Bivalent Brain Shuttle Increases Antibody Uptake by Monovalent Binding to the Transferrin Receptor

Abstract

The blood-brain barrier (BBB) is an obstacle for antibody passage into the brain, impeding the development of immunotherapy and antibody-based diagnostics for brain disorders. In the present study, we have developed a brain shuttle for active transport of antibodies across the BBB by receptor-mediated transcytosis. We have thus recombinantly fused two single-chain variable fragments (scFv) of the transferrin receptor (TfR) antibody 8D3 to the light chains of mAb158, an antibody selectively binding to Aβ protofibrils, which are involved in the pathogenesis of Alzheimer's disease (AD). Despite the two TfR binders, a monovalent interaction with TfR was achieved due to the short linkers that sterically hinder bivalent binding to the TfR dimer. The design enabled efficient receptor-mediated brain uptake of the fusion protein. Two hours after administration, brain concentrations were 2-3% of the injected dose per gram brain, comparable to small molecular drugs and 80-fold higher than unmodified mAb158. After three days, fusion protein concentrations in AD transgenic mouse brains were 9-fold higher than in wild type mice, demonstrating high in vivo specificity. Thus, our innovative recombinant design markedly increases mAb158 brain uptake, which makes it a strong candidate for improved Aβ immunotherapy and as a PET radioligand for early diagnosis and evaluation of treatment effect in AD. Moreover, this approach could be applied to any target within the brain.

Keywords: BBB shuttle /TfR /antibodies / Alzheimer's disease/ immunotherapy/PET..

Figure 1

Design and uptake mechanism of the BBB shuttle. (A). Transferrin is transported across the BBB by binding to iron and the TfR. The complex is endocytosed and with an altered pH in the endosomes the affinity changes so that transferrin and iron are released in the endosomes and will eventually reach the brain parenchyma. An antibody that binds monovalently to the TfR, will according to the same mechanism also be released in the endosomes. Bivalent TfR binders have higher affinity to the TfR and hence less antibody will be released, and as a consequence, more antibody will instead be degraded. When the antibody is released in the brain parenchyma it can find and bind to its intra-brain target. (B). Schematic picture of the RmAb158-scFv8D3 fusion protein design. RmAb158 binds to Aβ protofibrils, involved in AD pathogenesis. The scFv of 8D3, which binds to TfR, is attached to the C-terminus of the RmAb158 light chain with short linkers. (C). The short linker length between RmAb158 and scFv8D3 combined with the placement of scFv8D3 on the C-terminus of the light chains ensures that there cannot be bivalent binding to the TfR dimer (left). The presence of two rather than one scFv8D3 will increase the concentration of TfR binders and hence increase the likelihood of uptake. In contrast, 8D3 can bind bivalently to the TfR dimer (right).

Figure 2

Characterisation of the binding properties of the brain shuttle. (A). SDS-PAGE of RmAb158 and RmAb158-scFv8D3, displaying a single band of each antibody with an approximate size of 160 and 210 kDa, respectively. (B). TfR competition ELISA, demonstrating that 8D3, in line with the chemically conjugated fusion protein 8D3-F(ab')₂-h158 , binds bivalently to TfR, and displays 10-fold stronger binding compared with RmAb158-scFv8D3. A scFv of 8D3 displays an even weaker TfR binding due to its single binding site. (C). Inhibition ELISA, displaying that RmAb158-scFv8D3 retains a high, selective binding to Aβ protofibrils (PF) over monomers (M) comparable to RmAb158, whereas the control antibody 6E10 binds equally well to both Aβ species. Representative graphs from three independent experiments.

Figure 3

Increased brain uptake of the brain shuttle connected to the RmAb158 antibody in wt mice. (A). Ex vivo experiment displaying 80-fold difference in brain concentration of [¹²⁵I]RmAb158 and [¹²⁵I]RmAb158-scFv8D3 in wt mice 2 h post injection. When co-injected with 10 mg/kg 8D3, the difference is reduced to 3-fold. (B). Brain-to-blood concentration ratio in the same experiment. The ratio is 140-fold higher for [¹²⁵I]RmAb158-scFv8D3 than for [¹²⁵I]RmAb158, whereas only a 4-fold difference remains when 8D3 is co-administered. (n=3 for each group).

Figure 4

High brain retention of RmAb158-scFv8D3 in transgenic mice. (A). Ex vivo quantified brain concentration of [^124/125I]RmAb158-scFv8D3 in old (18-24 months) and young (8-9 months) wt and tg-ArcSwe mice 3 days post injection (n=3, 4, 4 and 6 for young wt, young tg-ArcSwe, old wt respective old tg-ArcSwe). (B). Comparison of ex vivo brain concentration of the chemically fused 8D3-F(ab')₂-h158 (n=5) and the recombinant RmAb158-scFv8D3 (n=6) in 18 months old tg-ArcSwe mice 3 days post injection. (C). Ex vivo quantified brain concentration of [^124/125I]RmAb158-scFv8D3 in old (18-24 months) tg-ArcSwe mice at 3, 6 and 10 days post administration in comparison with blood concentration over the same time period. Elimination from brain was much slower than elimination from blood (n=8).

Figure 5

Biodistribution of RmAb158-scFv8D3. Biodistribution of [^124/125I]RmAb158-scFv8D3 in different peripheral organs quantified as per cent of injected dose per gram organ at 3 (n=14), 6 (n=4) and 10 (n=10) days after administration. Brain concentrations in old tg-ArcSwe mice from fig 4C are included for comparison.

Figure 6

Improved sensitivity in PET imaging of Aβ pathology with RmAb158-scFv8D3 as a ligand. Sagittal PET images obtained after administration of [¹²⁴I]RmAb158-scFv8D3. (A). PET image in old tg-ArcSwe and wild-type (wt) mice obtained at 0-60 min post administration of [¹²⁴I]RmAb158-scFv8D3. Radioactivity concentration is similar in all brain regions. The same scale as in the other figures could not be used as the radioactivity was much higher during this early time point. (B). PET images obtained in old tg-ArcSwe, old wt and in young tg-ArcSwe mice at 3, 6 and 10 days post injection of [¹²⁴I]RmAb158-scFv8D3. (C). PET image images obtained from an old tg-ArcSwe injected with the previously generated chemically conjugated fusion protein using the same colour scale as in B to allow for comparison of brain concentrations. (D). PET image of a young tg-ArcSwe mouse at 6 days post injection (same as in B) displayed using a colour scale with a lower threshold. Cortical brain uptake of [¹²⁴I]RmAb158-scFv8D3 can be then visualized with PET also at this early stage of disease progression.

Figure 7

PET quantification of RmAb158-scFv8D3 concentration in wt and transgenic mouse brains. PET based brain region-to-cerebellum concentration ratios in hippocampus (A), thalamus (B), striatum (C), cortex (D) and whole brain (E). A comparison to chemically conjugated fusion protein is shown in (F). On day 0 some animal groups were not investigated and on day 10, cerebellum levels were below the limit of quantification in wt and young tg-ArcSwe mice, and hence, ratios could not be calculated. (n=3-5 per group except for 10 days when n=1. Total number of animals is 24 and each animal had one or two PET scans).

Figure 8

Uptake of the brain shuttle connected to the RmAb158 antibody at therapeutic doses. (A). Ex vivo experiment using therapeutic doses of RmAb158 and RmAb158-scFv8D3 displaying 10-fold difference in brain of the two ligands in wt mice 2 h post injection. (B). Brain-to-blood concentration ratio in the same experiment displaying a 20-fold difference between the two proteins. (n=5 for each group).