1 prevent the binding of the ligands (e.g EGF)

1Theepidermal growth factor receptor (EGFR) is a 170kD trans-membranetyrosine-kinase receptor of the ErbB family. This receptor has an intracellular domain that has tyrosine kinase activity, atrans-membrane domain and an extracellular ligand-binding domain. When itsligands, most notably epidermal growth factor (EGF) and transforming growthfactor-alpha (TGFa), bind to the extracellular domain, the EGFR is activated.These ligands are normally produced in the surrounding tissues as local growthfactors.

The activated EGFR forms homodimers or heterodimers bypairing with other receptors of the ErbBfamily. This dimerization induces the tyrosine kinase activity of theintracellular domain.  2Theoverexpression of EGFR is observed in a variety of epithelial cancers, such as breastcancer, non-small cell lung cancer (NSCLC), and colorectal cancer. 1This over expression cancause resistance to apoptosis, cancer proliferation, metastatic dissemination andneovascularization. It has been reported that EGFR is over-expressed in 14–91%of breast cancers. Because of these observations EGFR is an interesting targetfor diagnosis and therapeutic strategies. Twodistinct strategies have been applied to reduce and deactivate EGFR signaling.

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The first approach is to block the intercellular domain of the receptor byspecific tyrosine kinase inhibitors. 8Theseinhibitors 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 oneFDA-approved drug Osimertinib which is showing promising results.  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 bymonoclonal antibodies (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 concerning mAbs is that generation of new ormodified mAbs is costly and laborious.

Both problems can besolved by exploiting heavy chain only antibodies (HCAbs) from camelids(Hamers-Casterman et al., 1993; Muyldermans et al., 1994). Whereas the 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. This single Ig domain is stable and can be generated rapidly andcheaply with simple expression systems (Harmsen and De Haard, 2007). Single VHHdomains can be powerful diagnostic imaging tools, and are being developed for arange of research applications (Steyaert and Kobilka, 2011; Vaneycken et al.,2011). For therapeutic use, VHH domains (monomeric or multivalent) can bemodified to extend serum half-life and/or functionality (Saerens et al.

, 2008).Theclinical success of EGFR-targeted mAbs has caused significant interest indeveloping VHH domains that bind to and inhibit this receptor. SeveralEGFR-specific VHH domains have been reported (Roovers et al., 2007; Roovers etal., 2011) that have the potential to reproduce the clinical efficacy of mAbssuch as Cetuximab in an agent that is more stable and far less costly toproduce. Moreover, potent multivalent VHH molecules can be generated that binda number of targets (Emmerson et al., 2011; Jahnichen et al.

, 2010; Roovers etal., 2011), offering the potential to engineer multivalent agents that combinecetuximab-like EGFR inhibition with other modes of binding to EGFR or to othercancer targets.  7D12, a 133 amino acids VHH domain, is a selected nanobody withthe highest affinity binding to EGFR. ThisVHH domain competes with Cetuximab for EGFR binding (Roovers et al., 2011).

Although it is a much smaller VHH domain, it can block both Cetuximab andligand binding, which makes it a promising nanobodyagainst EGFR. 97D12 based nanobodies can also be used for imaging. For example,Gainkam et al. (2008) and van Dongen andVosjan (2010) used 99mTc-labelednanobody 7D12 to image the expression of EGFR in mice carcinomas. In anotherstudy, 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 (Vosjan et al.

, 2011). In another study(9), by using molecular dynamic (MD), we have madesuitable mutations in the selected key residues of 7D12 and designed a 7D12based nanobody with high binding affinity to EGFR. In comparison with wild-type7D12, these high affinity nanobodies are far more effective for therapeutic andbioimaging applications.

 9G8,a 136 amino acids 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. 4As statedbefore, the structure of 7D12 bound to EGFR shows how this smaller and readilyengineered binding unit can mimic inhibitory features of the intact monoclonalantibody drug cetuximab.

Multimerization of 7D12 with other VHH domains generatesa potent EGFR inhibitor (Roovers et al., 2011). 7D12 is thus a cassette thatcan be used to combine cetuximab-like inhibition with modules of synergisticand/or complementary inhibitory properties.  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.