P1‐072: pre‐clinical characterization of humanized, blood‐brain barrier (bbb)‐penetrating, amyloid‐β (aβ) oligomer‐targeting fusion protein

A 40-amino acid peptide (ABP) that selectively binds Aß1-42 oligomers and Aß deposits in brains of Alzheimer's disease (AD) patients and AD transgenic mice1,2, was shown to effectively lower CSF and brain Aß levels after systemic dosing in transgenic mice and rats when fused to the blood-brain barrier (BBB)-crossing single-domain antibody FC5. The fusion protein was subsequently humanized and further engineered for development as human therapeutic (KAL-ABP-BBB). Present studies show pre-clinical characterization of the KAL-ABP-BBB in transgenic mice and aged dogs. Recombinant non-humanized [KAL-ABP-BBB(M)] and humanized [KAL-ABP-BBB(H)] proteins were produced in CHO cells. BBB-permeability was assessed using in vitro BBB (formed by rat or human brain endothelial cells) and in vivo (rat and mouse) models. Aß binding was determined by ELISA, Western blot overlay and immunohistochemical methods. Serum, CSF and brain levels of systemically dosed KAL-ABP-BBB constructs and Aß were assessed by nanoLC-MRM, ELISA and Western blot. Both KAL-ABP-BBB bi-functional fusion proteins expressed in CHO cells retained Aß-oligomer binding activity and BBB-permeability in vitro. After systemic dosing, both KAL-ABP-BBB versions demonstrated identical long serum pharmacokinetics in rats and were detected in CSF, cortex and hippocampus of naïve and transgenic mice. In transgenic mice, treatment with either variant of KAL-ABP-BBB resulted in a significant (∼50%) reduction of Aß levels in the CSF suggesting in vivo target engagement. The ability of each variant to engage naturally occurring Aß was further shown after brain microinjection in live animals. Translational studies in aged dogs, which exhibit elevated levels of Aß (identical to human Aß), demonstrated similar serum PK to that observed in rats, >20-fold increased CSF exposure compared to control fusion protein, as well as a reduction in CSF levels of Aß, indicating target engagement. The study demonstrates translation of results (serum PK, enhanced CNS exposure, and lowering of Aß) obtained with KAL-ABP-BBB (M) in rodent species to aged dogs. 1Chakravarthy et al., J. Neurochem 126, 415, 2013. 2Biochem. Biophys. Res. Commun. 445, 656, 2014.