AbstractImmune checkpoint blockade of programmed death-1 (PD-1) by monoclonal antibody drugs has delivered breakthroughs in the treatment of cancer. Nonetheless, small-molecule PD-1 inhibitors could lead to increases in treatment efficacy, safety, and global access. While the ligand-binding surface of apo-PD-1 is relatively flat, it harbors a striking pocket in the murine PD-1/PD-L2 structure. An analogous pocket in human PD-1 may serve as a small-molecule drug target, but the structure of the human complex is unknown. Because the CC′ and FG loops in murine PD-1 adopt new conformations upon binding PD-L2, we hypothesized that mutations in these two loops could be coupled to pocket formation and alter PD-1’s affinity for PD-L2. Here, we conducted deep mutational scanning in these loops and used yeast surface display to select for enhanced PD-L2 binding. A PD-1 variant with three substitutions binds PD-L2 with an affinity two orders of magnitude higher than that of the wild-type protein, permitting crystallization of the complex. We determined the X-ray crystal structures of the human triple-mutant PD-1/PD-L2 complex and the apo triple-mutant PD-1 variant at 2.0 Å and 1.2 Å resolution, respectively. Binding of PD-L2 is accompanied by formation of a prominent pocket in human PD-1, as well as substantial conformational changes in the CC′ and FG loops. The structure of the apo triple-mutant PD-1 shows that the CC′ loop adopts the ligand-bound conformation, providing support for allostery between the loop and pocket. This human PD-1/PD-L2 structure provide critical insights for the design and discovery of small-molecule PD-1 inhibitors.Significance StatementImmune checkpoint blockade of programmed death-1 (PD-1) by monoclonal antibody drugs has transformed the treatment of cancer. Small-molecule PD-1 drugs have the potential to offer increased efficacy, safety, and global access. Despite substantial efforts such small-molecule drugs have been out of reach. We identify a prominent pocket on the ligand-binding surface of human PD-1 that appears to be an attractive small-molecule drug target. The pocket forms when PD-1 is bound to one of its ligands, PD-L2. Our high-resolution crystal structure of the human PD-1/PD-L2 complex facilitates virtual drug-screening efforts and opens additional avenues for the design and discovery of small-molecule PD-1 inhibitors. Our work provides a strategy that may enable discovery of small-molecule inhibitors of other “undruggable” protein-protein interactions.
Phase 1 Dose-Escalation Study of the Folic Acid-Tubulysin Small-Molecule Drug Conjugate Ec1456 in Patients (Pts) with Advanced Solid Tumors