title: 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.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands; Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Maarten
authors.initials: M
authors.lastname: Rotman
authors.affiliation: Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Mick M
authors.initials: MM
authors.lastname: Welling
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Marlinde L
authors.initials: ML
authors.lastname: van den Boogaard
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands; Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Laure Grand
authors.initials: LG
authors.lastname: Moursel
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands; Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Linda M
authors.initials: LM
authors.lastname: van der Graaf
authors.affiliation: Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Mark A
authors.initials: MA
authors.lastname: van Buchem
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Silvère M
authors.initials: SM
authors.lastname: van der Maarel
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands; Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: l.van_der_weerd@lumc.nl.
authors.forename: Louise
authors.initials: L
authors.lastname: van der Weerd
mesh_terms: D000544:Alzheimer Disease / Q000000981:diagnostic imaging / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000502:physiology*; D000818:Animals; D001812:Blood-Brain Barrier / Q000000981:diagnostic imaging* / Q000378:metabolism; D001921:Brain / Q000000981:diagnostic imaging* / Q000378:metabolism*; D004195:Disease Models, Animal; D005455:Fluorescent Antibody Technique; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D007205:Indium Radioisotopes / Q000493:pharmacokinetics; D007553:Isotope Labeling; D008657:Metabolic Clearance Rate; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D004369:Pentetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D049268:Positron-Emission Tomography / Q000379:methods; D053764:Presenilin-1 / Q000502:physiology*; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics*; D061905:Single-Domain Antibodies / Q000008:administration & dosage / Q000494:pharmacology*; D014018:Tissue Distribution
chemical_list: C545487:APP protein, human; D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007205:Indium Radioisotopes; D053764:Presenilin-1; D019275:Radiopharmaceuticals; D061905:Single-Domain Antibodies; C054343:1-(4-isothiocyanatobenzyl)diethylenetriaminepentaacetic acid; D004369:Pentetic Acid
keywords: (111)In labeling; APPswe/PS1dE9 mice; Biodistribution; Fc fusion; SPECT; VHH