Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging

Significance Programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) are key targets in the treatment of cancer, but current antibody-based drugs against this pathway have inherent drawbacks that may limit their effectiveness. We used directed evolution with yeast display to engineer a nonantibody biologic based on the ectodomain of PD-1. High-affinity PD-1 was more effective than anti–PD-L1 antibodies in the treatment of mouse tumor models and could additionally be used as a PET imaging tracer to noninvasively assess the PD-L1 expression status of tumors. This engineered protein thus represents an agent useful for clinical translation and highlights the paradigm of small protein biologics for future drug development. Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1–directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti–PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm3) and large tumors (150 mm3), whereas the activity of anti–PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1–positive and PD-L1–negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.