Selection of lead components for further pre clinical testin

Selection of ‘lead’ components for further (pre)clinical testing usually occurs on the basis of production yield, affinity and specificity, which should always be re-evaluated in their (radionuclide) labeled format. When internalization in cell line models is established, final validation is done in vivo with respect to tumor uptake, tumor-to-normal organ ratios and fast blood clearance. As such, selected components enable imaging as early as 1h post-injection, which contributes to patient safety. One drawback of this approach is the accumulation of radiolabeled nanobodies in the kidneys and bladder (Table 1), which is in fact a typical characteristic for small radiolabeled proteins and peptides. After glomerular filtration (cut-off of 60kDa), nonspecific reabsorption in the proximal tubuli may account for the residence in kidneys. Remarkably, D\'Huyvetter and co-workers showed that the highest accumulation occurs with Myc-His-tagged anti-HER2 nanobody, followed by His-tagged and finally untagged nanobody (70–88% less accumulation). The amino STA-4783 Supplier composition and polarity at the C-terminus thus predominantly affect kidney retention (D\'Huyvetter et al., 2014). This could be diminished with 90–95% by co-injecting gelofusine, an inhibitor of the megalin receptor responsible for tubular protein reabsorption. Similarly, Chatalic and co-workers showed that renal uptake of their His-tagged anti-PMSA nanobody could be reduced without loss of targeting by co-injecting a combination of gelofusine and the positively charged amino acid lysine or by removing the His-tag from the nanobody (Chatalic et al., 2015). Also the untagged 68Ga-labeled anti-HER2 nanobody showed a 50% drop in renal accumulation compared to its His-tagged equivalent (Xavier et al., 2013). Another determining parameter can be the type of chelating agent, as significantly higher kidney uptake was reported for the DOTA-based conjugates compared to the DTPA-based conjugates of anti-HER2 nanobody (D\'Huyvetter et al., 2012). Finally, albumin binding via a second nanobody could be a strategy to reduce renal retention, although this option is not preferred due to increased blood residence time and thus increased radiation exposure (Vosjan et al., 2012). For the case of 99mTc-labeled anti-MMR nanobodies to visualize tumor-associated macrophages, the accumulation was higher in liver and spleen (expressing MMR) as compared to the tumor (Movahedi et al., 2012). The authors solved this by administering an excess of unlabeled bivalent anti-MMR nanobody, which eliminates extratumoral signals while maintaining the targeting of tumor-associated MMR-positive cells. Currently, the best established nanobody-based imaging agent is the 68Ga-coupled anti-HER2 nanobody 2Rs15d for PET imaging (Xavier et al., 2013). Preclinical evaluation revealed a good toxicological profile and a low radiation burden, enabling the construct to enter phase I clinical trials on humans (Keyaerts et al., 2015). The construct scored well in terms of efficiency, tracer accumulation and safety as no adverse effects or antibodies against the administrated nanobody were detected, rendering this construct suitable to enter phase II clinical trials. One drawback however was the uptake of the agent in liver and intestines, next to its expected accumulation in the kidneys. This might obscure liver metastasis, although no such metastases were present in the patient group and the question could thus not be solved. As the liver uptake decreases over time, a later time point of imaging is proposed by the authors to improve signal-to-noise ratio at the liver if needed (Keyaerts et al., 2015). When using β-emitting radioisotopes such as 131I and 177Lu, a therapeutic effect can also be pursued by their ionization and DNA damaging activity. Such a radionuclide-based construct enabling both imaging and therapeutic use is termed ‘theranostic’. Gelofusin coinfusion with untagged 177Lu-DTPA-anti-HER2 nanobody almost completely blocked tumor growth in xenograft mouse models bearing small tumors, coinciding with increased event-free survival (D\'Huyvetter et al., 2014). Moreover, non-specific radiation to healthy tissues was absent. Although the system causes a comparable radiation level for both tumor and kidneys, histopathological analyses of renal tissue revealed no visible toxicity, although long-term events cannot be excluded (D\'Huyvetter et al., 2014). Characterization of this agent should be continued in clinical trials to highlight the potential of radiolabeled nanobodies as a valuable therapy. Also the radio-iodinated 131I-anti-HER2 nanobody shows potential for treatment of HER2-overexpressing malignancies (Pruszynski et al., 2013). Residualizing agents are used for radioiodination to avoid lysosomal degradation after receptor-mediated internalization, which would result in rapid loss of radioactivity. Such agents (for example containing guanidine or d-glutamates) render the construct ‘charged’ in lysosomes to prevent its diffusion across membranes and further avoid action of deiodinases. However, also very high radioactive levels were observed in the kidneys for the resulting constructs. The authors showed later on that radio-iodination via another method was superior in terms of tumor retention and coincided with lower uptake in normal tissues including the kidneys (Pruszynski et al., 2014), pointing to the importance of optimizing the labeling method.