Structural Optimization and Radiolabeling of EphA2-Targeted Bicyclic Peptides for Tumor Theranostics

Ephrin type-A receptor 2 (EphA2) has emerged as a promising theranostic target due to its prevalent overexpression in aggressive malignancies. In this study, we systematically optimized the BCY6088 bicyclic peptide scaffold through cyclization refinement and strategic amino acid substitutions that developed the high-affinity ligand Pep-34 exhibiting single-digit nanomolar binding affinity (K_D = 3.6 nM). The use of Pep-34 scaffold allowed the development of three structurally different DOTA-conjugated radiotracers: ⁶⁸Ga-Pep-36 (direct conjugation), ⁶⁸Ga-Pep-37 (PEGylated linker), and ⁶⁸Ga-Pep-38 (polysarcosine-Ala-Arg-Asp linker). Comprehensive evaluation identified ⁶⁸Ga-Pep-38 as the optimal diagnostic agent, demonstrating good serum stability and tumor targeting efficiency (SUV = 1.8 at 60 min postinjection in HeLa-EphA2^OE xenografts). Therapeutic translation using ¹⁷⁷Lu-Pep-38 resulted in a dose-dependent tumor growth inhibition (single dose: 83.6%; dual dose: 98.5%) while maintaining safety without detectable toxicity. Thus, Pep-38 is a promising bicyclic peptide that capable of precision molecular imaging and targeted radionuclide therapy for EphA2-overexpressed malignancies.