Immunomodulatory mAbs as Tools to Investigate on Cis-Interaction of PD-1/PD-L1 on Tumor Cells and to Set Up Methods for Early Screening of Safe and Potent Combinatorial Treatments

SIMPLE SUMMARY: A novel challenge in cancer immunotherapy is the identification of the most potent combinations of immunomodulatory mAbs that are capable of maximizing therapeutic benefits while minimizing irAEs. We set up an in vitro system to quickly predict the efficacy and eventual cardiotoxic side effects of combinatorial treatments, thus allowing for the early screening of most potent and safe combinatorial therapeutic regimens for both validated and emerging immunomodulatory mAbs against different immune checkpoints (ICs). Furthermore, we provide for the first time evidence on cis-interactions of ICs in tumor cells. ABSTRACT: Antibodies targeting Immune Checkpoints (IC) on tumor infiltrating lymphocytes improve immune responses against cancer. Recently, the expression of some ICs has also been reported on cancer cells. We used the clinically validated Ipilimumab and Nivolumab and other novel human antibodies targeting Cytotoxic T- lymphocyte-antigen 4 (CTLA-4), Programmed Death receptor-1 (PD-1) and Programmed Death Ligand 1 (PD-L1) to shed light on the functions of these ICs in cancer cells. We show here for the first time that all these antagonistic mAbs are able to reduce Erk phosphorylation and, unexpectedly, to induce a significant increase of ICs expression on tumor cells, involving a hyperphosphorylation of NF-kB. On the contrary, agonistic PD-L1 and PD-1 recombinant proteins showed opposite effects by leading to a significant reduction of PD-1 and PD-L1, thus also suggesting the existence of a crosstalk in tumor cells between multiple ICs. Since the immunomodulatory mAbs show their higher anti-tumor efficacy by activating lymphocytes against cancer cells, we also investigated whether it was possible to identify the most efficient combinations of immunomodulatory mAbs for achieving potent anti-tumor efficacy associated with the lowest adverse side effects by setting up novel simple and predictive in vitro models based on co-cultures of tumor cells or human fetal cardiomyocytes with lymphocytes. We demonstrate here that novel combinations of immunomodulatory mAbs with more potent anti-cancer activity than Ipilimumab and Nivolumab combination can be identified with no or lower cardiotoxic side effects. Thus, we propose these co-cultures-based assays as useful tools to test also other combinatorial treatments of emerging immunomodulatory mAbs against different ICs for the early screening of most potent and safe combinatorial therapeutic regimens.