Downregulation of EGFR by a novel multivalent nanobody-liposome platform

Abstract

The epidermal growth factor receptor (EGFR) is a recognized target for tumor therapy and monoclonal antibodies (mAbs, e.g. cetuximab) have been developed to inhibit receptor activation. Besides blocking ligand (e.g. EGF) binding to the receptor, reports have shown that mAbs promote slow receptor internalization and degradation in lysosomes, i.e. downregulation. The efficacy of receptor downregulation was recently shown to depend on the size of receptor clusters formed at the cell surface. In this study, a multivalent platform is presented, consisting of nanobodies recognizing the ectodomain of EGFR (EGa1) coupled to PEG-liposomes, and the in vitro and in vivo effects of this system on EGFR internalization and downregulation were investigated. Nanobodies are the smallest functional antigen-binding immunoglobulin fragments and the EGa1 nanobody has been described as an EGFR-antagonist. EGa1-liposomes (EGa1-L) induced a more than 90% removal of EGFR from the cell surface, as a result of receptor internalization. Furthermore, this massive sequestration of EGFR mediated by EGa1-L lead to receptor degradation, while no degradation was detected with the monovalent nanobody. The downregulatory capacity here reported was found to be independent of the epitope on EGFR recognized by the grafted nanobody, and exclusive to the nanobody-liposomes, as anti-EGFR single chain variable fragments (scFv) coupled to liposomes were unable to induce this effect. Importantly, EGa1-L induced a significant inhibition of tumor cell proliferation, in vitro, an effect likely mediated by the combination of receptor downregulation and receptor antagonism. Also in vivo, EGFR downregulation was observed in tumors of mice intravenously injected with EGa1-L, indicating that this multivalent platform blocks ligand binding to the receptor and simultaneously induces the downregulation of EGFR.

Introduction

The epidermal growth factor receptor (EGFR) has long been associated with tumor cell proliferation, metastasis and tumor angiogenesis and is nowadays a recognized target for therapy of many human epithelial tumors [1], [2]. The two main strategies employed to interfere with EGFR functioning are, at the intracellular level, with small tyrosine kinase inhibitors (e.g. erlotinib and gefitinib) that compete with ATP for binding to the active site of the receptor, and at the extracellular level with monoclonal antibodies (mAbs, e.g. cetuximab) that prevent ligand (e.g. EGF) binding to the receptor. Several successful clinical trials have been conducted with molecules from these two groups, also in combination with conventional chemotherapeutic agents (for overview see Ref. [3]), and newer anti-EGFR therapies are likely to arise from ongoing research. Besides ligand blocking, studies have shown that mAbs induce a slow (compared to EGF-induced) EGFR internalization and degradation in lysosomes, a process indicated as receptor downregulation [4]. Recently, an interesting synergistic downregulation of receptor tyrosine kinases (RTK) was reported by Freidman et al., mediated by a combination of mAbs that induced the formation of large receptor lattices at the cell surface [5]. Further development of multivalent antibody structures may lead to improved therapeutic efficacy of mAbs by further exploring the observed downregulatory effect on RTKs.

In this study, we present a novel multivalent antibody system and we have investigated whether this system was able to block ligand binding to the receptor and to induce EGFR internalization and downregulation, possibly by the formation of clusters of receptors due to its multivalency. To this aim, anti-EGFR nanobodies were grafted on the surface of PEG-liposomes, via a maleimide linker. Liposomes have been employed as a carrier system for a variety of molecules and different targeting moieties have been grafted to their surface [6]; however, in this study, liposomes are primarily used as a strategy to build a multivalent antibody platform. Nanobodies are small antibody fragments, derived from heavy chain-only antibodies discovered in the blood of camelids in 1993 [7]. In fact, nanobodies are the smallest functional antigen-binding immunoglobulin fragments (15 kDa, compared to 150 kDa for a conventional antibody), obtained after cloning of the variable domain of the heavy chain (VH) of these heavy (H) chain-only antibodies. Therefore, nanobodies are also referred to as VHHs [8]. Nanobodies possess several attractive characteristics when compared with whole antibodies and/or fragments thereof (e.g. antigen-binding fragments, Fabs of 55 kDa, or single chain variable fragments, scFv of 30 kDa). For instance, nanobodies re-fold very efficiently after heat-denaturation [9] and present better solubility (hydrophilicity) than Fabs and scFvs. Also, nanobodies show a similar degree of specificity and affinity towards their antigen as Fabs, having affinities in the low nanomolar to picomolar range. Together with the ability to be easily engineered and the rather cheap and fast production in bacteria or yeast, nanobodies show great potential for a wide range of applications [10], [11].

Our research group has previously used a phage display approach to select nanobodies that bind to the ectodomain of EGFR with high affinity. In vitro studies demonstrated that these nanobodies block EGF binding without stimulation of receptor kinase activity. Also in vivo, receptor antagonism mediated by anti-EGFR nanobodies has shown great potential by inhibiting the growth of human tumor xenografts [12]. In the present study, the EGFR-antagonist nanobody EGa1 [13] was grafted on the surface of PEG-liposomes (L), forming a new multivalent platform — i.e. nanobody-liposomes or EGa1-L — and the effects of EGa1-L on EGFR internalization and downregulation were investigated, both in vitro and in vivo.