Nanobodies are a novel type of immunoglobulinlike, antigenbinding protein with beneficial pharmacologic and pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. However, because of their camelid, nonhuman origin, the possible immunogenicity of Nanobodies when used in the clinic is a concern. Here we present a new strategy to quickly generate humanized Nanobodies for molecular imaging purposes. Methods: We genetically grafted the antigen-binding loops of NbCEA5, a Nanobody with specificity for the colon carcinoma marker carcinoembryonic antigen (CEA), onto the framework of a humanized Nanobody scaffold. This scaffold has been previously characterized in our laboratory as a stable Nanobody that can serve as a universal loop acceptor for antigen-binding loops from donor Nanobodies and has been additionally mutated at about 10 crucial surface-exposed sites to resemble the sequence of human variable immunoglobulin domains. The 3 recombinant Nanobodies (NbCEA5, humanized scaffold, and humanized CEA5 graft) were produced in bacteria and purified. Unlabeled and 99m Tc-labeled Nanobodies were biochemically characterized in vitro and tested as probes for SPECT/CT of xenografted tumors. Results: The success of loop-grafting was confirmed by comparing these Nanobodies for their capacity to recognize soluble CEA protein in enzyme-linked immunosorbent assay and by surface plasmon resonance and to bind to CEA-positive LS174T colon carcinoma cells and CEAtransfected but not untransfected Chinese hamster ovary cells in flow cytometry. Specificity of binding was confirmed by competition studies. All Nanobodies were heat-stable, could be efficiently labeled with 99m Tc, and recognized both soluble and membrane-bound CEA protein in binding studies. Finally, biodistribution experiments were performed with intravenously injected 99m Tc-labeled Nanobodies in LS174T tumor-bearing mice using pinhole SPECT/micro-CT. These in vivo experiments revealed specificity of tumor targeting and rapid renal clearance for all Nanobodies, with low signals in all organs besides the kidneys. Conclusion: This study shows the potency of antigenbinding loop-grafting to efficiently generate humanized Nanobodies that retain their targeting capacities for noninvasive in vivo imaging of tumors.

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The radiation dose of a standard micro-CT scan is relatively high and could influence the experimental outcome. We believe that the presented adaptation of the scan protocol allows for accurate imaging without the risk of interfering with the experimental outcome of the study.

Background: Nanobodies are single-domain antigen binding fragments derived from functional heavy-chain antibodies elicited in Camelidae. They are powerful probes for radioimmunoimaging, but their renal uptake is relatively high. In this study we have evaluated the role of megalin on the renal uptake of anti-EGFR 99m Tc-7C12 nanobody and the potency of gelofusine and/or lysine to reduce renal uptake of 99m Tc-7C12. Methods: First we compared the renal uptake of 99m Tc-7C12 in megalin-deficient and megalin-wild-type mice using pinhole SPECT/microCT and ex vivo analysis. The effect of gelofusine and lysine administration on renal accumulation of 99m Tc-7C12 was analyzed in CD-1 mice divided into lysine preload at 30 min before tracer injection (LysPreload), LysPreload R gelofusine coadministration (LysPreload R GeloCoad), lysine coadministration (LysCoad), gelofusine coadministration (GeloCoad) and LysCoad R GeloCoad. The combined effect of gelofusine and lysine on tumor uptake was tested in mice xenografts. Results: Renal uptake of 99m Tc-7C12 was 44.22 W 3.46% lower in megalin-deficient compared with megalinwild-type mice. In CD-1 mice, lysine preload had no effect on the renal retention whereas coinjection of lysine or gelofusine with the tracer resulted in 25.12 W 2.99 and 36.22 W 3.07% reduction, respectively. The combined effect of gelofusine and lysine was the most effective, namely a reduction of renal retention of 45.24 W 2.09%. Gelofusine and lysine coadministration improved tumor uptake. Conclusion: Megalin contributes to the renal accumulation of 99m Tc-7C12. Gelofusine and lysine coinjection with the tracer reduces the renal uptake while tumor uptake is improved. Although this methodology allows for optimization of imaging protocol using nanobodies, further improvements are needed before using these molecules for radionuclide therapy.

IV administration offers better repeatability and better sensitivity when compared to IP. In larger tumours, multiple factors may contribute to underestimation of tumour burden. It might, therefore, be beneficial to test novel therapeutics on small tumours to enable an accurate evaluation of tumour burden.