Development and preclinical evaluation of [¹¹¹In]In-DTPA-EphA2-57-1: a single-photon emission computed tomography...

Erythropoietin-producing hepatocellular receptor A2 (EphA2) is overexpressed in various malignant tumors and represents a promising target for cancer-specific imaging and therapy. However, many therapeutics (as well as imaging probes) targeting overexpressed EphA2 are limited by efficacy and safety concerns. We aimed to develop and preclinically evaluate a novel single-photon emission computed tomography (SPECT) probe, [¹¹¹In]In-DTPA-EphA2-57-1, based on a newly generated anti-EphA2 monoclonal antibody, EphA2-57-1. The antibody was conjugated with DTPA (p-SCN-Bn-DTPA) and radiolabeled with ¹¹¹In, achieving a radiochemical purity of 97.3%. In vitro, in EphA2-expressing cells (EphA2-positive U87MG glioblastoma cells), [¹¹¹In]In-DTPA-EphA2-57-1 demonstrated uptake and a high internalization fraction, reaching 74.9% at 24 h. Although DTPA conjugation reduced the apparent binding affinity compared with the native antibody, efficient receptor-mediated internalization contributed to sustained cellular retention. In vivo biodistribution studies in U87MG tumor-bearing mice revealed selective tumor accumulation (6.9-8.8%ID/g) and favorable tumor-to-blood and tumor-to-muscle ratios, which increased over time. Notably, the tumor-to-blood ratio reached 2.4 ± 0.8 and 3.4 ± 0.6 on Days 4 and 7, respectively, post-injection. Blocking with excess unlabeled antibody significantly reduced tumor uptake, confirming EphA2-specific targeting in vivo. SPECT enabled a clear and persistent visualization of EphA2-expressing tumors; however, a relatively high hepatic uptake was observed. [¹¹¹In]In-DTPA-EphA2-57-1 exhibited lower blood retention and higher tumor contrast at earlier time points than did the previously reported probe [¹¹¹In]In-BnDTPA-EphA2-230-1, reflecting improved imaging performance. These results indicate that [¹¹¹In]In-DTPA-EphA2-57-1 is a promising SPECT probe for the noninvasive assessment of EphA2 expression. Additionally, the findings may inform future EphA2-targeted theranostic applications.