Brain Penetrating Bifunctional Erythropoietin–Transferrin Receptor Antibody Fusion Protein for Alzheimer’s Disease

Abstract

Objective: Erythropoietin (EPO), a glycoprotein cytokine essential to hematopoiesis, has neuroprotective effects in rodent models of Alzheimer’s disease (AD). However, high therapeutic doses or invasive routes of administration of EPO are required to achieve effective brain concentrations due to low blood-brain barrier (BBB) penetrability, and high EPO doses result in hematopoietic side effects. These obstacles can be overcome by engineering a BBB-penetrable analog of EPO, which is rapidly cleared from the blood, by fusing EPO to a chimeric monoclonal antibody targeting the transferrin receptor (cTfRMAb), which acts as a molecular Trojan horse to ferry the EPO into the brain via the transvascular route. In the current study, we investigated the effects of the BBB-penetrable analog of EPO on AD pathology in a double transgenic mouse model of AD. Methods: 5.5 month old male APPswe PSEN1dE9 (APP/PS1) transgenic mice were treated with saline (n=10), or the BBB-penetrable EPO (n=10) 3 days/week intraperitoneally for 8 weeks, compared to same-aged C57BL/6J wild-type mice treated with saline (n=8) with identical regiment. At 9 weeks following treatment initiation, exploration and spatial memory were assessed with the open-field and Y-maze test, mice were sacrificed, and brains were evaluated for Aβ peptide load, synaptic loss, BBB disruption, microglial activation and microhemorrhages. Results: APP/PS1 mice treated with the BBB-penetrable cTfRMAb-EPO fusion protein had significantly lower cortical and hippocampal Aβ peptide number (p<0.05) and immune-positive area (p<0.05), a decrease in hippocampal synaptic loss (p<0.05) and cortical microglial activation (p<0.001), and improved spatial memory (p<0.05) compared with APP/PS1 saline controls. BBB-penetrating EPO was not associated with microhemorrhage development. Conclusions: The cTfRMAb-EPO fusion protein offers therapeutic benefits by targeting multiple targets of AD pathogenesis and progression (Aβ load, synaptic loss, microglial activation) and improving spatial memory in the APP/PS1 mouse model of AD.

Hematologic safety of chronic brain‐penetrating erythropoietin dosing in APP/PS1 mice

“…Behavior analysis was performed before and after 6 weeks of treatment. Locomotion and exploration were assessed by the open‐field test as described previously [13]. Movements of animals, after placing in a white open box (72 cm × 72 cm with 36 cm walls), were recorded for 5 min to measure (a) mean speed, (b) resting time, and (c) total distance [13].…”

“…Locomotion and exploration were assessed by the open‐field test as described previously [13]. Movements of animals, after placing in a white open box (72 cm × 72 cm with 36 cm walls), were recorded for 5 min to measure (a) mean speed, (b) resting time, and (c) total distance [13]. Time in the center was also recorded as an indicator of anxiety‐related behavior [16].…”

“…Spatial memory was assessed using the modified Y‐maze as previously described [13]. Briefly, during the training phase, one arm of the Y‐maze was blocked and the animal was placed in the start arm and was allowed to explore two open arms for 8 min.…”

“…Sections were washed in PBS, incubated in 70% formic acid for 10 min at room temperature (RT), and blocked with 0.5% bovine serum albumin in PBS containing 0.3% TritonX‐100 for 1 h at RT followed by overnight incubation with 1 μg/mL of Alexa Fluor 488‐conjugated 6E10 MAb (BioLegend) with 0.3% TritonX‐100 in PBS at 4°C [13]. Sections were cover‐slipped with VectaMount aqueous mounting media (Vector Laboratories) and imaged using the Leica TCS SP5 Confocal Microscope.…”

“…For long‐term treatment, the extravascular (EV) route, such as subcutaneous administration, is preferred over the IV route due to the ease of administration. However, lower bioavailability after EV dosing necessitates higher EV injection doses to achieve therapeutic brain concentrations comparable with IV dosing, and the typical EV dose of IgG‐based fusion proteins is higher and ranges between 3 and 5 mg/kg [13–15]. We recently showed that chronic treatment with a 3 mg/kg EV dose of cTfRMAb‐EPO reduces Aβ load, neuroinflammation, synaptic loss, and spatial memory deficits in the APPswe PSEN1dE9 (APP/PS1) mice [13].…”