Molecular imaging has played a role of great importance in noninvasive detection and quantification of EGFR expression in clinical research.(16, 42–44) PET is an imaging method that can provide superior sensitivity and quantitation accuracy compared to SPECT(45). Gallium-68 is a suitable positron-emitting radionuclide for clinical PET imaging. The merits of this radionuclide include the good availability of gallium-68 because of generator production and short half-life of 67.6 min, which results in low absorbed dose burden to patients. These advantages are conducive to clinical translation and underpin the remarkable expansion of clinical research with gallium-68 labeled radiopharmaceuticals, especially the ones based on rapidly cleared proteins(46).
We found that 68Ga-NOTA-ZEGFR:1907 only focused on the expression of EGFR protein and the uptake of that was merely correlated with total protein expression of EGFR (the order of uptake in tumor-bearing mice: HCC827 > H1975 > A549 > H358 > H520). However, the probe was not associated with EGFR mutants. Such results are consistent with the principle of targeted binding of 68Ga-NOTA-ZEGFR:1907. As we all know, EGFR is a single-chain transmembrane glycoprotein comprising an extracellular ligand-binding domain, a transmembrane region, and an intracellular tyrosine kinase domain. ZEGFR:1907 is specifically bound to the extracellular segment of EGFR, and it cannot bind to the intracellular tyrosine kinase domain. Therefore, the degree of intracellular mutation of EGFR could not be distinguished.
Anti-EGFR affibody ZEGFR:1907 has a similar molecular weight to epidermal growth factor (EGF) (7 kDa vs. 6.4 kDa). However, compared with 68Ga-labeled EGF-based radiotracers, 68Ga-NOTA-ZEGFR:1907 was higher in tumor uptake (1.51±0.16 vs. 2.07±0.73 %ID/g) at 30min p.i..(47) Moreover, tumor accumulations of 18F-FBEM-cEGF in UM-SCC1 xenografts were 1.87 ± 0.44 and 0.98 ± 0.33 %ID/g at 60 and 120 min p.i. respectively(14), while that of 68Ga-NOTA-ZEGFR:1907 were higher (2.58 ± 0.15 and 2.69 ± 0.14 %ID/g at 60 and 120 min p.i.). These results indicate that affibody-based radiotracer exhibits an extended retention time compared to EGF-based radiotracers.
In view of the biological half-life of antibodies is very long, such as that of cetuximab is 65 to 95 hours in the blood(48), a radioactive tracer with long half-life is needed to visualize its uptake, resulting in an increase in the patient's radiation dose. As for 68Ga-NOTA-ZEGFR:1907, both physical half-life of Gallium-68 and biological half-life of affibody are shorter and suitable for clinical application. Moreover, given that probe accumulation in tumors depends on physiological characteristics such as vascular permeability, tumors with high EGFR expression do not necessarily exhibit high uptake of radiolabeled antibodies.(49–51) In addition, 89Zr-labeled affibody demonstrated specific uptake in EGFR-expressing tissues, also tumor-to-organ ratio of that was higher compared with 89Zr-labeled antibody.(52)
The accumulations of 68Ga-NOTA-ZEGFR:1907 in most organs were lower except liver and kidney, mainly attributed to the fact that they are the major organs responsible for metabolism and clearance. More importantly, it is known that the high natural expression of EGFR in the liver. Liver accumulation of 68Ga-NOTA-ZEGFR:1907 was slightly lower in HCC827 tumor models compared to 64Cu-DOTA-ZEGFR:1907.(38) The unbound probe is rapidly cleared from blood via the kidneys, which makes it possible to obtain high contrast images after only a few hours after injection(53). Low 68Ga-NOTA-ZEGFR:1907 uptake in the lung region was detected, which was consistent with low endogenous EGFR expression reported in this organ.(54) Importantly, low accumulation in lung offers the considerable advantage of this probe in identifying primary or metastatic lung tumors expressing EGFR. Despite large injection mass of 68Ga-NOTA-ZEGFR:1907, no pathological changes were detected in the liver, kidneys, spleen, or lungs in biotoxicity assays, indicating that 68Ga-NOTA-ZEGFR:1907 is safe and therefore suitable for clinical applications.
Notably, 68Ga-NOTA-ZEGFR:1907 could not detect very low (0-1 point) EGFR-expressing tumors. In tumors with weak (2-3 points) EGFR expression, 68Ga-NOTA-ZEGFR:1907 accumulations were also slightly high, without completely correlation with IHC scores. Thus, it is possible that a minimum threshold of EGFR expression is required for reliably detecting EGFR levels with the radiotracer. In remaining moderate-high EGFR-expressing tumors (4-7 points), IHC scores and gray scale values were both significantly elevated. Taken together, our results indicate that a radiotracer-based gray scale value of ≤ 1.99 × 106 DLU/mm2 may be used as cutoff value to rule-out low EGFR-expressing tumors patient subgroups. Moreover, based on the IHC staining results, a cutoff value of ≥ 2.42 × 106 DLU/mm2 may identify moderate to high EGFR-expressing tumors. Besides, these cutoff values should be considered as exploratory, on account of that our study was based on a small sample. Finally, there were overlaps between the areas stained by EGFR antibodies and those detected by 68Ga-NOTA-ZEGFR:1907 in tissue samples, further demonstrating that the radiotracer specifically binds to EGFR in human tissues.
Although in vivo subcutaneous transplant tumor models provide extremely valuable information, future preclinical researches using orthotopic inoculation models, as well as clinical studies for 68Ga-NOTA-ZEGFR:1907 PET imaging in patients, are necessary for the clinical translation of this probe. Our data from this pilot study were limited, nevertheless, they provide a proof-of-principle of the clinical potential of 68Ga-NOTA-ZEGFR:1907 for PET imaging and quantifying EGFR expression in human tumors, and for monitoring the efficacy of EGFR-targeted therapy, such as anti-EGFR monoclonal antibodies cetuximab and panitumumab.(55)