[225Ac]Ac-Macropa-PEG4-HEA125 for Targeted α Therapy in Epithelial Cell Adhesion Molecule-Positive Tumors: Conjugation, Radiolabeling, and Efficacy.

Abstract

Introduction: Epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide range of epithelial malignancies, and thus is a potential target for antibody-based radiotherapy. This work describes the synthesis, labeling, and biological evaluation of an alpha-emitting radioconjugate, [²²⁵Ac]Ac-Macropa-PEG₄-HEA125, as a targeted alpha therapy candidate for EpCAM-positive tumors.

Materials and methods: The murine anti-EpCAM monoclonal antibody HEA125 was site-specifically conjugated to the chelator Macropa using a PEG₄-maleimide linker. The structural integrity and chelator-to-antibody (C/A) ratio of the conjugate were confirmed by SDS-PAGE and LC-MS. Radiolabeling with ²²⁵Ac was performed under mild conditions, and radiochemical purity was assessed using iTLC and radio-HPLC. In vitro studies included stability testing, immunoreactivity, and cytotoxicity assays using MCF-7 (EpCAM⁺) and CHO-K1 (EpCAM^-) cell lines. In vivo biodistribution and therapeutic efficacy were evaluated in MCF-7 xenograft-bearing female athymic nude mice (BALB/c nu/nu).

Results: Conjugation with HEA125 resulted in a C/A ratio of 4.2 ± 0.3, and SDS-PAGE proved integrity of antibodies to be preserved. Purity of radiolabeling was >98%, and >94% stability was retained for more than 120 h both in PBS and serum. Immunoreactive fraction was 86.2 ± 2.4%, and cytotoxicity assays showed, dose-dependent MCF-7 cell killing with minimal impact on EpCAM-negative controls. In vivo, [²²⁵Ac]Ac-Macropa-PEG₄-HEA125, exhibited significant tumor uptake (15.7 ± 2.3 %ID/g at 24 h), maintained retention (12.1 ± 1.9 %ID/g at 72 h), and minimal off-target accumulation. Therapeutic injection resulted in extensive tumor growth inhibition and long-term survival, with 60% of the mice surviving past day 30 with little overt toxicity.

Conclusions: [²²⁵Ac]Ac-Macropa-PEG₄-HEA125, establishes high radiochemical purity, in vitro stability, EpCAM specificity, and strong antitumor activity in preclinical models. These results warrant its advancement as a promising targeted alpha therapy candidate for EpCAM-expressing carcinomas.