The blood-brain barrier (BBB) presents a major obstacle in developing specific diagnostic imaging agents for many
neurological disorders. In this study we aimed to generate single domain anti-mouse
transferrin receptor antibodies (anti-mTfR VHHs) to mediate BBB transcytosis as components of novel MRI molecular contrast imaging agents. Anti-mTfR VHHs were produced by immunizing a llama with mTfR, generation of a VHH phage display library, immunopanning, and in vitro characterization of candidates. Site directed mutagenesis was used to generate additional variants. VHH fusions with
neurotensin (NT) allowed rapid,
hypothermia-based screening for VHH-mediated BBB transcytosis in wild-type mice. One anti-mTfR VHH variant was fused with an anti-
amyloid-beta (Aβ) VHH dimer and labeled with
fluorescent dye for direct assessment of in vivo target engagement in a mouse model of AD-related Aβ plaque pathology. An anti-mTfR VHH called M1 and variants had binding affinities to mTfR of <1nM to 1.52nM. The affinity of the VHH binding to mTfR correlated with the efficiency of the VHH-NT
induced hypothermia effects after
intravenous injection of 600 nmol/kg
body weight, ranging from undetectable for nonbinding mutants to -6°C for the best mutants. The anti-mTfR VHH variant M1P96H with the strongest
hypothermia effect was fused to the anti-Aβ VHH dimer and labeled with
Alexa647; the
dye-labeled VHH fusion construct still bound both mTfR and Aβ plaques at concentrations as low as 0.22 nM. However, after
intravenous injection at 600 nmol/kg
body weight into APP/PS1 transgenic mice, there was no detectible labeling of plaques above control levels. Thus, NT-
induced hypothermia did not correlate with direct target engagement in cortex, likely because the concentration required for NT-
induced hypothermia was lower than the concentration required to produce in situ labeling. These findings reveal an important dissociation between NT-
induced hypothermia, presumably mediated by hypothalamus, and direct engagement with Aβ-plaques in cortex. Additional methods to assess anti-mTfR VHH BBB transcytosis will need to be developed for anti-mTfR VHH screening and the development of novel MRI molecular
contrast agents.