Integrating PD-1 Blockade and OX40 Agonism: A Bispecific Fusion Protein for Comparative Cancer Immunotherapy

Abstract

Canine cancers represent a major clinical challenge, often recapitulating the immunosuppressive mechanisms observed in human oncology but lacking effective immunotherapeutic strategies. To address this gap, we developed a novel bispecific fusion protein, aPD1-Fc-OX40L, designed to simultaneously block PD-1/PD-L1 interactions and activate OX40 signaling, thereby reprogramming antitumor T-cell responses. Both murinized and caninized versions of the fusion protein were engineered, expressed, and purified with high structural integrity. In vitro binding assays confirmed high affinity and specificity for PD-1 and OX40 receptors, with minimal Fcγ receptor engagement to limit unintended effector functions. Functional studies demonstrated effective abrogation of PD-L1 binding to PD-1 and potent enhancement of OX40 signaling through PD-1 crosslinking. Ex vivo assays further revealed significant enhancement of canine T-cell proliferation, underscoring the molecule’s immune-activating potential In vivo, the murine construct (Mu-aPD1-Fc-OX40L) delayed tumor progression in the B16-F0 melanoma model, establishing proof-of-concept antitumor efficacy. The canine construct (K9-aPD1-Fc-OX40L) exhibited prolonged systemic persistence for up to 28 days in healthy beagle dogs and was well tolerated, with no evidence of sustained hepatic or renal toxicity or immune-related adverse events. To our knowledge, this work represents the first comprehensive development and characterization of a bispecific aPD-1/OX40 fusion protein specifically tailored for canine oncology. These findings highlight the therapeutic potential of targeting inhibitory and costimulatory immune pathways in parallel and illustrate the value of comparative oncology as a bridge toward translational cancer immunotherapy.