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This study investigated the in vivo properties of two heavy chain antibody fragments (VHH), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled VHH in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled VHH by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All VHH showed rapid renal clearance (10–20 min). Twenty-four hours post-injection 99mTc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for 99mTc-ni3A or DTPA(111In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled VHH, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both VHH showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that VHH detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits.

The interaction of ,3-D-galactosidase with its specific antibody does not lead to any significant change in enzymatic activity regardless of whether or not immune precipitation occurs.1 Cowie et al.2 found that ribosome-bound 13-D-galactosidase differed from soluble enzyme in that an increase in enzymatic activity occurred in the presence of anti-3-D-galactosidase serum. This was ascribed to the formation of new centers of fl-D-galactosidase activity on the ribosomes.3 We were interested in these observations for their potential use in a system to detect antibodies because the appearance of enzymatic activity would be a signal with inherent amplification capabilities. The antibody activation of ribosomal enzyme in the wild-type Escherichia coli was not readily amenable to our purposes because the average gain in activity was only threefold. We conducted a search for larger activations by screening a series of point mutants known to be defective in the z gene. Out of 47 tested, 2 were found. A biochemical characterization of an enzyme precursor found in the extract of one of these two mutants is presented below. This precursor is referred to as AMEF (antibody-mediated enzyme-forming substance). Materials and Methods.-(a) Purification of (3-D-galactosidase (3-D-galactoside galactohydrolase E.C. 3.2.1.23): Cells of K12 3300, grown in a 500-liter batch, were used to prepare purified O-D-galactosidase according to Craven et al.4 The specific activity of our enzyme was comparable to that of a sample (a gift of Dr. Steers) estimated to be homogenous by several criteria.4 The enzyme was stored in a refrigerator under a 40%

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.

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Accumulation and aggregation of the amyloid‐β (Aβ) peptide is associated with Alzheimer's disease (AD). Aβ is generated from the amyloid precursor protein by the successive action of two membrane‐associated processing enzymes: β‐secretase or β‐site of amyloid precursor protein cleaving enzyme 1 (BACE1) and γ‐secretase. Inhibition of one or both of these enzymes prevents Aβ generation and the accompanying Aβ accumulation. Antigen binding fragments from camelid heavy chain only antibodies (VHHs) were found to exert excellent enzyme inhibition activity. In the present study, we generated VHHs against BACE1 by active immunization of Lama glama with the recombinant BACE1 protein. Two classes of VHHs were selected from a VHH‐phage display library by competitive elution with a peptide encoding the Swedish mutation variant of the BACE1 processing site. One VHH was found to inhibit the enzyme activity of BACE1 in vitro and in cell culture, whereas two other VHHs were found to stimulate BACE1 activity under the same conditions in vitro. Furthermore, an in vivo study with a transgenic AD mouse model, using intracisternal injection of the inhibitory VHH, led to acute reduction of the Aβ load in the blood and brain. This inhibitory VHH may be considered as a candidate molecule for a therapy directed towards reduction of Aβ load and prevention of AD progression. Both the inhibitory and stimulatory VHH may be useful for improving our understanding of the structure–function relationship of BACE1, as well as its role in AD progression. Database The GenBank sequence accession numbers are http://www.ncbi.nlm.nih.gov/protein/KR363186 for VHH B1a; http://www.ncbi.nlm.nih.gov/protein/KR363187 for VHH B3a; and http://www.ncbi.nlm.nih.gov/protein/KR363188 for VHH B5a.

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