Novel, Stable, and Well-Secreted Single-Chain Fc Forms of Programmed Death-1 Immune Checkpoint Inhibitor Efficiently Block PD-1/PD-L1 Interaction

Immune checkpoint inhibitors (ICI), such as antibodies against Programmed Death 1 (PD-1), are widely used to treat different types of cancer. Unfortunately, only a small subset of patients benefits from such treatments, and the systemic administration of ICIs can cause severe immune-related adverse events. To overcome these hurdles, we performed an <i>in vitro</i> proof-of-concept investigation of recombinant adenoviral vectors encoding different variants of secreted PD-1 (sPD-1), differing in size, cysteine mutations, oligomerization, and effector functions. First, eight sPD-1 variants were screened, followed by the generation of recombinant adenoviruses with four of the highest-performing sPD-1 constructs based on qualitative analysis of sPD-1 secretion and ligand binding. The secretion of sPD-1 was analyzed using immunoblotting, while binding to PD-L1 and PD-L2 ligands was assessed using a pull-down assay and ELISA. In addition, the inhibition of PD-1:PD-L1 interaction was studied using cell-based signaling bioassays. It was demonstrated that the Fc-fusion sPD-1 variants resulted in the highest sPD-1 yields and were the most efficient in ligand binding. In particular, the single-chain Fc-fusion sPD-1 constructs were the most potent variants, as they effectively blocked PD-1-mediated signaling in T cells in two different coculture assays. The results of this <i>in vitro</i> proof-of-concept study indicate that stable and well-secreted sPD-1 constructs have significant potential for site-specific immune gene therapy.