A biparatopic anti‐EGFR nanobody efficiently inhibits solid tumour growth

Abstract

The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies (mAbs) have been successfully used, among which cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities. We have previously reported on the successful isolation of antagonistic anti-EGFR nanobodies. In the present study, we aimed to improve on these molecules by combining nanobodies with specificities similar to both cetuximab and matuzumab into a single bi-paratopic molecule. Carefully designed phage nanobody selections resulted in two sets of nanobodies that specifically blocked the binding of either matuzumab or of cetuximab to EGFR and that did not compete for each others binding. A combination of nanobodies from both epitope groups into the bi-paratopic nanobody CONAN-1 was shown to block EGFR activation more efficiently than monovalent or bivalent (monospecific) nanobodies. In addition, this bi-paratopic nanobody potently inhibited EGF-dependent cell proliferation. Importantly, in an in vivo model of athymic mice bearing A431 xenografts, CONAN-1 inhibited tumour outgrowth with an almost similar potency as the whole mAb cetuximab, despite the fact that CONAN-1 is devoid of an Fc portion that could mediate immune effector functions. Compared to therapy using bivalent, mono-specific nanobodies, CONAN-1 was clearly more potent in tumour growth inhibition. These results show that the rational design of bi-paratopic nanobody-based anti-cancer therapeutics may yield potent lead molecules for further development.

Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform

“…17 The heavy-chain only antibody-derived variable domain (VHH) has attracted much interest because of its stability, size (15 kDa), and cavity binding, as well as the ease with which it is engineered into manifold constructs with improved potency or broadened cross-reactivity profiles. 18,19 Due to the overwhelming attention that VHHs received over the last 2 decades, camelid conventional antibodies were overlooked.…”

Nanobodies Targeting the Hepatocyte Growth Factor: Potential New Drugs for Molecular Cancer Therapy

“…12, 13), and bispecific (Nanobodies binding to 2 different antigens; refs. 13,14) molecules. These formats are easy to construct and the modular proteins can often be expressed at high levels in bacteria or yeast.…”

“…What is more, biodistribution of aEGFRaEGFR-aAlb (50 kDa) was comparable with cetuximab (150 kDa), while it showed faster and deeper tumor penetration. The therapeutic potential of aEGFR-Nanobodies in comparison with conventional aEGFR mAbs was shown by Roovers and colleagues (13).…”

New Applications of Nanotechnology for Neuroimaging

“…They consist of heavy chains only and have desirable features such as small size, decreased immunogenicity, and high binding affinities. 56 Nanobodies have been designed for use in cancer therapy, 57,58 antivenoms, 59 inflammatory conditions, 60 and immunoimaging. [60][61][62][63][64] Nanobodies can detect vascular and parenchymal amyloid beta (A␤) deposits in vivo to differentiate cerebral ␤-amyloid indicative of Alzheimer disease and vascular A␤ plaques associated with cerebral amyloid angiopathy.…”