The activated. These ligands are normally produced in the

The epidermal
growth factor receptor (EGFR) is a 170kD trans-membrane tyrosine-kinase
receptor of the ErbB family. This receptor has an
intracellular domain that has tyrosine kinase activity, a trans-membrane domain
and an extracellular ligand-binding domain. When its ligands, most notably
epidermal growth factor (EGF) and transforming growth factor-alpha (TGFa), bind
to the extracellular domain, the EGFR is activated. These ligands are normally
produced in the surrounding tissues as local growth factors. The activated EGFR
forms homodimers or heterodimers by
pairing with other receptors of the ErbB
family. This dimerization induces the tyrosine kinase activity of the
intracellular domain (1).

 

The overexpression of EGFR is
observed in a variety of epithelial cancers, such as breast cancer, non-small
cell lung cancer (NSCLC), and colorectal cancer (2).

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This over expression can cause resistance to apoptosis,
cancer proliferation, metastatic dissemination and neovascularization. It has
been reported that EGFR is over-expressed in 14–91% of breast
cancers. Because of these observations
EGFR is an interesting target for diagnosis and therapeutic strategies (1).

 

Two
distinct strategies have been applied to reduce and deactivate EGFR signaling.
The first approach is to block the intercellular domain of the receptor by
specific tyrosine kinase inhibitors.

 

These
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).

 

The
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 antibodies (mAbs).

 

Cetuximab/ErbituxR,
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.

One
of the drawbacks of mAbs is their large size which limits tumor penetration,
and reduces their
effectiveness; another problem concerning mAbs is that generation of new or
modified mAbs is costly and arduous. Both problems can be
solved by exploiting heavy chain only antibodies (HCAbs) from camelids (4, 5).

 

Whereas
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
or nanobody(6).

 

VHHs 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).

 

Single
VHH domains can be powerful diagnostic imaging tools, and are being developed
for a range of research applications (Steyaert and Kobilka, 2011; Vaneycken et
al., 2011). For therapeutic use, VHH domains (monomeric or multivalent) can be
modified to extend serum half-life and/or functionality (Saerens et al., 2008).

The
clinical success of EGFR-targeted mAbs has caused significant interest in
developing VHH domains that bind to and inhibit this receptor. Several
EGFR-specific VHH domains have been reported (Roovers et al., 2007; Roovers et
al., 2011) that have the potential to reproduce the clinical efficacy of mAbs
such as Cetuximab in an agent that is more stable and far less costly to
produce. 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 acids VHH domain, is a selected nanobody with
the highest affinity binding to EGFR.

This
VHH domain competes with Cetuximab for EGFR binding (Roovers et al., 2011).
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 can also be used 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 (briefly

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(8).

 

In another study(8) ,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
applications.

 

9G8,
a 136 amino acids 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
receptor.

 

As
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).

 

 

The
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.

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