The development of in vitro potency assay for immune checkpoint regulators

Abstract

Background & Aim Cancer immunotherapy by aiding the immune system in recognition and destruction of tumor cells by enhancing its ability to react to tumor antigens is now been demonstrated to be effective in many instances. Genomic modifications of multiple gene loci to generate potent effector T cells resistant to multiple inhibitory pathways such as PD-1, CTLA-4 is one of major attractive strategies for cell-based immunotherapy. Both PD-1 and CTLA-4 are type I transmembrane T cell inhibitory molecules that express or being recruited on activated T cells. They interact with their ligands, such as PD-L1 and B7-1 respectively to induce co-inhibitory signals on T cells and suppress T-cell mediated immune response against tumors. Thus, the PD-1: PD-L1/PD-L2 and CTL-4: B7-1/B7-2 interactions are key regulators of the threshold of immune response and a consistent, quantitative in vitro potency assay would benefit the development of immune checkpoint-mediated cancer immunotherapy. Methods, Results & Conclusion Recombinant PD-1, CTLA-4 and their specific ligands were expressed in either HEK293 or CHO cells and purified. Purified molecules' biological activities were initially examined and confirmed using T cell-based proliferation assay where applicable. In this study, we would demonstrate how we used Octet instrument to determine the affinities and kinetics of recombinant PD-1 and CTLA-4 Fc chimera molecules’ bindings to their correspondent ligands, and further identified quantitatively different affinities and kinetics of PD-1 and CTLA-4 when bound to their different ligands. Our results indicate the Octet-based assay can serve as a rapid, label-free, quantitatively suitable in vitro potency analysis with very minimal interference for qualifying and studying immune checkpoint regulators.