Abstract 33P
Background
Immune checkpoint inhibitors (ICIs) are novel drugs with profound anti-cancer activity but also with adverse events. We investigated the cardiotoxic effects of ipilimumab (IPI, anti-CTLA-4), pembrolizumab (PEM, anti-PD-1) and avelumab (AVE, anti-PD-L1), in primary murine ventricular cardiomyocytes (mAVCs) and spleenocytes, which mainly consist of T- and B- lymphocytes.
Methods
C57BL6/J mice were sacrificed and primary mAVCs and spleenocytes were isolated. Primary cells were incubated with IPI, PEM and AVE at a concentration range of 0-100 μg/ml for 24h and cell viability was assessed by MTT assay. Furthermore, spleenocytes were treated with IPI, PEM and AVE for 24h and their conditioned medium was added onto the mAVCs for 24h. For the confirmation of PEM binding on the murine PD-1, human and murine PD-1 extracellular domains (ED) were produced and PEM binding was confirmed by circular dichroism (CD). Human IgG4 was used as a negative control.
Results
Only IPI led to cytotoxicity in primary spleenocytes and was therefore excluded from the study, as ICIs should not induce cytotoxicity in immune cells (IC). Incubation of mAVCs with PEM and AVE conditioned media, revealed that only PEM could induce IC-dependent cytotoxicity at 50 and 100 μg/ml. The latter was accompanied by an increase in Tnfa, Inos, Rela, Ifng mRNA expression in primary spleenocytes after PEM treatment and Tnfa, Tgfb, Inos, Rela, Ifng as well as autophagy markers Lc3b, Lc3a, Becn2, Atg5 and Endoplasmic Reticulum (ER) stress markers Canx and Ddit3 increase in the primary mAVCs after treatment with PEM conditioned medium. The latter were confirmed with Western Blot analysis. We then investigated whether PEM binds on the murine PD-1 or whether the observed effects are due to an off-target effect. CD experiments showed that PEM binds on the murine PD-1 ED in a similar manner as with the human PD-1 ED used as the positive control, which supports the use of the murine model for the extraction of translational results. The binding of PEM on the murine PD-1 ED was also confirmed by in silico protein-protein docking experiments.
Conclusions
PEM induces IC-dependent cytotoxicity on primary mAVCs via induction of inflammation, autophagy and ER-stress.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
National and Kapodistrian University of Athens.
Disclosure
All authors have declared no conflicts of interest.