Biomedical Engineering Reference
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in parallel signaling pathways. In the case of glioblastoma
multiforme it has been reported that tumor tissues, but not
normal brain tissues, can be associated with activation of
multiple kinase receptors, including EGFR, PDGFR-
a
,
RET, and CSF-1R [7]. Based on these observations and
supporting in vitro data, it has been proposed that such
receptor tyrosine kinase coactivation is responsible for the
poor clinical responses observed with monotherapies. More-
over, evidence is emerging to suggest that the treatment of
cancer patients with a monotherapy may actually drive
additional alterations in other pathways that ultimately
lead to acquired drug resistance. In one example, selection
of tumor cells resistant to the EGFR inhibitor cetuximab was
associated with significant increases in phospho-ErbB2,
phospho-ErbB3, and phospho-cMet [21]. It was noted
that cMet in these resistant cells, but not in the parental
cells, could be activated with EGF, suggesting that receptor
tyrosine kinases may act in a cooperative manner to over-
come drug-mediated inhibition. In a similar case, when a
non-small cell lung carcinoma (NSCLC) cell line HCC827
was rendered resistant to the EGF receptor inhibitor gefitinib
through prolonged low-dose exposure significantly height-
ened levels of phospho-ErbB3 and phospho-cMet were
found. Notably, these cells were resistant to inhibitors of
EGFR or cMet when used alone but were sensitive when a
combination of inhibitors was used [22]. This result was
further supported by an analysis of NSCLC patients' tumors.
Those tumors that were originally sensitive to EGFR inhibi-
tors but subsequently became resistant to these drugs had a
high incidence of MET gene amplification. Other groups
have found that combining inhibitors of IGF-1 receptors
with EGF receptor antagonists could also result in enhance-
ment of antiproliferative and proapoptotic activity in
NSCLC cell lines [23]. In aggregate, numerous preclinical
and clinical reports support the notion that the targeting of
multiple pathways may be critical in affecting strong clinical
responses in complex diseases such as cancer. As a result of
such observations, testing combinations of drugs that target
cooperative pathways is becoming increasingly common.
Based on preclinical evidence demonstrating functional
synergy, combinations of EGFR inhibitors and other tar-
geted therapeutics such as mTOR inhibitors are progressing
into clinical trials [24].
While the rationale for combining different targeted
therapeutics continues to grow stronger, the regulatory
process for the approval of investigational drug combina-
tions, particularly where the individual drug candidates may
have only marginal efficacy when used alone, remains
difficult. Moreover, the relatively high price of biotherapeu-
tics, including antibodies, can become prohibitively expen-
sive when used as combinations. As a result, a number of
groups have looked into the possibility of creating single
molecules that can simultaneously inhibit multiple path-
ways. Within this new category of drug candidates are the
antibody-like molecules engineered to have multiple bind-
ing specificities. With the recent approval of the Catumax-
omab, a bispecific antibody derived from a mixed-specificity
quadroma, and promising clinical results with the
CD19/CD3 bispecific tandem binder, Blinatumomab, multi-
specific antibody-like molecules appear to have a very
promising future. Although these particular molecules act
through recruitment and engagement of acquired immune
cells with one binding arm together with tumor cell targeting
with the other arm, other publications have demonstrated
enhanced preclinical activity in which the targets involved
are members of parallel activation pathways. For example,
as both osteopontin (OPN) and vascular endothelial growth
factor (VEGF) play a role in tumor angiogenesis, a bispecific
antibody was produced that inhibited the biological activi-
ties of both of these soluble factors [25]. This engineered
antibody demonstrated much greater antiangiogenic activity
in vitro and in vivo than either conventional parental anti-
body when tested individually. A similar observation was
made by another group using a bispecific antibody targeting
VEGF together with the receptor for PDGF-
b
[26]. In
addition, Robinson et al. [27] created a bispecific antibody
that bound both of the cancer-associated EGF receptor
family members, ErbB2 and ErbB3. In this instance, the
bispecific antibody achieved selective binding to cells that
coexpressed both target proteins, presumably because of the
increased avidity of this molecule for these cells rather than
those that only expressed one of the target proteins. Owing to
this selective targeting, it was hypothesized that such a
molecule may have an improved safety profile versus mono-
specific antibodies.
One of the key technological hurdles in the development
of clinical-grade bispecific antibodies is random pairing of
heavy and light chains when chains comprising different
specificities are expressed within the same cell. Maintaining
the proper light and heavy chain association is necessary to
retain the target specificity of the original antibodies. To
ensure the correct pairing of heavy and light chains in a
bispecific antibody, several strategies have been developed
over the years. Creation of single chain variable fragments
(scFv), formed by linking the heavy chain to the light chain
with a flexible peptide linker, has proven to be a valuable
method of generating a single binder with high heavy and
light chain pairing fidelity [28]. While scFvs have reportedly
suffered from stability and expression liabilities, improve-
ments in the understanding of molecular interactions
between the variable region domains has permitted this
technology to advance to a clinically acceptable state, as
exemplified by Blinatumomab currently in Phase II trials.
Another solution to the chain-pairing problem takes advan-
tage of the preferential association of mouse and rat light
chains for their respective heavy chain counterparts [29].
Quadromas generated from a fusion of mouse and rat
hybridomas have species-specific heavy and light chain
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