Development of radiopharmaceuticals for cancer treatment

Introduction:
Nanobodies (Nbs) are small (15 kDa) antibody fragments with beneficial pharmacokinetic properties, and those targeted to HER2 can be used for the molecular imaging of HER2 overexpressing breast cancer. Labelled with a therapeutic radionuclide, these Nanobodies may be used for the treatment of HER2 overexpressing cancer. However, long term tumour exposure is limited, which makes them suboptimal for therapeutic applications. Binding kinetics of Nanobodies can be improved by developing bivalent constructs, which may enhance cellular retention. During the past year, the monovalent Nanobodies 2Rb17c and 2Rs15d were selected to develop the bivalent constructs 2Rb17c-2Rb17c, 2Rs15d-2Rb17c and 2Rb17c-2Rs15d. Furthermore, we recently observed that the C-terminal sequence has an important role in the extent of renal retention of Nbs, labeled with radiometals. By removing this sequence, renal retention was lowered by >50%. Consequently, we evaluated the difference in renal retention between the his6-tag and the notag construct of the radioiodinated monovalent 2Rs15d nanobody
Materials and Methods:
The Nanobody constructs were designed and produced according to a previously established protocol. They were labeled with radioiodine using the residualizing prosthetic group N-succinimidyl 4-guanidinomethyl-3-iodobenzoate ([*I]SGMIB). Improved cellular retention and in vivo behavior have been demonstrated when antibodies and their fragments were radioiodinated using [*I]SGMIB. Affinities of the radiolabeled Nanobody constructs were obtained on HER2+ BT474/M1 cells. Internalization assays were performed in a pair-label format at 37°C over 0, 1, 2, 4, 6, 24h, using Nanobodies labeled with either [125I]SGMIB or [131I]SGMIB. The constructs, labeled with AlexaFluor488, were analyzed for internalization using adherent HER2+ BT474/M1 cells. After DAPI staining, fluorescence images were obtained at incubation time points (37°C) 0-2-6-24h with a Zeiss Axio Observer widefield fluorescence microscope, through a 63x oil immersion objective lens. A preliminary biodistribution experiment in normal mice was performed to compare the monovalent [*I]SGMIB-Nanobody with and without the hexahistidine C-terminal tag. Animals were killed at 1, 4, 12, and 24h.
Results:
KD values of 0.17 ± 0.02, 0.58 ± 0.15 and 1.05 ± 0.1 nM were obtained for radiolabeled 2Rb17c-2Rb17c, 2Rb17c-2Rs15d and 2Rs15d-2Rb17c compared to 4.73 ± 0.4 and 1.7 ± 0.2 nM for 2Rs15d and 2Rb17c, respectively. The internalization assays with the [*I]SGMIB-Nanobody constructs revealed a significant higher degree of cellular retention (membrane bound + internalized fraction) for all three bivalent constructs at all time points compared to their monovalent counterparts, with values after 24h incubation of about 60% of the bound activity for the bivalent Nanobody constructs versus about 25% of the bound activity for the monovalent Nanobodies. The fluorescent images gave a visual confirmation of the higher cellular retention of the bivalent Nanobody constructs. Biodistribution results with the monovalent [*I]SGMIB-Nanobdy with and without the his6 C-terminal tag for major organs. Both exhibited fast washout from the blood due to their small size, with low accumulation in liver and spleen. The low thyroid uptake values suggest a low level of dehalogenation of the [*I]SGMIB-Nb conjugate. An unexpected observation was the rapid washout of radioactivity from the kidneys, with only about 2%ID/g remaining at 4h post injection; by 12 h, less than 0.5%ID/g remained in the kidneys. No significant differences in kidney retention were observed between the 2Rs15d Nb with and without the -his6tag.
Conclusion:
The generation of hetero- and homobivalent dimers from HER2 targeted Nb monomers increased affinity and also resulted in an up to 3.5 fold increase in cellular retention of radioactivity. The latter characteristic should be particularly important for therapeutic applications. Although the mechanism for this behavior is not known at this time, SGMIB labeling resulted in lower initial kidney levels and more rapid renal clearance in healthy mice than observed previously with any other labeling method-radionuclide combination. A planned in vivo analysis in HER2+ tumor bearing mice will generate additional data to select the optimal Nanobody construct for a therapeutic evaluation.