The epidermalgrowth factor receptor (EGFR) is a 170kD trans-membrane tyrosine-kinasereceptor of the ErbB family. This receptor has anintracellular domain that has tyrosine kinase activity, a trans-membrane domainand an extracellular ligand-binding domain. When its ligands, most notablyepidermal growth factor (EGF) and transforming growth factor-alpha (TGFa), bindto the extracellular domain, the EGFR is activated. These ligands are normallyproduced in the surrounding tissues as local growth factors. The activated EGFRforms homodimers or heterodimers bypairing with other receptors of the ErbBfamily. This dimerization induces the tyrosine kinase activity of theintracellular domain (1). The overexpression of EGFR isobserved in a variety of epithelial cancers, such as breast cancer, non-smallcell lung cancer (NSCLC), and colorectal cancer (2).
This over expression can cause resistance to apoptosis,cancer proliferation, metastatic dissemination and neovascularization. It hasbeen reported that EGFR is over-expressed in 14–91% of breastcancers. Because of theseobservations EGFR is an interesting targetfor diagnosis and therapeutic strategies (1).
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Twodistinct strategies have been applied to reduce and deactivate EGFR signaling.The first approach is to block the intercellular domain of the receptor byspecific tyrosine kinase inhibitors. Theseinhibitors bind to the ATP-binding site of the EGFR tyrosine-kinase domain. Theliterature and the clinical trials of this approach mainly focus on NSCLCbecause of the promising results. Gefitinib and Erlotinib have resulted in asignificant improvement in patients overall conditions. However, after a periodof time patients develop tumor resistance due to the emergence of theresistance mutations. Another complication is dose-limiting toxicity in drugslike Afatinib due to simultaneous inhibition of wild-type EGFR. There is one FDA-approveddrug Osimertinib which is showing promising results(3).
Thesecond strategy, which is our focus of the current study, is to prevent thebinding of the ligands (e.g EGF) to the extracellular domain of the EGFR by monoclonalantibodies (mAbs). Cetuximab/ErbituxR,is an FDA-approved antibody with these properties in current use in the clinic. Whereasantibodies that bind EGFR and other targets have shown promise in the clinic, there arelimitations to their effective application and future development.Oneof the drawbacks of mAbs is their large size which limits tumor penetration,and reduces theireffectiveness; another problem regarding mAbs is that generation of new mAbs iscostly and difficult. Bothproblems can be solved by developing heavy chain only antibodies(HCAbs) from camelids(4,5). Whereasthe antigen recognitionregion in conventional antibodies comprises the variable regions of both theheavy and the light chains (VH and VL respectively), the antigen recognitionregion of HCAbs comprises a single variable domain, referred to as a VHH domainor nanobody(6). VHHsare thermo- and pH-stable proteins that are well tolerated by the human immunesystem and can be generated rapidly and cheaply with simple expression systems (7).
SingleVHH domains are being used for research and diagnostic applications. For therapeuticuse they can be modified to extend serum half-life and functionality (8).Theclinical success of EGFR-targeted mAbs has caused significant interest indeveloping VHH domains that bind to and inhibit this receptor. VHH domains thatspecifically bind to EGFR have the potential to reproduce the clinical effectivenessof mAbs such as Cetuximab. Furthermore they are more stable and far less costlyto produce (9).
Moreover,potent multivalent VHH molecules can be generated that bind a number of targets(Emmerson et al., 2011; Jahnichen et al., 2010; Roovers et al., 2011), offeringthe potential to engineer multivalent agents that combine cetuximab-like EGFRinhibition with other modes of binding to EGFR or to other cancer targets. 7D12, a 133-amino acid VHH domain, is a selected nanobody with thehighest affinity binding to EGFR.
ThisVHH domain competes with Cetuximab for EGFR binding (10). Although itis a much smaller VHH domain, it can block both Cetuximab and ligand binding, which makes it a promising nanobody against EGFR. 7D12 based nanobodies are also a good tool for imaging. Forexample, Gainkam et al.
(2008) and van Dongen and Vosjan (2010) used99mTc-labeled nanobody 7D12 to image the expression of EGFR in mice carcinomas.In another study, bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine(brieflyDf-Bz-NCS) was conjugated with nanobody 7D12 and then labeled by89Zr (t1/2, 78.4 h). This combination (89Zr-Df-Bz-NCS-7D12) was applied toimage the expression of EGFR in carcinomas(11). In another study (11),by using molecular dynamic (MD), we have made suitable mutationsin the selected key residues of 7D12 and designed a 7D12 based nanobody withhigh binding affinity to EGFR. In comparison with wild-type 7D12, these highaffinity nanobodies are far more effective for therapeutic and bioimagingapplications.
9G8,a 136-amino acid VHH domain, is another nanobody that binds to a differentepitope on EGFR. Interestingly, unlike 7D12, 9G8 do not compete with Cetuximabfor binding to EGFR (Rooverset al., 2011). Instead, this VHH domain binds to anepitope that is inaccessible to Cetuximab and that undergoes largeconformational changes during EGFR activation, sterically inhibiting thereceptor. Asstated before, the structure of 7D12 bound to EGFR shows how this smaller andreadily engineered binding unit can mimic inhibitory features of the intactmonoclonal antibody drug cetuximab. Multimerization of 7D12 with other VHHdomains generates a potent EGFR inhibitor (Roovers et al., 2011).
7D12 is thusa cassette that can be used to combine cetuximab-like inhibition with modulesof synergistic and/or complementary inhibitory properties(9). Theaim of the current study was to fuse 7D12 and 9G8 with a linker and determinetheir synergistic binding potential by MD methods. We compared the potency ofthe 7D12 inhibitory effects individually and while coupled with 9G8.
In 2011, Roovers et al.(10) showed that thebi-paratopic anti-EGFR nanobody 7D12-9G8 is very potent in inhibiting EGFRsignalling. The length and the composition ofthe connecting linker are important contributes to the characteristics of the7D12-9G8 molecule. This linker must provide sufficient space/length and freedomto allow the two nanobodies to bind simultaneously to the same EGFR molecule.(10)