The Global Cancer Statistics report estimated that there were 18.1 million new cancer cases and 9.6 million cancer deaths in 2018, with over 50% of the cancer deaths in occurring in Asia . A new report predicts a 60% increase in the global number of cancer cases within the next two decades, with lung cancer continuing to be the leading cause of cancer deaths . Immunotherapies, especially inhibitors targeting programmed cell death protein 1 (PD-1) or its ligand (PD-L1), have become a focus of cancer research in recent years. However, the PD-L1 status of the tumor is correlated with the outcome of PD-1:PD-L1 blockade occurring outside the tumor microenvironment.
In recent years, tracers for radioimmune-imaging have provided a non-invasive alternative to traditional immunohistochemical (IHC) staining for monitoring of PD-L1 expression. Although 18F-fluorodeoxyglucose (18F-FDG) is the most commonly used radioactive tracer, its uptake is not tumor cell-specific and it can also be taken up by activated immune cells . Thus, several specific imaging agents, including monoclonal antibodies (mAbs; such as 89Zr-avelumab, 89Zr-atezolizumab, 89Zr-nivolumab), mAb fragments (such as minibodies and nanobodies [7, 8]), small proteins (such as 18F-BMS-986192 [6, 9]), and peptides (such as 64Cu-WL12 ), have been developed and investigated in preclinical models in addition to some early clinical studies.
Although the high specificity, affinity, and ready availability of full-length IgG antibodies provides some feasibility for imaging, the characteristics of mAb metabolism in liver lead to high uptake of radioactivity in the liver, and their large size (approximately 150 kD) limits tissue penetration, tumor retention and clearance from the circulation. Furthermore, the long half-lives of these proteins means that high-contrast images cannot be obtained in a short timeframe (several days are required) [11, 12]. To overcome these challenges, peptides and smaller antibody fragments (approximately 10 kD) have been developed for in vivo imaging. These agents provide superior imaging characteristics, such as rapid clearance from the circulation, higher tissue penetration, and higher signal-to-background ratios .
The protein dose has an important influence on imaging. The uptake of 68Ga-DOTA-TOC improved in the neuroendocrine tumour and decreased in liver and spleen as the peptide dose increased to 50 µg, whereas the uptake decreased in the lesions and healthy organs with further elevation of the peptide dose to 500 µg . The ratio of the lesion-to-liver uptake of 68Ga-ABY-025 was higher with a high peptide dose (427 µg) than with the low peptide dose (78 µg). The spleen, blood and tumor uptake of 111In-DTPA-anti-PD-L1 were significantly altered in the presence of excess (30- or 100-fold) unlabeled anti-PD-L1 mAb (spleen and blood, P ≤ 0.0002; tumor, P ≤ 0.05; all relative to the unblocked control) . In a study of the PD-L1 targeting tracer 64Cu-WL12, the imaging and biodistribution data showed high uptake in the tumor after pretreatment with low doses (0.06 mg/kg) of the anti-PD-L1 mAb, atezolizumab, compared with that at the higher doses of the mAb (0.6 and 3.6 mg/kg) . These findings indicate that the signal-to-background uptake was high relative to the unlabeled dose of the protein.
Nanobodies, also known as single domain antibodies, are small proteins (approximately 15 kDa). As a result of the suitable configuration of The conformation of the complementarity determining regions confer nanobodies with many features that are for imaging applications, such as rapid targeting and blood clearance, high solubility, and stability, ease of cloning, and the capacity to bind to cavities and difficult-to-access antigens .
Nb109 is a non-blocking nanobody with high specific affinity for PD-L1 (with an equilibrium dissociation constant (KD) of 2.9 × 10− 9 M) [18, 19]. The epitope to which by Nb109 binds differs from those bound by the therapeutic PD-1 and PD-L1 antibodies. After conjugation with the chelator 1,4,7- triazacyclononane-1,4,7-triacetic acid (NOTA) and labeling with the radionuclide 68Ga, 68Ga-NOTA-Nb109 can bind with PD-L1 in vivo, and accumulates specifically in locations with high PD-L1 expression, such as the A375-hPD-L1 tumor. The uptake ratio is associated with the PD-L1 expression level. In addition to the uptake in the tumor, the kidney showed relatively high uptake compared with the liver and other organs (33.7% vs. 1.1 % and < 1.5% ID/g, where ID%/g refers as the percentage injected dose per gram in each organ tissue).
In this study, we explored the suitable dose of 68Ga-NOTA-Nb109 by comparing the effect of the injected mass on the biodistribution in two strains of mice (including humanized mice), and conducted positron emission tomography/computed tomography (PET/CT) imaging in non-human primates to further verify the dose of 68Ga-NOTA- Nb109 based on the protein mass dose range determined. The biodistribution data of cynomolgus monkey were extrapolated to estimate the radiation dose for the adult male.