inhibitors bind to the ATP-binding site of the EGFR tyrosine-kinase domain. The
literature and the clinical trials of this approach mainly focus on NSCLC
because of the promising results. Gefitinib and Erlotinib have resulted in a
significant improvement in patients overall conditions. However, after a period
of time patients develop tumor resistance due to the emergence of the
resistance mutations. Another complication is dose-limiting toxicity in drugs
like Afatinib due to simultaneous inhibition of wild-type EGFR. There is one FDA-approved
drug Osimertinib which is showing promising results(3).
second strategy, which is our focus of the current study, is to prevent the
binding of the ligands (e.g EGF) to the extracellular domain of the EGFR by monoclonal
is an FDA-approved antibody with these properties in current use in the clinic. Whereas
antibodies that bind EGFR and other targets have shown promise in the clinic, there are
limitations to their effective application and future development.
of the drawbacks of mAbs is their large size which limits tumor penetration,
and reduces their
effectiveness; another problem regarding mAbs is that generation of new mAbs is
costly and difficult. Both
problems can be solved by developing heavy chain only antibodies
(HCAbs) from camelids
the antigen recognition
region in conventional antibodies comprises the variable regions of both the
heavy and the light chains (VH and VL respectively), the antigen recognition
region of HCAbs comprises a single variable domain, referred to as a VHH domain
are thermo- and pH-stable proteins that are well tolerated by the human immune
system and can be generated rapidly and cheaply with simple expression systems (7).
VHH domains are being used for research and diagnostic applications. For therapeutic
use they can be modified to extend serum half-life and functionality (8).
clinical success of EGFR-targeted mAbs has caused significant interest in
developing VHH domains that bind to and inhibit this receptor. VHH domains that
specifically bind to EGFR have the potential to reproduce the clinical effectiveness
of mAbs such as Cetuximab. Furthermore they are more stable and far less costly
to 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), offering
the potential to engineer multivalent agents that combine cetuximab-like EGFR
inhibition with other modes of binding to EGFR or to other cancer targets.
7D12, a 133-amino acid VHH domain, is a selected nanobody with the
highest affinity binding to EGFR.
VHH domain competes with Cetuximab for EGFR binding (10). Although it
is 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. For
example, Gainkam et al. (2008) and van Dongen and Vosjan (2010) used
99mTc-labeled nanobody 7D12 to image the expression of EGFR in mice carcinomas.
In another study, bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine
Df-Bz-NCS) was conjugated with nanobody 7D12 and then labeled by
89Zr (t1/2, 78.4 h). This combination (89Zr-Df-Bz-NCS-7D12) was applied to
image the expression of EGFR in carcinomas(11).
In another study (11),by using molecular dynamic (MD), we have made suitable mutations
in the selected key residues of 7D12 and designed a 7D12 based nanobody with
high binding affinity to EGFR. In comparison with wild-type 7D12, these high
affinity nanobodies are far more effective for therapeutic and bioimaging
a 136-amino acid VHH domain, is another nanobody that binds to a different
epitope on EGFR. Interestingly, unlike 7D12, 9G8 do not compete with Cetuximab
for binding to EGFR (Rooverset al., 2011). Instead, this VHH domain binds to an
epitope that is inaccessible to Cetuximab and that undergoes large
conformational changes during EGFR activation, sterically inhibiting the
stated before, the structure of 7D12 bound to EGFR shows how this smaller and
readily engineered binding unit can mimic inhibitory features of the intact
monoclonal antibody drug cetuximab. Multimerization of 7D12 with other VHH
domains generates a potent EGFR inhibitor (Roovers et al., 2011). 7D12 is thus
a cassette that can be used to combine cetuximab-like inhibition with modules
of synergistic and/or complementary inhibitory properties(9).
aim of the current study was to fuse 7D12 and 9G8 with a linker and determine
their synergistic binding potential by MD methods. We compared the potency of
the 7D12 inhibitory effects individually and while coupled with 9G8. In 2011, Roovers et al.(10) showed that the
bi-paratopic anti-EGFR nanobody 7D12-9G8 is very potent in inhibiting EGFR
The length and the composition of
the connecting linker are important contributes to the characteristics of the
7D12-9G8 molecule. This linker must provide sufficient space/length and freedom
to allow the two nanobodies to bind simultaneously to the same EGFR molecule.(10)