Bound to succeed
MPI Research and Perfinity Biosciences are working
in partnership to streamline and
validate bioanalysis of antibody drug conjugates.
Vice President of Bioanalytical/Analytical
Services Alan Breau and Senior Scientist Kevin
Meyer come together to explain the process.
Antibody drug conjugates (ADCs) link precisely
targeted antibodies to cytotoxic small-molecule
drugs,
with the goal of improving both the safety and
efficacy of chemotherapeutics. Pivotal clinical
trials of
trastuzumab-DM1 for women with treatment-resistant
HER2-
positive breast cancer and of SGN-35 for relapsed/refractory
Hodgkin’s lymphoma represent the vanguard
of several dozen
ADCs currently in various stages of clinical development.
In
ADCs, the antibody acts primarily as a carrier
to deliver the
potent drug to the target tissue, thus limiting
total body
exposure to the free and potent small molecule,
a strategy that
can mitigate toxic side effects. However, ADCs
pose several
challenges, including the tendency for the covalently-bound
small molecules to detach and circulate freely
in the body, the
difficulty of manufacturing conjugates reproducibly
and the
metabolic fate of the conjugate and the antibody.
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"We are proud to be
leading the
scientific advances necessary to allow
ADCs to achieve commercialisation
and widespread clinical application."
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Currently the analysis of ADCs requires four
distinct assays on each plasma sample, two small
molecule assays and two ELISA assays. The small
molecule assays detect freely circulating drug
and bound drug that is chemically released from
circulating ADCs.
Free or bound?
MPI Research and Perfinity Biosciences are currently
collaborating
to streamline and validate this process. One option
is to measure the
free and bound small-molecule levels by using
immunosorbent
technology to trap free drug for analysis, while
the proteincontaining
eluate is collected and hydrolysed to liberate
bound drug
for analysis. Thus, free and bound small molecules
could be analysed
from the same serum sample rather than from duplicates.
The proteins could be measured using two different
ELISA
assays: the small-molecule free antibody could
be measured
by conventional ELISA with an antibody that recognises
the
antibody/linker and the ADC, and the antibody-containing
drug
molecule could be measured by an ELISA that utilises
an antibody
that recognises the small molecule/linker portion
of the ADC.
To further complicate analysis, ADCs are not
homogenous
products, but rather a mixture containing differing
numbers of
potent small molecules attached to each antibody.
Therefore, the
batch-to-batch distribution differences and the
kinetics of the
changing small-molecule distribution in vivo may
impact the clinical
effectiveness of the compounds. Currently only
gross averaged data
are obtained, which may confound an accurate pharmacokinetic/
pharmacodynamic prediction or batch potency determination.
Simultaneous quantification of total ADC, in
vivo fragments of the
ADC and drug conjugated at each site in the ADC
can be achieved
using MRM technology and carbon 13-labelled isopotomers
of
peptides and drug-bearing peptide conjugates.
This approach
quantifies small molecules linked at specific
locations on the
antibody and tracks their binding on the antibody
to provide a more
accurate indicator of the potency of each batch,
as well as its
potential in vivo performance.
High performance
Relative binding affinity with receptors also
affects how well ADCs
perform. For example, if antibody without any
drug has a higher
affinity for the receptor than antibody with drug,
then
the presence of drug-free antibody would block
the desired binding
of antibody-containing drug, potentially attenuating
the ADC’s
efficacy. We believe an assay that traps the ADC
using
an immobilised receptor protein or antibody, followed
by
deglycosylation and enzymatic digestion of the
protein, would yield
a series of peptides that could be analysed by
liquid chromatography
and mass spectrometry. The peptides that contain
drug/linker would
differ in retention and molecular weight from
the analogous peptides
that did not contain a linked small molecule.
If tracked sites that
were more labile to in vivo release could be identified,
then product
specifications could limit the amount of ADCs
containing linked
drug at these labile sites. The result would be
a safer, more potent
ADC mixture.
ELISA assay sensitivity need not be an issue.
For example, the
published assay of Herceptin – which has
which has an average
molecular weight of 145,531 daltons – has
a validated range of range
from 5-100 ng/ml. The samples in the published
ELISA are diluted
one to two-thousand-fold to correspond to an assay
of 10-200 ìg/ml.
The published Cmax values for Herceptin are in
the ìg/ml range,
suggesting that the sensitivity of many ELISA
assays is too low for
protein therapeutics.
We believe that new bioanalytical and analytical
processes will be
necessary to support emerging ADC therapeutics.
To that end, MPI
Research and Perfinity Biosciences, in consultation
with Professor Fred
Regnier who founded Perfinity Biosciences, are
actively investigating
innovative bioanalytical/analytical methods for
ADCs. We are proud to
be leading the scientific advances necessary to
allow ADCs to achieve
commercialisation and widespread clinical application.
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