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Page 750 Lyons et al. Cancer Drug Resist 2021;4:745-54 https://dx.doi.org/10.20517/cdr.2021.37

induced mice will become antigen positive and show ADC accumulation.

It is significantly easier and faster to manipulate gene expression in cell line-based cancer models than in
transgenic mice. Therefore, it is exciting that the advances in tumor modeling discussed above are to some
extent tissue-culture based, as this opens up many possibilities for their efficient genetic manipulation and
thus thorough and rigorous in vivo experimentation.

ADVANCES IN PRECLINICAL IMAGING
Depending on the nature of the conjugated moiety, ADC molecules can be considered “theranostic”, a term
used to describe a molecule with both therapeutic and diagnostic properties. Once a candidate ADC
molecule has satisfied stringent in vitro and tissue-histology performance criteria, preclinical imaging can be
used to dynamically measure both aspects of this, i.e., to visualize the biodistribution of an ADC molecule in
the context of the whole body over time and to accurately measure the anticancer effects of ADC treatment.
Critically, imaging can be used to standardize the timing of ADC administration (e.g., on the basis of tumor
size) across experimental cohorts of mice and can provide meaningful “before and after” ADC treatment
measurements of the same tumor in the same individual. Collectively, these experimental advantages serve
to reduce the number of assumptions made in an in vivo study, greatly improving study robustness and
reducing animal cohort size. Non-invasive imaging is also particularly pertinent in the context of the tumor
organoid model advances discussed above, as tumors development in orthotopic and deep tissue locations
are otherwise not visible externally.

Rather than comprehensively review the field of preclinical imaging, we instead mention here several key
imaging practices that we believe are particularly impactful for current and future ADC development.

Approaches to image antibody biodistribution in vivo
ADCs are extremely versatile tumor targeting molecules due to the broad variety of functional groups that
can be attached to them, whether therapeutic “warheads” used to treat cancer or diagnostic ones for tumor
imaging. All preclinical imaging modalities have relative strengths and weaknesses and so the conjugated
imaging moiety should be selected based on the nature of the experimental goal. It should also be noted that
different conjugates will influence the in vivo biodistribution of an ADC and that not all variant ADC can be
presumed to perform equally.

[21]
In the preclinical space, the attachment of a fluorescent moiety (e.g., an Alexa Fluor dye ) to a candidate
ADC can uniquely enable direct visualization and accurate quantitation of antibody binding to target cells
by microscopy or flow cytometry in vitro. However, fluorescence imaging is in general poorly suited for
whole-body in vivo imaging, as visible wavelengths of light are poorly tissue penetrant and prone to scatter
[22]
and absorption from overlying tissues . Autofluorescence generated from surrounding gram amounts of
non-labeled tissue can also prove troublesome in the context of attempting to detect a signal above
background from small, milligram-sized tumors. Intravital microscopy, however, is a unique in vivo
fluorescence-based imaging approach that allows the researcher to look at the accumulation of labeled
antibody at the site of tumor development at sub-cellular resolution in vivo . No other mainstream in vivo
[23]
imaging modality can offer this kind of imaging scale, but it should be noted that IVM only offers a small
field of view with limited depth of tissue penetration (0.5 mm) and is only suitable for imaging through an
implanted “window” at a single location in the body. Fluorescent immunopeptides are also of additional
clinical interest and are being developed for guided intraoperative imaging purposes, to assist surgeons to
[24]
both locate tumors and set resection margins in theatre . We speculate that recent advances in tissue
clearing techniques and light sheet microscopy may also enable visualization of fluorescent ADC binding at