Dual-Receptor-Targeted (DRT) Radiation Nanomedicine Labeled with Lu Is More Potent for Killing Human Breast Cancer Cells That Coexpress HER2 and EGFR Than Single-Receptor-Targeted (SRT) Radiation Nanomedicines.

Resistance to HER2-targeted therapies in breast cancer (BC) is associated in some cases with an increased expression of epidermal growth factor receptors (EGFR). We describe a dual-receptor-targeted (DRT) radiation nanomedicine for local intratumoral (i.t.) treatment of BC composed of 15 nm sized gold nanoparticles (AuNPs) modified with trastuzumab (TmAb) to target HER2 and panitumumab (PmAb) to target EGFR. The AuNPs were modified with poly(ethylene glycol) (PEG) linked to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelators to complex the β-particle emitter, Lu. Our aim was to compare the properties of these DRT-AuNP-Lu with single-receptor-targeted (SRT)-TmAb-AuNP-Lu or PmAb-AuNP-Lu or nontargeted (NT)-AuNP-Lu using human BC cells that expressed HER2, EGFR, or both receptors. To construct these radiation nanomedicines, PEG was linked to TmAb or PmAb, while PEG was linked to DOTA. These polymers were conjugated to AuNP via two Au-thiol bonds using a terminal lipoic acid (LA) group on the polymers. NT-AuNP-Lu were constructed without modification with TmAb or PmAb. MDA-MB-231-H2N, MDA-MB-468, and BT-474 human BC cells were designated as HER2/EGFR, EGFR/HER2, and HER2/EGFR, respectively, based on the expression of these receptors. Specific binding to HER2 and/or EGFR was assessed by incubating BC cells with DRT-AuNP-Lu or TmAb-AuNP-Lu or PmAb-AuNP-Lu, or NT-AuNP-Lu in the absence or presence of an excess of TmAb or PmAb or both competitors. Binding and internalization of AuNP by BC cells were assessed by dark-field microscopy. Cell fractionation studies were conducted to quantify AuNP-Lu bound and internalized. The cytotoxicity of DRT-AuNP-Lu was determined in clonogenic survival (CS) assays after an exposure of 5 × 10 BC cells to 3 MBq (1.4 × 10 AuNP) for 16 h and then seeding and culturing the cells for 7-15 days. CS was compared to exposure to TmAb-AuNP-Lu and PmAb-AuNP-Lu or NT-AuNP-Lu. The absorbed doses to the nucleus in these CS assays were estimated. DRT-AuNP-Lu were specifically bound by BC cells that expressed HER2 or EGFR or both receptors. In contrast, SRT-TmAb-AuNP-Lu and PmAb-AuNP-Lu were bound and internalized only by BC cells that expressed HER2 or EGFR, respectively. NT-AuNP-Lu exhibited very low binding to BC cells. DRT-AuNP-Lu and SRT-TmAb-AuNP-Lu or PmAb-AuNP-Lu were internalized by BC cells in accordance with the receptor expression. Importantly, DRT-AuNP-Lu were more potent in vitro than PmAb-AuNP-Lu for killing MDA-MB-231-H2N cells that coexpress HER2 and EGFR (CS = 18.8 ± 1.0 vs 51.5 ± 10.4%; = 0.006). Furthermore, DRT-AuNP-Lu were more potent for killing BT-474 cells with high HER2 but low EGFR expression than TmAb-AuNP-Lu (CS = 8.9 ± 3.3 vs 20.7 ± 2.4%; = 0.007) or PmAb-AuNP-Lu (CS = 63.9 ± 1.7%; < 0.0001). Even for MDA-MB-468 cells that overexpress EGFR but have negligible HER2, DRT-AuNP-Lu were more potent for cell killing than PmAb-AuNP-Lu (CS = 3.2 ± 3.0 vs 7.5 ± 1.8%; = 0.001) or TmAb-AuNP-Lu (63.2 ± 3.2%; = 0.0002). All targeted forms of AuNP-Lu were more cytotoxic to BC cells than those of NT-AuNP-Lu. High absorbed doses (36-119 Gy) were deposited in the nucleus of BC cells by DRT-AuNP-Lu. We conclude that a DRT radiation nanomedicine is more potent for killing BC cells that coexpress HER2 and EGFR than SRT radiation nanomedicines. These results are promising for further evaluation of these DRT-AuNP-Lu in vivo for the local radiation treatment of human BC tumors that coexpress HER2 and EGFR in mice following i.t. injection, especially tumors that are resistant to HER2-targeted therapies.