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Supplemental material, sj-pdf-1-jcb-10.1177_0271678X211035625 for Preclinical in vivo longitudinal assessment of KG207-M as a disease-modifying Alzheimer’s disease therapeutic by Min Su Kang, Monica Shin, Julie Ottoy, Arturo Aliaga Aliaga, Sulantha Mathotaarachchi, Kely Quispialaya, Tharick A Pascoal, D Louis Collins, M. Mallar Chakravarty, Axel Mathieu, Åsa Sandelius, Kaj Blennow, Henrik Zetterberg, Gassan Massarweh, Jean-Paul Soucy, A Claudio Cuello, Serge Gauthier, Michael Waterston, Nathan Yoganathan, Etienne Lessard, Arsalan Haqqani, Kerry Rennie, Danica Stanimirovic, Balu Chakravarthy and Pedro Rosa-Neto in Journal of Cerebral Blood Flow & Metabolism

We report preclinical study results of a novel anti-amyloid agent (KAL-ABP). KAL-ABP includes an Aβ oligomer binding protein (ABP) fused to “carrier” module that allows the delivery of the active compound through the blood-brain barrier (KAL-ABP-BBB). KAL-ABP has been reported to selectively bind Aβ1-42 oligomer in vitro and ex vivo and prevent its toxicity. Here, using in vivo longitudinal biomarker acquisitions and McGill-R-Thy1-APP transgenic rat model (Tg), we further replicate and extend these results by quantifying KAL-ABP-BBB cerebrospinal fluid concentrations; target engagement via changes in amyloid load with [18F]NAV4694 positron emission tomography scans (NAV-PET); therapeutic efficacy via changes in hippocampal volumetry (MRI-HV) and vasculature. A total of 17 rats (wild type (WT-ABP), N=4; Tg-ABP, N=9; Tg-SAL, N=4). They had an initial dose of either KAL-ABP-BBB 30mg/Kg/IV followed by 15mg/Kg/IV/week, or placebo on the subsequent 4 weeks. 60-minute NAV-PET dynamic scan was acquired concomitantly with a bolus tail vein injection of the radiotracer after a 9-minute transmission scan. The images were reconstructed using Maximum a posteriori algorithm with scatter, dead time, and decay correction. Non-displaceable binding potential (BPND) was generated using cerebellar grey matter as a reference region based on simplified reference tissue model (SRTM). 60-minute scan via Fast Imaging with Steady-state Precession sequence with TE/TR of 2.5/5.0ms was used to generate structural images. Then, MRI-HV (mm3) was acquired via manual segmentation. Susceptibility MRI were acquired using echo planar acquisitions for vasculature. For image processing and statistical analysis, MINC tools, VoxelStats, R, and Prism7 were used. Linear Mixed effect regression model (corrected by sex, age, weight) and simple t-test were used to estimate the target engagement and therapeutic efficacy, respectively. KAL-ABP-BBB reached CSF concentrations of 300 ng/mL. KAL-ABP-BBB intervention reduced global [18F]NAV4694 BPND(27%; p=0.0442) and rectified baseline genotype observation (Tg vs Wt; p=0.0087). KAL-ABP-BBB intervention increased MRI-HV (8%; p=0.0099). No vascular abnormality was observed during KAL-ABP-BBB intervention. This study provides strong evidence that KAL-ABP-BBB crosses the BBB, safely lowers the amyloid burden and mitigates hippocampal atrophy in vivo. Overall, these results invite further clinical study in humans.

We have identified an amyloid-ß (Aß) binding peptide (ABP) that selectively bound pathological Aß1-42 oligomers and reduced Aß- mediated cell toxicity1. ABP also bound Aß deposits in brains of AD transgenic mice and Alzheimer’s disease (AD) patients in vitro, and when directly injected into live transgenic mice (APPSwe and ΔPS1) brain2, suggesting that, when delivered to the brain, ABP can engage Aß and facilitate its clearance. Present study shows brain-delivery of ABP by a novel BBB-crossing domain antibody, and targeting and clearance of Aß in transgenic mice. Recombinant ABP-BBB carrier fusion construct (ABP-BBB) were produced in CHO cells. BBB-permeability of the construct 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 and Western blot overlay assays. For PK/PD studies, serum, CSF and brain levels of ABP-BBB construct and Aß were assessed following iv injection by nanoLC- MRM, ELISA and Western blot methods. ABP-BBB bi-functional fusion protein was successfully expressed in CHO cells. The fusion protein retained both Aß-oligomer binding activity and BBB-permeability in vitro. ABP-BBB transmigrated the in vitro BBB, in contrast to ABP without BBB carrier. When injected iv into rats, ABP-BBB appeared in the CSF in a dose- and time-dependent manner indicating transport of ABP across BBB in vivo. Apparent CNS exposure (Expapp), calculated as CSF[AUC]/serum[AUC], indicated a 15-20-fold increase in Expapp for ABP-BBB compared to non-BBB permeable ABP. This was further confirmed in mouse model wherein following iv injection, the fusion protein was detected in the CSF, and cortical and hippocampal regions of wild type and Tg mice. More importantly, in Tg mice ABP treatment resulted in a significant (∼50%) reduction of Aß levels in the CSF, and brain cortex and hippocampus within 4-weeks.