Background: Ephrin type-A receptor 2 (EphA2) is a glycoprotein of the ephrin receptor subfamily. EphA2 is primarily involved in tissue patterning during embryonic development, and its expression levels are low or absent in normal adult tissues. However, EphA2 overexpression has been observed in multiple malignant tumors such as bladder, cervical, ovarian, colorectal, lung and esophageal cancers. In addition to being a tumor biomarker, EphA2 plays an active role in tumor survival, metastasis and neo-angiogenesis, which can lead to poor prognosis for cancer patients. The broad overexpression in solid tumors and relatively low expression in normal adult tissues make EphA2 attractive for targeted radiopharmaceutical therapy (RPT). Methods: RAYZ-6114 is comprised of a macrocyclic peptide binder to EphA2, a linker, and DOTA chelator which can be complexed with different radiometals. RAYZ-6283 shares the same peptide binder and chelator with RAYZ-6114 but differs in the linker. The binding affinity, selectivity and cross-species reactivity to EphA2 and other Ephrin proteins were determined by surface plasma resonance (SPR). Target-mediated internalization was measured using flow cytometry. In vivo biodistribution and anti-tumor efficacy studies were performed in tumor-bearing athymic nude mice. A coagulopathy study was performed in Sprague Dawley rats. For tumor type identification, EphA2 immunohistochemistry (IHC) was performed on tumor microarrays (TMA) representing diverse tumor types. Results: IHC analyses of TMAs confirmed the expression of EphA2 in a multitude of solid tumors, with the highest positivity rates in cervical, pancreatic, bladder, colorectal, esophageal, and non-small cell lung cancers. RAYZ-6114 showed high binding affinity to human EphA2 with a K_D of 0.03 nM. High-affinity binding was conserved across mouse, cynomolgus monkey, and human EphA2. No binding to other Ephrin type-A or Ephrin type-B proteins was detected, nor to EphA2-knockout cells. The binder rapidly and efficiently internalized in EphA2-positive H1299 cells upon target engagement, with ~75% internalized by 1 hour. In PC3 xenograft mice, ¹⁷⁷Lu-RAYZ-6283 showed sustained tumor uptake (~25% ID/g) for up to 48 hours and tumor/kidney ratios of 2.7, 3.3, and 5.9 at 24h, 48h, and day 7, respectively. Low uptake was seen in other normal tissues. Both ¹⁷⁷Lu- and ²²⁵Ac- labelled RAYZ-6114 significantly inhibited tumor growth. Particularly, durable tumor regression and survival benefit were achieved by a single dose of ²²⁵Ac-RAYZ-6114 (3 uCi), out-performing ¹⁷⁷Lu-RAYZ-6114 dosed at 3 mCi. All treatments were well tolerated. Conclusions: RAYZ-6114 and RAYZ-6283 are first-in-class, highly potent and selective macrocyclic peptide binders. Preclinical pharmacodynamic, pharmacokinetic, biodistribution and efficacy data demonstrated their potential for treatment of patients with EphA2-positive tumors.