The site of the EGFR tyrosine-kinase domain. The literature

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. While
antibodies that bind to EGFR 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 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
(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 are being used for research and diagnostic applications. For therapeutic
use they can be modified to extend serum half-life and functionality (8).

The
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). Furthermore, multivalent
VHH molecules that bind more than one targets can be developed, offering the
potential to engineer multivalent agents that combine cetuximab-like EGFR
inhibition with other modes of binding to (10,
11).

 

7D12, a 133-amino acid VHH domain, is a selected nanobody with the
highest affinity binding to EGFR.

This
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 (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(12).

 

In another study (12),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 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
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 (10). 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 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
signalling.

 

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).