Selection of single domain anti-transferrin receptor antibodies for blood-brain barrier transcytosis using a neurotensin based assay and histological assessment of target engagement in a mouse model of Alzheimer’s related amyloid-beta pathology

Abstract

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.

Enhancedin VivoBlood Brain Barrier Transcytosis of Macromolecular Cargo Using an Engineered pH-sensitive Mouse Transferrin Receptor Binding Nanobody

“…Development of mouse transferrin receptor binding nanobodies. The production and development of the lead candidate nanobody NIH-TfR-M1 was described previously [29]. Briefly, an adult llama was immunized with a recombinant protein consisting of the extracellular domain of the mouse transferrin receptor.…”

“…Expression in E. coli and purification: Expression of 6xHis tagged nanobodies in E. coli was described previously [29]. Briefly, nanobody constructs under the control of the lacZ promotor and an N-terminal pelB periplasmic translocation sequence were cloned into the pHEN2 phagemid vector and transformed into TG-1 competent E. coli.…”

“…Transferrin receptor ELISA with pH-dependent wash steps: Plate-based measurements of nanobody construct affinities were performed as described previously [29]. Briefly, 96-well plates were coated with 2 µg/mL recombinant mouse transferrin receptor extracellular domain (amino acids 89-763) overnight at 4°C, followed by blocking with 1% (w/v) bovine serum albumin (BSA) in 1x Phosphate-Buffered Saline (PBS) for 1 hour and incubated for 1 hour at room temperature with varying concentration of nanobody constructs.…”

“…We showed that this nanobody binds independently of the presence or absence of mouse holo-(iron bound) transferrin with relatively high (5 nM) affinity [29]. The M1 nanobody showed modest BBB transcytosis in vivo as measured using a neurotensin (NT)-induced hypothermia assay [29]. Others have reported that antibodies with lower affinities or monovalent binding mediate improved BBB transcytosis [30] [31] [32].…”

“…As an initial foray, we previously reported that introduction of a single histidine mutant at residue 96 imparts enhanced unbinding at pH 5.5 and modestly improved in vivo BBB transcytosis as measured using the NT-induced hypothermia assay. However, the M1P96H mutant nanobody was still not effective as a carrier of amyloid-beta binding nanobodies across the BBB in a mouse model of Alzheimer Disease-related amyloid plaque pathology [29]. Therefore, we endeavored to further engineer the M1 nanobody for more extensive pH-dependent unbinding while retaining high affinity at pH 7.4.…”