Nanobody-based canine PD-L1-targeting immune checkpoint inhibitors for cancer therapy in dogs

Although the demand for novel immunotherapies to treat companion dogs with spontaneously developing cancer is increasing in high-income countries, most options remain inaccessible. Dogs host a complete and functional immune system reacting to the presence of their tumor. As for humans, many canine neoplasms were shown to overexpress programmed death-ligand 1 (PD-L1), an immune checkpoint inhibitor (ICI) known to downregulate cytotoxic T cell activity upon interaction with its ligand PD-1. In this study, we used alpaca-derived single domain antibodies (sdAbs), also known as nanobodies (Nbs), to develop new ICI targeting canine PD-L1. We selected several clones binding to both recombinant soluble and cell membrane-anchored cPD-L1 forms. Next, their cPD-L1-binding affinities, cPD-1/cPD-L1-blocking abilities and epitope relationships were determined. Most effective Nbs binding to non-overlapping epitopes were combined as biparatopic Fc fusions to provide additional functionalities and improve their efficacy. Remarkably, all engineered Nb constructs efficiently interfered with the cPD-1/cPD-L1-induced signaling pathway, with some multivalent molecules displaying inhibitory concentrations reaching low picomolar range. Moreover, Fc-competent Nb constructs were also shown to induce tumor cell death by antibody-dependent cell-mediated cytotoxicity using human or canine models. Finally, using donor canine peripheral blood mononuclear cells (PBMCs), best candidates were favorably compared with atezolizumab in a Staphylococcal enterotoxin B (SEB)-based interferon-γ (IFNγ) secretion assay.