Application of a newly-developed cynomolgus macaque BiTE-mediated cytotoxic T-lymphocyte activity assay to various immunomodulatory agents in vitro

Abstract

The immunotoxic potential of drug candidates is assessed through the examination of results from a variety of in vitro and in vivo immunophenotyping and functional study endpoints in pre-clinical studies. CD8+ cytotoxic T-lymphocyte (CTL) activity impairment by immunosuppressive agents is recognized to be a potentiating factor for decreased antiviral defense and increased cancer risk. A bi-specific T-cell engager (BiTE®)-mediated CTL activity assay that applies to ex vivo experimentation in non-human primates in the context of toxicology studies was successfully developed and applied in cynomolgus monkey regulatory studies. While an ex vivo analysis conducted in the context of repeat-dose toxicology studies focuses on the long-term impact on CTL function, an in vitro assay with the same experimental design captures acute effects in the presence of the test article. Here, the in vitro assay was applied to a list of drugs with known clinical immunomodulatory impact to understand the applicability of the assay. The results showed this assay was sensitive to a wide range of immunosuppressants directly targeting cell-intrinsic signaling pathways in activated CTL. However, agents executing immuno-modulation through inhibiting cytokines/cytokine receptors, co-stimulatory molecules, and cell adhesion and migration pathways did not impair the CTL activity in this short-term in vitro culture. In addition, anti-PD-1/PD-L1 immune checkpoint blockers enhanced the CTL activity. Taken together, the results here demonstrate that in concordance with their mechanism of action, the in vitro BiTE®-mediated CTL assay is applicable and sensitive to immunomodulatory agents acting via a variety of mechanisms.

Keywords

• bite
• immunomodulation; in vitro
• ctl activity
• cynomolgus