111In-Labeled Trastuzumab (Herceptin) Modified with Nuclear Localization Sequences (NLS): An Auger Electron-Emitting Radiotherapeutic Agent for HER2/neu-Amplified Breast Cancer

Abstract

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...

Nanomolecular HLA-DR10 Antibody Mimics: A Potent System for Molecular Targeted Therapy and Imaging

“…4 Further, a radionuclide localized inside the cell has improved efficacy and specificity, if its emission ranges are short; Auger electrons inside the cell are efficient for inducing lethal effects. 30 If radionuclides that emit both photon and Auger radiations are used, both imaging and radiotherapy can be achieved. The 111 In, used in this study, is such a radionuclide and, like the Ct containing SHALs, is also capable of residualizing inside the cell.…”

“…Additionally, the lysine residues in the linkers can also be used to attach other radionuclides and functionalities. 30 All of these mAb mimetics have exhibited selectivity for the target protein, have been labeled with radionuclides in high yield and purity, and are expected to have a long shelf-life and potential for oral administration. Gram amounts can be synthesized for a fraction of the cost of biologicals.…”

Nuclisome—targeting the tumor cell nucleus

“…Proteins or peptides tagged with NLS can be transported form cytoplasmic compartments to the nucleus and if such proteins or peptides are radiolabeled and targeted to tumor markers a successful delivery of radionuclides to tumor-cell nuclei can potentially be achieved. Encouraging results from studies employing NLS-tagged radiolabeled peptides and proteins verify an increased therapeutic effect compared to that obtained with the corresponding radiolabeled targeting agent lacking NLS [6]. A possible drawback of the NLS-based strategy is the absence of DNA targeting.…”

“…Focus has been on delivery of 111 In utilizing nuclear-localization sequences (NLS) [5][6][7][8]. Proteins or peptides tagged with NLS can be transported form cytoplasmic compartments to the nucleus and if such proteins or peptides are radiolabeled and targeted to tumor markers a successful delivery of radionuclides to tumor-cell nuclei can potentially be achieved.…”

Possibilities of Therapy of Her-2-Positive Regional Breast Cancer

“…Комбинация лучевой и таргетной терапии В настоящее время активно изучается радиочувст-вительность опухолей, обладающих гиперэкспрессией HER-2 / neu [20,21]. Клинических исследований по изучению безопасности и эффективности таргетной и лучевой терапии в лечении РМЖ единицы.…”