Cancer Genetics and Epigenetics 2017, Vol.5, No.7, 33-38
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results of PD-L1 staining can be affected by differential sensitivity towards different antibodies, difficulty in
interpreting positive stainingvalue, uneven expression of PD-L1 in tumors, sampling time and sampling position,
etc., thus decreasing the reliability and accuracy of immunohistochemical methods for detecting PD-L1. Therefore,
dynamic assessments of tumor microenvironment invivo, and the use of biological or radioactive markers to
perform live imaging will allow for an improved selection of patients that are likely to benefit from immune
checkpoint therapy (Wang et al., 2016). Immuno-PET imaging, on the other hand, is a non-invasive method used
to evaluate the pharmacokinetics of immune checkpoint mAbs and quantification the expression levels of tumor
markers (Mayer et al., 2016). Immuno-PET imaging uses radiolabeled antibodies to target and identify cells;
however, designing immuno-PET imaging tracers is not an easy task. Radiotracer options, antibody design, and
imaging kinetics, etc., are factors that need to be taken into consideration.
3.2 Choosing tracers for immuno-PET imaging of immune checkpoints
Immuno-PET is an advantageous imaging method, in that it is both high-resolution and high-sensitivity, and can
be used to quantify the invivobehaviorof administered mAbsand their interactions with target tissues. Determining
an ideal immune checkpoint tracer involves many factors pertaining to the cancer itself, such as its localization,
size, morphology, physiology, and sensitivity to radiation. Under normal circumstances, optimal
tumor-to-background ratio can be obtained 2-4 days after the injection of mAbs, which have limited the
application of long-lived radionuclides (van Dongen and Vosjan, 2010). Common metal positrons such as 99mTc,
86Y, 68Ga, or 64Cu have half-life duration of 15 hours, and will decay well before the radionuclide-tagged mAbs
have reached optimal tumor-to-background ratios. However, recently, theimaging of 64Cu and 68Ga-tagged mAbs
1 hour following injection have achieved some success in animal research. The use of long-lived radionuclides
will allow the imaging to be performed at optimal tumor-to-background ratios before decay of the mAb tag.
Examples of radionuclides with long half-life duration are 124I and 89Zr, which have half-lives of 4.18 and 3.27
days, respectively, making those better choices for use as tracers in Immuno-PET imaging. Different nuclides have
their own respective shortcomings; for instance, iodine (I) markers are relatively simple to use and to acquire, but
its decay process involves the activity of high energy positrons, which decreases its image resolution. Zirconium
(Zr) is a residual radionuclide which remains stable inside the body, and remains in target cells after catabolism.
As a result, 89Zr markers are often used with mAbs in Immuno-PET imaging. At present, 89Zr-tagged mAbs have
been used in many preclinical studies, include cetuximab, ibritumomabtituxetan, rituximab, bevacizumab, and
trastuzumab, etc (Wang and Yang, 2016). Choosing an ideal radionuclide is an important step while performing
immuno-PET; in addition, to allow the radionuclide to more efficiently localize and reach target tissues,
researchers have invented engineered antibody fragments, dual-function antibodies, small-molecule antibodies,
and other mAb fragments. In the future, studies will use improved antibody fragments to target proteins and
increase imaging resolutions (Wright and Lapi, 2013).
3.3 Preliminary advances in PET, SPECT, and optical imaging methods in the visualization of immune
checkpoint molecules
At Stanford University, Dr. Mayer A.T. and colleagues (Mayer et al., 2017) have developed 64Cu and 68Ga
radionuclide-tagged murine PD-1 mAbs, and imaged the expression of PD-1 on tumor-infiltrating lymphocytes in
murine CT26 colon carcinoma models, using Immuno-PET methods. Targeting these types of high-affinity protein
scaffolds (HAC-PD1), the group designed 6 HAC-PD1 radiotracer variants, and imaged tumor regions 1, 2, 4, 18
and 24 h after the injection of radioactive tracers, reporting that imaging tumor-bearing CT26 colon carcinoma
cell lines 1h postinjection resulted in the most prominent signal. These small proteins not only increase tumor
penetration, but also have biological characteristics that allow them to be used in conjunction easily with other
immunotherapy agents. For instance, the effects of co-administration of Nivolumab (a PD-1 blocker) and
Ipilimumab (a CTLA-4 blocker) have been proven in patients with melanoma (Wolchok et al., 2013). In addition,
many preclinical studies have been done to support their targeted synergistic effects on multiple types of immune
checkpoints, such as PD-L1, TIM-3, LAG-3, etc. These results shed light on the potential of small-protein mAbs
in research on immune checkpoint blockades, as well as their applicability in the modulation of the immune
