The trans-membrane domain and an extracellular ligand-binding domain.

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 these observationsEGFR is an interesting target for 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 oneFDA-approved drug 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 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 arduous. Both problems can besolved by exploiting 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).  VHHs are thermo- and pH-stable proteins that are well toleratedby the human immune system and can be generated rapidly andcheaply with simple expression systems (7).

 SingleVHH domains can be powerful diagnostic imaging tools, and are being developedfor a range of research applications (Steyaert and Kobilka, 2011; Vaneycken etal., 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.   7D12 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(8). In another study(8) ,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 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.

 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 thus acassette that can be used to combine cetuximab-like inhibition with modules ofsynergistic 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.

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