Preclinical targeted alpha therapy of B7-H3-positive tumors using [²²⁵Ac]Ac-Macropa-PEG₂-enoblituzumab via EDC/NHS-mediated conjugation.

Abstract

Targeted alpha therapy (TAT) has the advantage of localized cytotoxicity using the high linear energy transfer capabilities of alpha particles. Actinium-225 ([225Ac]Ac) has been shown to be suitable for antibody-based targeting but lacks chelate stability and conjugation reproducibility. CD276 (B7-H3) has been shown to be broadly overexpressed in various solid tumors and thus presents as a suitable therapeutic target. We developed and evaluated a well-defined [225Ac]Ac-Macropa-PEG2-Enoblituzumab radioimmunoconjugate prepared using thiol-maleimide-based PEG2-Macropa conjugation strategy. The Enoblituzumab was activated (thiolated) using Traut's reagent followed by conjugation with activated Macropa-PEG2 using EDC/NHS chemistry. The Macropa-PEG2-Enoblituzumab conjugate was characterized by SEC-HPLC and MALDI-TOF MS. Radiolabeling with [225Ac]Ac- was achieved under mild conditions and the [225Ac]Ac-Macropa-PEG2-Enoblituzumab assessed with iTLC and radio-SEC-HPLC. Functional studies involved saturated binding & internalization analyses using CD276-positive DU145-B7-H3 cells and CD276-negative CHO-K1 controls. Biodistribution, dosimetry, and therapeutic effectiveness were assessed using DU145-B7-H3 xenograft-bearing mice model with excess Enoblituzumab blocking and non-target CHO-K1 controls. The conjugate revealed reproducible chelator incorporation (CAR = 3.4 ± 0.12), efficient radiolabeling (>95%), and in vitro stability (>90% intact up to 7 days). High-affinity binding (KD = 0.18 ± 0.05 nM) and time-dependent internalization were observed in DU145-B7-H3 cells, with minimal uptake in CHO-K1 controls. In vivo studies showed gradual tumor accumulation (15.7 ± 1.1%ID/g at 72 h) with sustained retention and favorable tumor-to-background ratios. CHO-K1 xenografts showed minimal uptake, and blocking significantly reduced tumor accumulation, confirming receptor-mediated targeting. Therapy studies demonstrated significant tumor growth inhibition and prolonged survival with good tolerability. [225Ac]Ac-Macropa-PEG2-Enoblituzumab shows strong stability, specificity, and therapeutic efficacy, supporting further development as a promising translational candidate for targeted alpha therapy of B7-H3-expressing malignancies.