообразований

Abstract

Aim. To generate anti-PD-L1 CAR-T effectors carrying extracellular domain PD-1 as antigen-recognizing site and to study their cytolytic activity as well as to functionally assess the anti-PD-L1 CAR-T effectors in vitro with a view to apply them in multi-targeted tumor therapy. Materials & Methods. Chimeric antigen receptor PD-1 was constructed using molecular cloning of PD-1 antigen-recognizing region (12–170 amino acids) into mammalian expression plasmid vector adding activation and co-stimulatory domains. Primary Т-lymphocytes of healthy donor peripheral blood mononuclear fraction were derived by expanding monoclonal antibody combination on surface markers CD3/CD28. Anti-PD-L1 CAR-T effectors were obtained by lentiviral transduction of primary T-lymphocyte genome of a healthy donor. Chimeric antigen receptor PD-1 expression and transduction efficiency were assessed by flow cytofluorometry. Specific cytotoxicity of the anti-PD-L1 CAR-T effectors was analyzed in vitro by means of real-time cytotoxicity assay (RTCA) with HeLa_PD-L1 target cell line co-cultivation. The level of cytokines IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A was assessed by flow cytofluorometry using Human Th1/Th2/Th17 CBA Kit (BD, USA). Results. The efficiency of lentiviral transduction and the proportion of the anti-PD-L1 CAR-T effectors were 42 %. The specificity of cytotoxic response of the anti-PD-L1 CAR-T effectors with a low effector/tumor ratio (1:20) was verified during HeLa_PD-L1 co-cultivation by a 1.5-fold decrease in the cell index (CI = 0.738) versus control (CI = 1.0645). The increase in synthesis of cytokines IL-2 (1000 pg/mL), IL-6 (438.5 pg/mL), TNF-α (44 pg/mL), and IFN-γ (1034 pg/mL) during HeLa_PD-L1 target cell line co-cultivation confirms the functionality of the analyzed effector cells. Conclusion. Anti-PD-L1 chimeric antigen receptor was constructed and tested in vitro. Anti-PD-L1 CAR-T lymphocytes specifically recognize and promote the cytolysis of tumor target cells by increased secretion of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6, and IL-2. Chimeric antigen receptor PD-1 can be modified into chimeric switch receptor (CSR) by deleting CD3ζ-domain and can be used together with other CARs without predicted non-specific toxicity.