Tumor-targeted nanobullets for anti-cancer combination therapy

Background: The epidermal growth factor receptor (EGFR) is a well established target for anti-cancer therapy and several EGFR-targeted therapeutics are used in the clinic. However, crosstalk between EGFR and other receptor signaling networks can contribute to accelerated tumorigenesis and even induce resistance to EGFR-directed therapies. Combined inhibition of EGFR and insulin-like growth factor 1 receptor (IGF- 1R) signaling is a rational strategy to enhance anti-cancer treatment and possibly delay resistance development. We have encapsulated the anti- IGF-1R kinase inhibitor AG538 in anti-EGFR nanobody-liposomes. The efficacy of this targeted dual-active nanomedicine was assessed on EGFR- (over)expressing tumor cells in vitro. Material and Methods: Anti-EGFR nanobodies (EGa1) were coupled to PEGylated liposomes. AG538 was encapsulated in the EGa1-liposomes by remote loading with a calcium acetate gradient. EGa1-AG538-liposomes were characterized according to size, surface charge and encapsulation efficiency. Cellular assays were performed with EGFR-(over)expressing UM-SSC- 14C human head and neck cancer cells (14C) and EGFR-/- NIH 3T3 clone 2.2 murine fibroblasts (3T3 2.2). Cell association of rhodaminelabeled EGa1-AG538-liposomes was determined by flow cytometry and cell uptake was visualized by confocal microscopy. Inhibition of EGFR and IGF-1R signaling was investigated by Western Blotting with phosphospecific antibodies directed at the targeted pathways. Inhibition of tumor cell proliferation was determined by the sulforhodamine B (SRB) assay and the BrdU-colorimetric immunoassay. These assays were conducted upon continuous exposure to the nanomedicine for 48 h and short-term exposure for 4 h followed by a 44 h treatment-free period to mimic in vivo drug exposure. Results: EGa1-AG538-liposomes associated with EGFR-positive 14C cells in an EGFR-specific manner and were internalized by 14C cells but not by EGFR-negative 3T3 2.2 cells. EGa1-AG538-liposomes inhibited both EGFR and IGF-1R signaling activation and induced downregulation of EGFR. The dual-active nanomedicine strongly inhibited 14C proliferation in short-term exposure assays while control formulations did not. Similar inhibitory effects of the nanomedicine on cell proliferation were observed with EGFR(over)expressing MDA-MB-468 human breast cancer cells. Conclusions: Anti-EGFR nanobody liposomes loaded with anti-IGF1- R kinase inhibitor simultaneously block activation of EGFR and IGF- 1R signaling. In short-term exposure assays, EGa1-AG538-L induces strong inhibition of tumor cell proliferation. This targeted nanomedicine is a promising anti-cancer therapy for tumors that are dependent on (over)expression of EGFR and IGF-1R and their in vivo applicability is currently under investigation.