Development and preclinical characterization of a novel radiotheranostic EphA2-targeting bicyclic peptide

Erythropoietin-producing hepatocellular receptor A2 (EphA2), is a receptor tyrosine kinase involved in cell-cell interactions. It is known to be overexpressed in various tumors and is associated with poor prognosis. EphA2 has been proposed as a target for theranostic applications. Low molecular weight peptide-based scaffolds with low nanomolar affinities have been shown to be ideal in such applications. Bicyclic peptides have emerged as an alternative to traditional peptides for this purpose, offering affinities comparable to antibodies due to their constrained nature, along with high tissue penetration, and improved stability compared to linear counterparts. This study presents the development and comprehensive <i>in vitro</i> and <i>in vivo</i> preclinical evaluation of BCY18469, a novel EphA2-targeting bicyclic peptide-based radiotheranostic agent. <b>Methods:</b> The EphA2-targeting Bicycle^® peptide BCY18469 was identified through phage-display and chemically optimized. BCY18469 was radiolabeled with ⁶⁸Ga, ¹⁷⁷Lu and ¹¹¹In. The physicochemical properties, binding affinity and internalization as well as specificity of the peptide were evaluated <i>in vitro</i>. <i>In vivo</i> PET/MR and SPECT/CT imaging studies were performed using [⁶⁸Ga]Ga-BCY18469 and [¹¹¹In]In-BCY18469, respectively, along with biodistribution of [¹⁷⁷Lu]Lu-BCY18469 up to 24 h post injection in HT1080- and PC-3-tumor bearing BALB/c nu/nu EphA2-overexpressing xenograft mouse models. <b>Results:</b> The EphA2-targeting bicyclic peptide BCY18469 showed high binding affinity toward human and mouse EphA2 (1.9 and 3.8 nM, respectively). BCY18469 specifically bound and internalized into EphA2-expressing HT1080 cells. Imaging studies showed high tumor enrichment at early time-points (SUV of 1.7 g/mL at 1 h p.i. and 1.2 g/mL at 2 h p.i. in PET/MRI, HT1080 xenograft) with tumor contrast as early as 5 min p.i. and kidney-mediated clearance. Biodistribution studies revealed high early tumor uptake (19.5 ± 3.5 %ID/g at 1 h p.i., HT1080 xenograft) with SPECT/CT imaging further confirming these findings (5.7 ± 1.5 %ID/g at 1 h p.i., PC-3 xenograft). <b>Conclusion:</b> BCY18469 demonstrated high affinity, specific targeting of EphA2, a favorable biodistribution profile, and clearance through renal pathways. These findings underscore the potentially important role of bicyclic peptides in advancing radiotheranostic approaches and encourage additional translational research.