Abstract

Immune checkpoint inhibitors targeting the PD-1/PD-L1 pathway have improved for a number of solid tumors. Unfortunately, ovarian cancer represents a major clinical hurdle for immune checkpoint blockade (ICB) with reported low patient response rates. Using IHC staining, we find that PD-L2 is highly expressed in ovarian cancers and other malignancies with sub-optimal response to ICB, and is expressed at low levels in cancers responsive to ICB. Based on this observation, we hypothesized that the elevated expression of PD-L2 produced by both tumor and surrounding stromal cells contributes to immune-suppression. Since PD-L2 has been reported to have a 6- to10-fold higher native binding affinity to PD-1 compared with PD-L1, we hypothesized that high levels of PD-L2 can lead to insufficient blockade of the PD-1 signaling pathway. To overcome the immune repressive activity of PD-L2, we engineered a soluble PD-1 decoy molecule (sPD-1 mutant) that binds and neutralizes both PD-L1 and PD-L2 with a 10,000- and 200- fold improvement in binding affinity, respectively, when compared to wild-type binding to these same molecules. Such enhancement in binding affinity is facilitated by amino acid mutations both within and outside of the binding interface. Furthermore, this high affinity sPD-1 mutant molecule demonstrates superior in vivo efficacy in multiple cancer models including ovarian cancer where PD-L2 is highly expressed on the cell surface. One Sentence SummaryDual Inhibition of PD-L1 and PD-L2 using an affinity enhanced sPD-1 decoy molecule delivers superior antitumor activity when compared with PD-1 and PD-L1 antibodies in ovarian cancer.