Genetically-encoded discovery and development of peptide-macrocycle imaging agents for PD-L1

The unique cell surface composition of tumor cells forms the molecular basis for many targeting and cell-based therapies. Here, we describe the development of novel peptide-based targeting agents for programmed death ligand 1 (PD-L1). Molecular imaging by peptide agents, coupled with therapeutic intervention using the same modality, represents a critical advancement in cancer management. Whole-body PET imaging of PD-L1 expression offers a superior alternative to traditional immuno-histochemistry, making PD-L1 radiodiagnostic imaging a highly sought-after modality. PD-L1 targeting modalities developed for clinical imaging to date can be divided into antibodies, protein domains, and small macrocyclic peptides with fewer than 20 amino acids. The latter modalities can address many challenges seen in antibody-based targeting vectors. All potent PD-L1 targeting peptide modalities reported to date rely extensively on non-canonical amino acids (ncAAs). Here, we report a comprehensive structure-activity relationship (SAR) analysis of a family of macrocycles discovered from an Sx2Cx8Cx2 phage-display library composed entirely of natural amino acids (x represents 19 natural amino acids excluding Cys). Using >10,000 variants in ''focused'' phage-display libraries, we optimized these macrocycles to achieve single-digit-nanomolar potency in protein- and cell-based assays. En route to this optimization, the activity of 216 synthetic macrocycles towards PD-L1 was measured in five distinct assays; two leads have been evaluated by imaging in tumor xenografts in mice, and the X-ray structure of one advanced lead in complex with PD-L1 has been determined at 2.78 [A] resolution. This publication demonstrates the development potential of PD-L1-targeting macrocycles that do not require extensive incorporation of ncAAs and the democratization of discovery by mapping the optimization path to single-digit-nanomolar assets for targeted radiopharmaceuticals via canonical phage-display technology.