Preclinical in vivo longitudinal assessment of KG207-M as a disease-modifying Alzheimer's disease therapeutic

Abstract

In vivo biomarker abnormalities provide measures to monitor therapeutic interventions targeting amyloid-β pathology as well as its effects on downstream processes associated with Alzheimer's disease pathophysiology. Here, we applied an in vivo longitudinal study design combined with imaging and cerebrospinal fluid biomarkers, mirroring those used in human clinical trials to assess the efficacy of a novel brain-penetrating anti-amyloid fusion protein treatment in the McGill-R-Thy1-APP transgenic rat model. The bi-functional fusion protein consisted of a blood-brain barrier crossing single domain antibody (FC5) fused to an amyloid-β oligomer-binding peptide (ABP) via Fc fragment of mouse IgG (FC5-mFc2a-ABP). A five-week treatment with FC5-mFc2a-ABP (loading dose of 30 mg/Kg/iv followed by 15 mg/Kg/week/iv for four weeks) substantially reduced brain amyloid-β levels as measured by positron emission tomography and increased the cerebrospinal fluid amyloid-β_42/40 ratio. In addition, the 5-week treatment rectified the cerebrospinal fluid neurofilament light chain concentrations, resting-state functional connectivity, and hippocampal atrophy measured using magnetic resonance imaging. Finally, FC5-mFc2a-ABP (referred to as KG207-M) treatment did not induce amyloid-related imaging abnormalities such as microhemorrhage. Together, this study demonstrates the translational values of the designed preclinical studies for the assessment of novel therapies based on the clinical biomarkers providing tangible metrics for designing early-stage clinical trials.

Keywords: Alzheimer’s disease; anti-amyloid-beta therapeutic; longitudinal in vivo biomarkers; preclinical study; protein-based treatment.

Figure 1.

Baseline measurements including PET [¹⁸F]AZD4694, sMRI, and rs-fMRI were acquired at 21 months of age. Then, weekly KG207-M injection via tail vein from 22.5-23.5 months (30 mg/Kg/iv initial dose following 15 mg/Kg/iv dose) was followed by a serial CSF collection at the indicated times via cisterna magna after fixing the animals in a stereotaxic apparatus with isoflurane anesthesia. Then, serial blood was collected at the indicated times via the subclavian vein and separated into the plasma via centrifuge. On Week 6 at 24 months old, the follow-up measurements were acquired including PET [¹⁸F]AZD4694, sMRI, rs-fMRI, susceptibility MRI, and CSF.

Figure 2.

This is a schematic representation of KG207-M. The FC5 segment is in red, ABP is in blue, and the mouse IgG Fc fragment is in grey.

Figure 3.

(a, b & c) Observed (filled circles) and simulated (solid line) KG207-M (a & b) or total Aβ. (Aβ_total) (c). The KG207-M concentration in plasma (a) or CSF (b & c) vs time following administration of 30 mg/kg/iv bolus followed by four 15 mg/kg/iv bolus doses every 7 days for 28 days to Tg-ABP rats are shown. (d) Aβ_total concentration vs KG207-M concentration in CSF.

Figure 4.

The gray shadow represents the 95% confidence intervals of models in (a and b). (a) The linear mixed-effect model of global [¹⁸F]AZD4694 BP_ND showed a significant increase in Tg from 10 to 24 months old (solid green line; t(21) = 2.437, p = 0.0238; adjusted for sex, weight, and cohort variability) but a significant decline in Tg-ABP between 21 to 24 months old (solid red; t(7.4) = −2.657, p = 0.03112; adjusted for sex and weight). The interaction between treatment and disease progression showed that KG207-M significantly modified global [¹⁸F]AZD4694 BP_ND (cohort 1 and Tg-SAL vs Tg-ABP = 21 vs 16, M/F = 16/21, t(17.3) = −3.321, p = 0.00397; adjusted for sex, weight, and cohort variability). (b) The linear mixed-effect model showed a significant decline in CSF Aβ_42/40 ratio in Tg rat model from 10 to 19 months old (solid blue; t(114.4) = −2.971, p = 0.00362, adjusted for sex and weight). A box whisker plot based on Tukey method represented a paired t-test, which revealed that CSF Aβ_42/40 ratio increased (solid red; t(13) = 4.373, p = 0.0008) in Tg-ABP animals as compared with a population-based prediction by the model while no differences were found in Tg-SAL (t(3) = 0.4869, p = 0.6597). (c) A voxel-wise effect size map showed up to 47% decline in [¹⁸F]AZD4694 BP_ND prefrontal region (baseline-follow-up/baseline). (d) A voxel-wise linear mixed-effect model analysis showed a significant [¹⁸F]AZD4694 BP_ND clearance in the prefrontal cortex, cingulate cortex, entorhinal cortex, hippocampus, and basal forebrain in Tg-ABP compared to Tg-SAL (cohort 1 and Tg-SAL vs Tg-ABP = 21 vs 16, M/F = 16/21, adjusted for sex, weight, and cohort variability). The interaction term was corrected for multiple comparisons based on RFT at p < 0.05. (e, f) Campbell-Switzer silver staining displayed widespread Aβ plaques throughout the cortical regions in Tg-SAL animals while relatively few plaques were seen in Tg-ABP brains collected at 26.7 months old (Tg-SAL vs Tg-ABP = 2 vs 8, M/F = 7/5, t(24) > 4, p < 0.001). Here, one brain image from each group was included but comparable results were seen in all animals from each group.

Figure 5.

(a) The gray shadow represents the 95% confidence intervals of the model per subject. The linear mixed-effect model showed a significant increase in CSF NFL_log concentrations, represented after log transformation, in the Tg rat model from 10 to 19 months old (solid blue; t(67.6) = 4.819, p < 0.0001; adjusted for sex and weight). A box whisker plot based on Tukey method represented a paired t-test, which revealed that CSF NFL_log concentrations decreased (solid red; t(9) = 2.6286, p = 0.0481) in Tg-ABP as compared to a population-based prediction by the model while Tg-SAL showed no difference (t(3) = 1.208, p = 0.3137). (b) The figure represents a significant group contrast in resting-state blood-oxygen-level-dependent (BOLD) signal from cingulate to the rest of the brain between WT (n = 8) and McGill-R-Thy1-APP (n = 8) at 16-19 months. The voxel-wise linear mixed-effect analysis showed a significant reduction in resting-state cingulate connectivity in the basal forebrain, parietal associative cortex, and hippocampus in Tg (reproduced from Parent et al.) The result images were adjusted for multiple comparisons based on RFT at p < 0.05. (c) The figure represents a significant contrast in the resting-state BOLD signal from cingulate to the brain in Tg-ABP (n = 9) following KG207-M treatment compared to baseline at 21 months of age. The voxel-wise linear mixed-effect analysis showed a significant increase in resting-state cingulate connectivity in the basal forebrain, parietal associative cortex, and hippocampus when the network is compared before and after KG207-M treatment. The result images were adjusted for multiple comparisons based on RFT at p < 0.05. (d) The adjusted hippocampal volume in Tg-SAL and Tg-ABP before and after KG207-M treatment are displayed as mean ± standard deviation. The linear mixed-effect analysis revealed a significant increase in hippocampal volume in Tg-ABP (t(7) = 3.554, p = 0.00397; adjusted for sex and weight), however, there was no significant hippocampal volume change in Tg-SAL (t(1) = 0.1694, p = 0.8932).

Figure 6.

(a) The susceptibility MRI in the coronal view of Tg-SAL in the upper corner image and Tg-ABP in the lower corner. Both images do not show any evidence of ARIA-H. (b) Histological staining using Perls/DAB of the same animals in the corresponding section from the susceptibility images are shown. These sections were taken adjacent to the previous Campbell-Switzer silver staining section. In both Tg-SAL and Tg-ABP, there is no evidence of ARIA-H. (c) Susceptibility signal contrast was converted into volume and displayed in mean ± standard deviation in Tg-SAL (n = 3) and Tg-ABP (n = 9). The contrast signal implies that there is a deposit of iron (ARIA-H), vasculatures, or ventricles. There was no significant difference in susceptibility signal contrast volume between Tg-SAL and Tg-ABP after five weeks of KG207-M treatment (unpaired t-test; Tg-SAL vs Tg-ABP = 3 vs 9, M/F = 7/5, t(2.59) = 0.455, p = 0.685).