APR January/February 2022 - 22

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FORMULATION AND DEVELOPMENT
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that can be gathered preclinically, the better scientists will be able to
select the correct compounds.
In Vitro Assays Only Show Part of the Picture
In vitro cytokine release assays involve culturing human peripheral
blood mononuclear cells (PBMCs)-including T cells, B cells, and
natural killer cells-with the drug of interest in a closed, controlled
environment. This approach allows scientists to measure cytokine
production, cytotoxicity, and targeted efficacy against cancer cells.
These assays are especially useful for drug target validation because
they are generally quick and inexpensive.
However, CRS is impossible to measure completely or accurately in
vitro because it is a systemic phenomenon that affects far more than
just the immune cells and cancer cells that interact with the substance
in the blood. Studies have also shown that in vitro assay results can
include false positives or negatives, and can also vary significantly by
drug mechanism of action (MoA). For example, in vitro assessments
of the TGN1412 monoclonal antibody yielded false negatives, failing
to predict the strong, dangerous CRS response that the human trial
participants experienced.2
How In Vivo CRS Evaluation Studies Capture
Systemic Information
Scientists create humanized mice by grafting adult human PBMCs
into immunodeficient mice, creating a functioning human immune
system circulating through and interacting with mouse vasculature
and organs. The grafted immune system is established after six days,
at which point researchers can inject a drug to study its effects.3
Monitoring for CRS over the following one to seven days involves
serially measuring body weight and temperature, quantifying human
cytokines at key time points, and performing flow cytometry and
other assays.
In addition to evaluating CRS, researchers can use serum analysis
and histology to investigate additional effects such as downstream
organ infiltration and toxicity. It is also possible to engraft tumors to
evaluate on-target efficacy. These assays are more predictive because
they capture human-specific immune responses within a complex
biological system. Furthermore, direct comparisons have shown that
in vivo CRS assays deliver more higher-quality data than in vitro assays.3
Decreasing Reliance on Non-Human Primate Trials
Non-human primates were previously the animal model that gave the
closest preclinical approximation of human physiology and disease
progression for drug safety and efficacy testing. However, studies in
these animals are extremely expensive, time-consuming, and ethically
weighty, and the approximation may not be as close as once thought.
More recent studies of TGN1412 concluded that the CRS response in
clinical trial participants had a direct connection to the CD28 receptor
on human CD4+ effector memory T cells. None of the animals used
for preclinical tests express this receptor, which is why the risk of CRS
went undetected.4
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Using Humanized Mice to Gather
New Data Types Preclinically
To maximize the potential of in vivo CRS evaluation studies, researchers
should not limit themselves to the same investigations performed
with in vitro assays and non-human primate models. With more robust
preclinical assessments, patients can receive safer, more effective
therapeutics even in early phases of clinical trials.
Early Dose Ranging
By engrafting tumors into humanized mice and measuring tumor
shrinkage following therapeutic administration, researchers can
compare efficacy and safety not only of different drugs but also of
different doses of the same drug. Using a single PBMC donor to avoid
confounding variables, scientists can perform dose ranging studies
that reveal the therapies and dosages that strike the ideal balance
of maximal tumor shrinkage with minimal toxicity. These preclinical
studies will allow for more strategic investments and have the
potential to accelerate final dose determination processes once the
therapeutics begin moving through clinical trials.
Modeling Diversity for More Accurate Safety and
Efficacy Predictions
Different individuals can have radically different responses to the
same therapeutic, even at the same dose. This diversity extends
to both efficacy and toxicities like CRS. The more diverse the study
population, the more likely a drug is to be accessible and beneficial
to the population as a whole. Fortunately, researchers have found
that humanized mice show reproducible, representative variation
in cytokine release levels depending on the PBMC donor used to
generate their human immune systems.3
Modeling the diversity of a patient population through precharacterized
PBMC donors provides drug developers with preclinical
results that may increase the chances of success for new therapies.
Some individuals may be highly sensitive and more likely to
experience toxic CRS from a given therapeutic. The inclusion of such
individuals in early clinical trials might cause the drug's maximum
tolerated dose to be set at a level below that which would be required
to produce efficacy in a broader population. With a representative in
vivo preclinical study, pharmaceutical developers can pinpoint factors
that increase sensitivity and risk of toxicity and use that information to
pre-screen high-risk individuals-who are unlikely to benefit from the
positive effects of the drug-out of the clinical trial. The trial can then
move forward and potentially yield a higher maximum tolerated dose,
increasing potential efficacy.
Ultimately, in vivo CRS evaluation studies with humanized mice
could enable a precision medicine approach to therapeutic
selection. Scientists could humanize mice using an individual
patient's PBMCs and perform testing that would allow them to
predict in a matter of weeks what treatments will be safest and
most effective for that patient.

APR January/February 2022

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