The cytotoxicity and tumor-targeting properties of the anti-HER2/neu monoclonal antibody trastuzumab modified with peptides (CGYGPKKKRKVGG) harboring the nuclear localization sequence ([NLS] italicized) of simian virus 40 large T-antigen and radiolabeled with 111 In were evaluated. Methods: Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) for reaction with NLSpeptides and labeled with 111 In using diethylenetriaminepentaacetic acid (DTPA). The immunoreactivity of 111 In-NLS-trastuzumab was determined by its ability to displace the binding of trastuzumab to SK-BR-3 human breast cancer (BC) cells. Cellular uptake and nuclear localization were evaluated in SK-BR-3, MDA-MB-361, and MDA-MB-231 BC cells, expressing high, intermediate, or very low levels of HER2/neu, respectively, by cell fractionation and confocal microscopy. Biodistribution and nuclear uptake were compared in athymic mice bearing MDA-MB-361 xenografts. The cytotoxicity of 111 In-trastuzumab and 111 In-NLS-trastuzumab was studied by clonogenic assays, and DNA damage was assessed by probing for phosphorylated histone H2AX (gH2AX) foci. Results: The dissociation constant for binding of 111 In-NLS-trastuzumab to SK-BR-3 cells was reduced ,3-fold compared with that of 111 In-trastuzumab, demonstrating relatively preserved receptorbinding affinity. The receptor-mediated internalization of 111 Intrastuzumab in SK-BR-3, MDA-MB-361, and MDA-MB-231 cells increased significantly from 7.2% 6 0.9%, 1.3% 6 0.1%, and 0.2% 6 0.05% to 14.4% 6 1.8%, 6.3% 6 0.2%, and 0.9% 6 0.2% for 111 In-NLS-trastuzumab harboring 6 NLS-peptides, respectively. NLS-trastuzumab localized in the nuclei of BC cells, whereas unmodified trastuzumab remained surface-bound. Conjugation of 111 In-trastuzumab to NLS-peptides did not affect its tissue biodistribution but promoted specific nuclear uptake in MDA-MB-361 xenografts (2.4-2.9 %ID/g [percentage injected dose per gram] for 111 In-NLS-trastuzumab and 1.1 %ID/g for 111 In-trastuzumab). 111 In-NLS-trastuzumab was 5-and 2-fold more potent at killing SK-BR-3 and MDA-MB-361 cells than 111 In-trastuzumab, respectively, whereas toxicity toward MDA-MB-231 cells was minimal. 111 In-NLS-trastuzumab was 6-fold more effective at killing SK-BR-3 cells than unlabeled trastuzumab. The development of recombinant antibodies for cancer therapy has emerged as one of the most promising areas in oncology (1). Trastuzumab (Herceptin; Hoffmann-La Roche), in particular, is a humanized monoclonal antibody (mAb) directed against the human epidermal growth factor receptor-2 (HER2/neu), a transmembrane receptor tyrosine kinase that is overexpressed in 25%-30% of breast cancers (BCs) and distant metastases (2). Trastuzumab shows clinical activity in women with HER2/neu-overexpressing metastatic BC and exhibits synergistic antitumor effects when combined with paclitaxel or anthracyclines, achieving overall response rates of 40%-60% (2). Despite its effectiveness in combination regimens, the response rate...