Multiplexing Phenotype and Function for More Biologically Relevant Insights - 32

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Although the cell-based assay was deemed less suitable for the trial, it is still a
source of pride for Dr. Wu: "To our knowledge, this is the first cell-based NAb assay
for a monoclonal antibody with ADCC as a mechanism of action. The assay development approach, optimization, characterization, and strategy for comparative
assessment of NAb assays may shed light on NAb assessment for other biopharmaceuticals with ADCC activities."
Optimizing Bioassays for Biosimilars
Despite the outcome of Dr. Wu's comparative study, ligand binding assays may not
always provide a suitable alternative to functional cell-based assays. Alpana Prasad,
Ph.D., product manager, DiscoverX, argued that for biosimilar molecules "the focus
needs to be functional-it needs to provide evidence of functional equivalency
between the originator molecule and the biosimilar."
Assay design can probe signaling pathways relevant to drug mechanism at
different stages. These stages, Dr. Prasad noted, could be categorized as the
"binding event," the "molecular event," and the "phenotypic event."
Dr. Prasad illustrated these stages with a relevant example: bevacizumab, which is
a monoclonal antibody that impedes tumor angiogenesis by inhibiting activation
of the vascular endothelial growth factor (VEGFR-2) receptor. The growth factor
VEGF-A, he explained, binds to the receptor (binding event), activating VEGFR-2
and causing dimerization (molecular event), which initiates blood vessel growth
and angiogenesis (phenotypic event).
DiscoverX optimized its functional cell-based assays by focusing on the molecular
event, or, in the case of their PathHunter® bevacizumab bioassay, VEGFR-2 receptor
dimerization. The bioassay uses the company's enzyme-fragment complementation technology, which results in a chemiluminescent signal when two enzyme
fragments, a donor-acceptor pair, come together. Thus, functional activation of
VEGFR-2 receptors tagged with the enzyme fragments will emit a signal upon
dimerization. However, if bevacizumab, or a biosimilar, blocks VEGF-A binding, it
inhibits dimerization and the resulting chemiluminescent signal.
DiscoverX has complemented the simplicity of its chemiluminescent readout with
the adoption of cryopreserved, ready-to-use cells-a growing trend in the industry.
In addition to decreasing cell-culture variability, the use of cryopreserved cells has
32

| January, 2019

Assay design can
probe signaling
pathways
relevant to drug
mechanism at
different stages.
These stages,
Dr. Prasad
noted, could
be categorized
as the "binding
event," the
"molecular
event," and the
"phenotypic
event."

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Multiplexing Phenotype and Function for More Biologically Relevant Insights

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