Abstract 6025: Discovery of novel macrocyclic peptide radioligands for tumor therapy by mRNA display

Abstract Radioligand therapeutics (RLT) aim to selectively deliver radioisotopes to cancer tissues to eradicate tumor cells while limiting the damage to surrounding tissues. They are composed of a tumor-targeting ligand and a radioactive payload. The therapeutic potential of RLT strongly depends on their biostability and residence time in tumor and healthy tissue after injection. Macrocyclic peptides are effective tumor-targeting ligands because their size and molecular characteristics confer optimal pharmacologic properties that can be fine-tuned during the development process. mRNA display is a powerful platform for macrocyclic peptide drug discovery that identifies high affinity peptide ligands through in vitro selection. Here, we describe the selection and initial characterization of macrocyclic peptides discovered by mRNA display that bind to the extracellular domain of a tumor target. Enrichment of the library for peptides that bound to the recombinant target protein was observed after four rounds of selection with no further enrichment in round five. The library of round 5 was sequenced by Illumina sequencing and the top 1, 000 sequences (highest counts) were used to perform cluster analysis. We tested the top sequences of 43 different clusters in in vitro translation binding assays to validate the selection output and identify sequences for hit confirmation. Most of the sequences tested showed clear binding to the target protein and only a few peptides showed binding to a closely related protein from the same protein family. No non-specific binding of the discovered peptides to an irrelevant protein was observed showing the high specificity of the generated library. From this confirmatory selection data, we chose a subset of peptides for chemical synthesis. Binding affinity measurements were determined by surface plasmon resonance (SPR) and cellular binding of selected candidates to target-positive and -negative tumor cells was analyzed by flow cytometry. From this hit confirmation data, in vitro internalization and in vitro anti-tumor efficacy of radiolabeled peptide candidates for RLT are assessed. Our data highlights the potential of mRNA display platforms for fast and efficient discovery of highly specific macrocyclic peptides with optimal binding properties suitable for RLT. Citation Format: Anika Jaekel, Kathryn Sherry, Prachi Desai, David Fox, Tang Guo-Qing, Manuel Sturzbecher-Hoehne, Dennis Mewis, Kaori Noridomi, Liam Rayman, Ping Ye. Discovery of novel macrocyclic peptide radioligands for tumor therapy by mRNA display abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 6025.