Preclinical characterization of novel peptide binders for EphA2-targeted radiopharmaceutical therapy

EphA2 is a transmembrane glycoprotein, primarily involved in tissue patterning during embryonic development, and its expression levels are low or absent in normal adult tissues. EphA2 overexpression has been observed in multiple cancers, and is often associated with poor prognosis. The broad overexpression in solid tumors and relatively low expression in normal adult tissues make EphA2 attractive for targeted radiopharmaceutical therapy. RAYZ-6114 is comprised of a peptide binder to EphA2, a linker, and DOTA chelator that can be complexed with different isotopes. RAYZ-6283 differs from RAYZ-6114 only in the linker. The binding affinity, selectivity and cross-species reactivity to EphA2 and other Ephrin proteins were determined by SPR. Internalization was measured using flow cytometry. In vivo biodistribution and anti-tumor efficacy studies were performed in nude mice. For tumor type identification, EphA2 IHC was performed on tumor microarrays representing diverse tumor types. IHC analyses confirmed EphA2 expression in a multitude of tumors, with the highest positivity rates in cervical, pancreatic, bladder, colorectal, esophageal and lung cancers. RAYZ-6114 showed potent binding to human EphA2 (KD=0.03 nM), which was conserved across mouse and cynomolgus monkey EphA2. No binding to other Ephrin type-A or Ephrin type-B proteins was detected. The binder internalized in EphA2+ H1299 cells, 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. 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.