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Lyons et al. Cancer Drug Resist 2021;4:745-54 https://dx.doi.org/10.20517/cdr.2021.37 Page 751

[25]
cellular resolution in reconstructed 3D images of tissue .

Optoacoustic (or photoacoustic) imaging is similar in nature to fluorescence-based approaches, but it is
better suited for deep tissue imaging. Instead of attaching a fluorophore, this approach relies upon the
conjugation of a light absorbing or quenching moiety [26,27] , i.e., the attachment of a molecule with a high
molecular extinction co-efficient and low quantum yield. Unlike a strong fluorescent label, whereby
absorbed excitation energy is largely emitted as red-shifted light, a strong photoacoustic label
predominantly emits absorbed excitation energy as heat. When the excitation light is pulsed in nanosecond
bursts, the resulting pulses of heat (and associated pulses of thermal tissue expansion) generate an
ultrasound signal that can be readily detected. Most imaging modalities offer either high resolution images
with poor image sensitivity (e.g., MRI) or highly sensitive and poorly resolved images (e.g., PET). While not
yet offering subcellular image resolution similar to fluorescence imaging, optoacoustic imaging is a good
compromise whole-body and clinically translatable imaging modality, offering both reasonable imaging
resolution and sensitivity at deeper tissue sites.

Arguably the most sensitive and quantifiable way to non-invasively track the biodistribution of an ADC
candidate molecule throughout the whole body is by PET (positron emission tomography). Full-size
antibodies have a relatively long serum half-life in vivo and so conjugation of the positron emitting isotope
Zirconium 89 (t = 3.3 days) allows follow up biodistribution scans for more than a week after
1/2
administration . Unlike optical signals, the gamma rays detected by PET are much less prone to scatter or
[28]
attenuation from overlying tissue. Accordingly, resultant tomographic images are not surface-weighted and
allow for the detection and discrimination of individual lesions in relatively close proximity, irrespective of
depth in tissue. Derivative antibody fragments, such as Fabs or diabodies, have a significantly shorter serum
half-life, often clearing the body within 24 h, and so can be adequately imaged by PET with more
conventional and shorter-lived radioisotopes, such as Fluorine 18 .
[29]
In the absence of a formal biodistribution study, whole-body PET images of an antibody can be used to
estimate where in the body the same ADC with a different therapeutic radioisotope attached [such as
Yttrium 90 (β-emitter) or Actinium 225 (α-emitter)] would accumulate in the body. As mentioned above,
ADCs with different conjugated moieties cannot be assumed to behave in an identical fashion in vivo.
Matched pairs of imaging and diagnostic radioisotopes [such as Scandium 44 (positron emitter for PET
imaging) and Scandium 47 (β-emitter for therapy)] are being developed to circumvent such issues, lending
greater predictive power to the PET scan .
[30]

Advances in imaging the therapeutic effects of an ADC
In addition to being able to non-invasively track the biodistribution of a candidate ADC molecule,
preclinical imaging can be equally useful in providing a dynamic and quantifiable measure of tumor cell
cytotoxicity and therapeutic effect.

The most obvious preclinical modality to mention in this context is bioluminescence imaging (BLI). For
reasons of relative sensitivity, speed, safety (does not involve ionizing radiation), and affordability, this
[31]
optical imaging modality has been widely adopted by the research community . The BLI signal is reliant
upon first introducing the expression of a luciferase transgene into the tumor model of interest. An
expanding list of candidate enzymes and substrates suitable for BLI have now been described ; however,
[32]
enzymes that rely upon ATP to generate light are the most useful for assessing relative tumor cell viability in
vivo. A highly relevant feature of these enzymes is that dead cells no longer emit light and so the cytotoxic
effects of any drug or ADC can be readily assessed following treatment. This viability measure is often