Receptor tyrosine kinase-like orphan receptor 1 (RORl) is a cell surface protein that mediates signaling upon binding of its ligand, Wnt5a. High levels of RORl expression are observed in invasive malignancies such as hematological and solid tumors, but not in normal adult tissues, offering an attractive target for tumor-specific therapy. In this presentation, we will describe the discovery of macrocyclic peptides as binders of the extracellular Cysteine-Rich Domain (CRD) of human ROR1, and <i>in vitro </i>methods used to characterize binding and cellular activities.  The initial hits, comprising three unique macrocyclic series, were identified through mRNA display, a powerful <i>in vitro</i> selection technique.  Individual peptides were synthesized off-RNA and evaluated by a combination of biophysical methods using recombinant proteins, and cell-based assays.  Positional scanning of key residues led to optimization of the preferred peptide series, with representatives showing single digit nM affinity in a cell-based assay.  Both receptor internalization and apoptosis experiments performed with ROR1-positive tumor lines will be discussed for key exemplars of the peptide series, including fluorescently labeled analogs, and a representative Peptide Drug Conjugate (PDC).

Macrocyclic peptides bind to the extracellular CRD domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1)

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