A 177Lu-labeled Inetetamab theranostic conjugate with dual cytotoxic and stromal-depleting activities for targeted radionuclide therapy of HER2-positive breast cancer.
Abstract
Purpose
HER2-positive breast cancer requires targeted therapies that overcome resistance and desmoplastic barriers. This study aimed to develop and biologically evaluate a 177Lu-labeled Inetetamab conjugate (177Lu-Inetetamab) with dual cytotoxic and stromal-depleting activities for targeted radionuclide therapy.
Methods
177Lu-Inetetamab was synthesized using p-SCN-Bn-DOTA as the bifunctional chelator. Radiochemical purity, stability, and HER2-specific binding were assessed in vitro. In vivo SPECT/CT imaging, biodistribution, and therapeutic efficacy were evaluated in BT-474 xenograft-bearing mice. Tumor growth, survival, immunohistochemistry (Ki67, BCL-2, γ-H2AX, TNF-α), stromal remodeling (99mTc-FAPI-04 SPECT/CT, Masson's trichrome), and transcriptomic profiling were performed.
Results
177Lu-Inetetamab was successfully synthesized using p-SCN-Bn-DOTA as the bifunctional chelator, achieving a conjugation efficiency of 40.3 ± 2.5% at pH 9.0 and a chelate-to-antibody ratio of approximately 3.3 DOTA per antibody. The radiochemical purity (> 95%) was confirmed by radio-HPLC and radio-TLC and remained stable in PBS and mouse serum at 37 °C for up to 168 h. HER2-specific cellular uptake was confirmed, with an immunoreactive fraction of 75.5 ± 1.9% and a dissociation constant (KD) of 0.772 nM. SPECT/CT showed sustained tumor retention (1.45 ± 0.96 %ID/mL at 168 h) and favorable hepatobiliary clearance. Treatment with 177Lu-Inetetamab significantly suppressed tumor growth (no net growth by day 10, whereas control tumors increased by >150%) and prolonged survival compared to controls. IHC revealed reduced Ki67 and BCL-2, increased γ-H2AX and TNF-α. The reduced FAPI-04 uptake (1.05 ± 0.33 vs. 2.35 ± 0.45%ID/mL) and Masson's staining indicated CAF loss, to which β-emission cross-fire may have contributed. Transcriptomics showed activation of p53-mediated apoptosis and downregulation of profibrotic genes (TGFB2, CCN2, FN1).
Conclusion
177Lu-Inetetamab exhibits high HER2 specificity, potent antitumor efficacy, and stromal-modulating activity, supporting its potential as a theranostic agent for resistant or stroma-rich HER2-positive breast cancer.