Brain pharmacokinetics of two BBB penetrating bispecific antibodies of different size

Abstract

Background Transferrin receptor (TfR1) mediated enhanced brain delivery of antibodies have been studied extensively in preclinical settings. However, the brain pharmacokinetics, i.e. brain entry, distribution and elimination are still not fully understood for this class of antibodies. The overall aim of the study was to compare the brain pharmacokinetics of two BBB-penetrating bispecific antibodies of different size (210 vs 58 kDa). Specifically, we wanted to investigate if the faster systemic clearance of the smaller non-IgG antibody di-scFv3D6-8D3, in comparison with the IgG-based bispecific antibody mAb3D6-scFv8D3, was also reflected in the brain. Methods Wild-type (C57/Bl6) mice were injected with 125I-iodinated ([125I]) mAb3D6-scFv8D3 (n = 46) or [125I]di-scFv3D6-8D3 (n = 32) and euthanized 2, 4, 6, 8, 10, 12, 16, or 24 h post injection. Ex vivo radioactivity in whole blood, peripheral organs and brain was measured by γ-counting. Ex vivo autoradiography and nuclear track emulsion were performed on brain sections to investigate brain and parenchymal distribution. Capillary depletion was carried out at 2, 6, and 24 h after injection of [125I]mAb3D6-scFv8D3 (n = 12) or [125I]di-scFv3D6-8D3 (n = 12), to estimate the relative levels of radiolabelled antibody in brain capillaries versus brain parenchyma. In vitro binding kinetics for [125I]mAb3D6-scFv8D3 or [125I]di-scFv3D6-8D3 to murine TfR were determined by LigandTracer. Results [125I]di-scFv3D6-8D3 showed faster elimination from blood, lower brain Cmax, and Tmax, a larger parenchymal-to-capillary concentration ratio, and a net elimination from brain at an earlier time point after injection compared with the larger [125I]mAb3D6-scFv8D3. However, the elimination rate from brain did not differ between the antibodies. The study also indicated that [125I]di-scFv3D6-8D3 displayed lower avidity than [125I]mAb3D6-scFv8D3 towards TfR1 in vitro and potentially in vivo, at least at the BBB. Conclusion A smaller size and lower TfR1 avidity are likely important for fast parenchymal delivery, while elimination of brain-associated bispecific antibodies may not be dependent on these characteristics.

Transferrin Receptor Binding BBB-Shuttle Facilitates Brain Delivery of Anti-Aβ-Affibodies

“…This confirms that TfR1 mediated transport is a robust strategy for enhancing brain delivery of affibody molecules (37). Di-scFv3D6-8D3 binds monovalently to mTfR1 (50), which has been reported to increase brain uptake (10,51).…”

“…Di-scFv3D6-8D3 binds monovalently to mTfR1 (50), which has been reported to increase brain uptake (10,51). We have previously shown that di-scFv3D6-8D3 displayed higher brain parenchyma-to-capillary ratio compared to the 210 kDa bivalent IgG-based antibody, mAb3D6-scFv8D3, potentially due to the monovalent TfR1 interaction of di-scFv3D6-8D3 (37). The impact of TfR1 affinity on therapeutic antibodies has also been studied, where a moderate affinity of 50 nM-100 nM was found optimal for brain delivery (52).…”

“…The affibody binding to Aβ protofibrils and mTfR1 before and after radiolabeling was assessed with indirect ELISA in comparison with di-scFv3D6-8D3 (37). In short, 96-well half area plates (Corning Inc.) were coated with 250 nM Aβ-PF (Innovagen) in PBS or 2 µg/ml mTfR1 (BioArctic, Stockholm, Sweden) in PBS and incubated at 4°C overnight.…”

“…We have previously developed preclinical positron emission tomography (PET) ligands based on antibodies, able to visualize intra-brain Aβ-pathology in vivo (33)(34)(35)(36). A small (58 kDa) antibody-based fusion protein, di-scFv3D6-8D3, showed more favorable imaging pharmacokinetics compared to an IgG-like bispecific antibody (33,37). However, biological circulation time of antibodies are typically too long to match the half-life of radioisotopes relevant for clinical usage (38).…”

PET Imaging in Preclinical Anti-Aβ Drug Development

“…Although their slow pharmacokinetics is a clear draw-back, their ability to detect Aβ beyond amyloid is interesting. Smaller bispecific formats appear to show more favorable and PET compatible pharmacokinetics than large antibody-like formats (35,92,93). Further, bispecific antibodies have also been labelled with the clinically preferred radionuclide 18 F (94).…”