Bent to Bind: Exploiting the Programmed Cell Death-1 (PD-1) Receptor Plasticity to Design Pembrolizumab H3 Loop Mimics

Checkpoint blockade of the Programmed cell Death-1 (PD-1) immunoreceptor with its ligand 1 (PD-L1) by the monoclonal antibody pembrolizumab provided compelling clinical results among various cancer types, yet the molecular mechanism by which this drug blocks the PD-1:PD-L1 binding interface and reactivates exhausted T cells remains unclear. To address this question, we examined the conformational motion of PD-1 associated with the binding of pembrolizumab. The largely overlooked innate plasticity of both PD-1 C’D and FG loops appears crucial to closing in the receptor edges on the drug. Herein, we describe how PD-1 bends to initiate the formation of a deep binding groove (371 ų) across several epitopes while engaging pembrolizumab. Our analysis ultimately provided a rational design for mimicking the pembrolizumab H3 loop [RDYRFDMGFD] as a PD-1 inhibitor. A series of H3 loop mimics were synthesized and their folding characterized by CD and NMR spectroscopy. As a result, a first-in-class b-hairpin peptide inhibitor of the PD-1/PD-L1 interface was identified (IC₅₀ of 0.6 ± 0.2 μM). Overall, this study demonstrates that the dynamic groove formed between the C’D and FG loops of PD-1 is an attractive target for the development of peptide-based PD-1 inhibitors.