Dual-Receptor-Targeted (DRT) Radiation Nanomedicine Labeled with ¹⁷⁷Lu 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_3k) linked to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelators to complex the β-particle emitter, ¹⁷⁷Lu. Our aim was to compare the properties of these DRT-AuNP-¹⁷⁷Lu with single-receptor-targeted (SRT)-TmAb-AuNP-¹⁷⁷Lu or PmAb-AuNP-¹⁷⁷Lu or nontargeted (NT)-AuNP-¹⁷⁷Lu using human BC cells that expressed HER2, EGFR, or both receptors. To construct these radiation nanomedicines, PEG_5K was linked to TmAb or PmAb, while PEG_3k 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-¹⁷⁷Lu 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^mod/EGFR^mod, EGFR^high/HER2^neg, and HER2^high/EGFR^low, respectively, based on the expression of these receptors. Specific binding to HER2 and/or EGFR was assessed by incubating BC cells with DRT-AuNP-¹⁷⁷Lu or TmAb-AuNP-¹⁷⁷Lu or PmAb-AuNP-¹⁷⁷Lu, or NT-AuNP-¹⁷⁷Lu 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-¹⁷⁷Lu bound and internalized. The cytotoxicity of DRT-AuNP-¹⁷⁷Lu was determined in clonogenic survival (CS) assays after an exposure of 5 × 10⁵ BC cells to 3 MBq (1.4 × 10¹² AuNP) for 16 h and then seeding and culturing the cells for 7-15 days. CS was compared to exposure to TmAb-AuNP-¹⁷⁷Lu and PmAb-AuNP-¹⁷⁷Lu or NT-AuNP-¹⁷⁷Lu. The absorbed doses to the nucleus in these CS assays were estimated. DRT-AuNP-¹⁷⁷Lu were specifically bound by BC cells that expressed HER2 or EGFR or both receptors. In contrast, SRT-TmAb-AuNP-¹⁷⁷Lu and PmAb-AuNP-¹⁷⁷Lu were bound and internalized only by BC cells that expressed HER2 or EGFR, respectively. NT-AuNP-¹⁷⁷Lu exhibited very low binding to BC cells. DRT-AuNP-¹⁷⁷Lu and SRT-TmAb-AuNP-¹⁷⁷Lu or PmAb-AuNP-¹⁷⁷Lu were internalized by BC cells in accordance with the receptor expression. Importantly, DRT-AuNP-¹⁷⁷Lu were more potent in vitro than PmAb-AuNP-¹⁷⁷Lu for killing MDA-MB-231-H2N cells that coexpress HER2 and EGFR (CS = 18.8 ± 1.0 vs 51.5 ± 10.4%; P = 0.006). Furthermore, DRT-AuNP-¹⁷⁷Lu were more potent for killing BT-474 cells with high HER2 but low EGFR expression than TmAb-AuNP-¹⁷⁷Lu (CS = 8.9 ± 3.3 vs 20.7 ± 2.4%; P = 0.007) or PmAb-AuNP-¹⁷⁷Lu (CS = 63.9 ± 1.7%; P < 0.0001). Even for MDA-MB-468 cells that overexpress EGFR but have negligible HER2, DRT-AuNP-¹⁷⁷Lu were more potent for cell killing than PmAb-AuNP-¹⁷⁷Lu (CS = 3.2 ± 3.0 vs 7.5 ± 1.8%; P = 0.001) or TmAb-AuNP-¹⁷⁷Lu (63.2 ± 3.2%; P = 0.0002). All targeted forms of AuNP-¹⁷⁷Lu were more cytotoxic to BC cells than those of NT-AuNP-¹⁷⁷Lu. High absorbed doses (36-119 Gy) were deposited in the nucleus of BC cells by DRT-AuNP-¹⁷⁷Lu. 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-¹⁷⁷Lu 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.