Fusion of hIgG1-Fc to 111In-anti-amyloid single domain antibody fragment VHH-pa2H prolongs blood residential time in APP/PS1 mice but does not increase brain uptake.

Llama single domain antibody fragments (VHH), which can pass endothelial barriers, are being investigated for targeting amyloid plaque load in Alzheimer's disease (AD). Contrary to conventional human or murine antibodies consisting of IgG or F(ab')2 antibody fragments, VHH are able to effectively pass the blood brain barrier (BBB) in vitro. However, in earlier in vivo studies, anti-amyloid VHH showed poor BBB passage due to their short serum half-lives. It would be of interest to develop a VHH based protein with elongated serum half-life to enhance BBB passage, allowing the VHH to more easily reach the cerebral amyloid deposits. To increase serum persistence, the Fc portion of the human IgG1 antibody (hinge plus CH2 and CH3 domains) was fused to the C-terminus of the VHH (VHH-pa2H-Fc). To determine the pharmacokinetics and biodistribution profile of the fusion protein, the chelator p-SCN-Bz-DTPA was linked to the protein and thereafter labeled with radioactive indium-111 ((111)In). Double transgenic APPswe/PS1dE9 and wild type littermates were injected with 20 μg VHH-pa2H-Fc-DTPA-(111)In (10-20 MBq). Pharmacokinetics of the tracer was determined in blood samples at 10 intervals after injection and imaging using microSPECT was performed. The biodistribution of the radioactivity in various excised tissues was measured at 48 h after injection. We succeeded in the expression of the fusion protein VHH-pa2H-Fc in HEK293T cells with a yield of 50mg/L growth medium. The fusion protein showed homodimerization - necessary for successful Fc neonatal receptor recycling. Compared to VHH-pa2H, the Fc tailed protein retained high affinity for amyloid beta on human AD patient brain tissue sections, and significantly improved serum retention of the VHH. However, at 48 h after systemic injection of the non-fused VHH-DTPA-(111)In and the VHH-Fc-DTPA-(111)In fusion protein in transgenic mice, the specific brain uptake of VHH-Fc-DTPA-(111)In was not improved compared to non-fused VHH-DTPA-(111)In. Using VHH-Fc conjugates increases the blood half-life of the protein. However, purely extending the time window for brain uptake does not increase BBB passage. Nevertheless, VHH-Fc holds promise for therapeutic applications where a sustained systemic circulation of VHH is advantageous.

Rotman M ，Welling MM ，van den Boogaard ML ，Moursel LG ，van der Graaf LM ，van Buchem MA ，van der Maarel SM ，van der Weerd L ... -  《-》

Enhanced glutathione PEGylated liposomal brain delivery of an anti-amyloid single domain antibody fragment in a mouse model for Alzheimer's disease.

Treatment of neurodegenerative disorders such as Alzheimer's disease is hampered by the blood-brain barrier (BBB). This tight cerebral vascular endothelium regulates selective diffusion and active transport of endogenous molecules and xenobiotics into and out of the brain parenchyma. In this study, glutathione targeted PEGylated (GSH-PEG) liposomes were designed to deliver amyloid-targeting antibody fragments across the BBB into the brain. Two different formulations of GSH-PEG liposomes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and egg-yolk phosphatidylcholine (EYPC) were produced. Both formulations encapsulate 15kDa amyloid beta binding llama single domain antibody fragments (VHH-pa2H). To follow the biodistribution of VHH-pa2H rather than the liposome, the antibody fragment was labeled with the radioisotope indium-111. To prolong the shelf life of the construct beyond the limit of radioactive decay, an active-loading method was developed to efficiently radiolabel the antibody fragments after encapsulation into the liposomes, with radiolabeling efficiencies of up to 68% after purification. The radiolabeled liposomes were administered via a single intravenous bolus injection to APPswe/PS1dE9 double transgenic mice, a mouse model of Alzheimer's disease, and their wildtype littermates. Both GSH-PEG DMPC and GSH-PEG EYPC liposomes significantly increased the standard uptake values (SUV) of VHH-pa2H in the blood of the animals compared to free VHH-pa2H. Encapsulation in GSH-PEG EYPC liposomes resulted in the highest increase in SUV in the brains of transgenic animals. Overall, these data provide evidence that GSH-PEG liposomes may be suitable for specific delivery of single domain antibody fragments over the BBB into the brain.

Rotman M ，Welling MM ，Bunschoten A ，de Backer ME ，Rip J ，Nabuurs RJ ，Gaillard PJ ，van Buchem MA ，van der Maarel SM ，van der Weerd L ... -  《-》

Pharmacokinetic analysis of the blood-brain barrier transport of 125I-amyloid beta protein 40 in wild-type and Alzheimer's disease transgenic mice (APP,PS1) and its implications for amyloid plaque formation.

Kandimalla KK ，Curran GL ，Holasek SS ，Gilles EJ ，Wengenack TM ，Poduslo JF ... -  《JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS》

In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease.

Nabuurs RJ ，Rutgers KS ，Welling MM ，Metaxas A ，de Backer ME ，Rotman M ，Bacskai BJ ，van Buchem MA ，van der Maarel SM ，van der Weerd L ... -  《PLoS One》

PET imaging of brain with the beta-amyloid probe, [11C]6-OH-BTA-1, in a transgenic mouse model of Alzheimer's disease.

Toyama H ，Ye D ，Ichise M ，Liow JS ，Cai L ，Jacobowitz D ，Musachio JL ，Hong J ，Crescenzo M ，Tipre D ，Lu JQ ，Zoghbi S ，Vines DC ，Seidel J ，Katada K ，Green MV ，Pike VW ，Cohen RM ，Innis RB ... -  《EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING》