Tumor-targeted Nanobullets: Anti-EGFR nanobody-liposomes loaded with anti-IGF-1R kinase inhibitor for cancer treatment

Abstract

The epidermal growth factor receptor (EGFR) is a validated target for anti-cancer therapy and several EGFR inhibitors are used in the clinic. Over the years, an increasing number of studies have reported on the crosstalk between EGFR and other receptors that can contribute to accelerated cancer development or even acquisition of resistance to anti-EGFR therapies. Combined targeting of EGFR and insulin-like growth factor 1 receptor (IGF-1R) is a rational strategy to potentiate anti-cancer treatment and possibly retard resistance development. In the present study, we have pursued this by encapsulating the kinase inhibitor AG538 in anti-EGFR nanobody-liposomes. The thus developed dual-active nanobody-liposomes associated with EGFR-(over)expressing cells in an EGFR-specific manner and blocked both EGFR and IGF-1R activation, due to the presence of the EGFR-blocking nanobody EGa1 and the anti-IGF-1R kinase inhibitor AG538 respectively. AG538-loaded nanobody-liposomes induced a strong inhibition of tumor cell proliferation even upon short-term exposure followed by a drug-free wash-out period. Therefore, AG538-loaded nanobody-liposomes are a promising anti-cancer formulation due to efficient intracellular delivery of AG538 in combination with antagonistic and downregulating properties of the EGa1 nanobody-liposomes.

Introduction

The epidermal growth factor receptor (EGFR) can contribute to tumorigenesis and metastasis and is a well established target for anti-cancer therapy [1], [2]. Overexpression of EGFR occurs in a number of epithelial tumors such as head and neck squamous-cell carcinoma (HNSCC), non-small-cell lung cancer, colorectal cancer and pancreatic cancer [1], [2]. EGFR targeted therapeutics that have been approved by the FDA include the tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib and the monoclonal antibodies (mAbs) cetuximab and panitumumab [2], [3]. Despite good initial clinical response, many tumors eventually develop acquired resistance to EGFR-targeted therapies [4]. One of the underlying mechanisms of acquired tumor resistance is crosstalk between EGFR and insulin-like growth factor 1 receptor (IGF-1R) signaling [4], [5]. Interaction of EGFR and IGF-1R signaling pathways can occur directly by heterodimerization of the receptors, or indirectly via common downstream signaling molecules [5]. Similar to EGFR, IGF-1R can stimulate tumor progression and development by activating signaling cascades regulating processes such as cell proliferation, apoptosis and survival [6]. Upregulated expression of IGF-1R has been described in a variety of human cancers and several IGF-1R inhibitors are currently undergoing clinical trials [7]. Combinations of EGFR/IGF-1R inhibitors have been utilized to demonstrate enhanced inhibition of tumor cell proliferation when compared to mono-targeted therapies, underlining the importance of crosstalk between these signaling networks in tumor progression and resistance [5]. We have previously reported on anti-EGFR nanobody-liposomes that are able to inhibit ligand binding to EGFR and to induce its downregulation [8]. Anti-EGFR nanobodies or VHHs are small antigen-binding fragments derived from heavy chain-only antibodies which occur naturally in the blood of camelidae [9]. Anti-EGFR nanobody-liposomes demonstrated increased tumor cell proliferation inhibition compared to free nanobody in vitro and downregulation of EGFR in vivo [8]. In the present study, we employed these anti-EGFR nanobody-liposomes for the development of a targeted dual-active nanomedicine that simultaneously inhibits both EGFR and IGF-1R. To accomplish this, AG538, a potent inhibitor of IGF-1R kinase [10], was encapsulated in anti-EGFR nanobody-liposomes by remote loading. We assessed the efficacy of AG538-loaded nanobody-liposomes on EGFR-(over)expressing UM-SCC-14C tumor cells in vitro by investigating the capability of the liposomal system to deliver its cargo within the targeted tumor cells and their effects on the targeted pathways as well as tumor cell proliferation. AG538-loaded nanobody-liposomes showed strongly enhanced inhibition of tumor cell proliferation after short-term exposure to tumor cells, demonstrating the beneficial effects of anti-EGFR/IGF-1R combination therapy.