Biologic TNF-α inhibitors reduce microgliosis, neuronal loss, and tau phosphorylation in a transgenic mouse model of tauopathy

Abstract

Background Tumor necrosis factor-α (TNF-α) plays a central role in Alzheimer’s disease (AD) pathology, making biologic TNF-α inhibitors (TNFIs), including etanercept, viable therapeutics for AD. The protective effects of biologic TNFIs on AD hallmark pathology (Aβ deposition and tau pathology) have been demonstrated. However, the effects of biologic TNFIs on Aβ-independent tau pathology have not been reported. Existing biologic TNFIs do not cross the blood–brain barrier (BBB), therefore we engineered a BBB-penetrating biologic TNFI by fusing the extracellular domain of the type-II human TNF-α receptor (TNFR) to a transferrin receptor antibody (TfRMAb) that ferries the TNFR into the brain via receptor-mediated transcytosis. The present study aimed to investigate the effects of TfRMAb-TNFR (BBB-penetrating TNFI) and etanercept (non-BBB-penetrating TNFI) in the PS19 transgenic mouse model of tauopathy. Methods Six-month-old male and female PS19 mice were injected intraperitoneally with saline (n = 12), TfRMAb-TNFR (1.75 mg/kg, n = 10) or etanercept (0.875 mg/kg, equimolar dose of TNFR, n = 10) 3 days/week for 8 weeks. Age-matched littermate wild-type mice served as additional controls. Blood was collected at baseline and 8 weeks for a complete blood count. Locomotion hyperactivity was assessed by the open-field paradigm. Brains were examined for phosphorylated tau lesions (Ser202, Thr205), microgliosis, and neuronal health. The plasma pharmacokinetics were evaluated following a single intraperitoneal injection of 0.875 mg/kg etanercept or 1.75 mg/kg TfRMAb-TNFR or 1.75 mg/kg chronic TfRMAb-TNFR dosing for 4 weeks. Results Etanercept significantly reduced phosphorylated tau and microgliosis in the PS19 mouse brains of both sexes, while TfRMAb-TNFR significantly reduced these parameters in the female PS19 mice. Both TfRMAb-TNFR and etanercept treatment improved neuronal health by significantly increasing PSD95 expression and attenuating hippocampal neuron loss in the PS19 mice. The locomotion hyperactivity in the male PS19 mice was suppressed by chronic etanercept treatment. Equimolar dosing resulted in eightfold lower plasma exposure of the TfRMAb-TNFR compared with etanercept. The hematological profiles remained largely stable following chronic biologic TNFI dosing except for a significant increase in platelets with etanercept. Conclusion Both TfRMAb-TNFR (BBB-penetrating) and non-BBB-penetrating (etanercept) biologic TNFIs showed therapeutic effects in the PS19 mouse model of tauopathy.

Efficacy and Safety of a Brain-Penetrant Biologic TNF-α Inhibitor in Aged APP/PS1 Mice

“…Recombinant TfRMAb-TNFR was produced via transient expression in Chinese hamster ovary (CHO)-K1 cells, sequentially purified by protein A and size exclusion chromatography (WuXi Biologics, Cranbury, NJ, USA), and verified by immunoblot as described previously [ 17 , 18 ]. The affinity of TfRMAb-TNFR to the mouse TfR and human TNF-α was confirmed by enzyme-linked immunoassays (ELISAs) [ 18 ].…”

“…Recombinant TfRMAb-TNFR was produced via transient expression in Chinese hamster ovary (CHO)-K1 cells, sequentially purified by protein A and size exclusion chromatography (WuXi Biologics, Cranbury, NJ, USA), and verified by immunoblot as described previously [ 17 , 18 ]. The affinity of TfRMAb-TNFR to the mouse TfR and human TNF-α was confirmed by enzyme-linked immunoassays (ELISAs) [ 18 ]. Both the TfRMAb-TNFR fusion protein and etanercept (International Laboratory USA, San Francisco, CA, USA) were formulated in 100 mM glycine, 150 mM NaCl, 28 mM Tris, and 0.01% Polysorbate 80, pH = 6.4, sterile filtered, and stored at −80 °C until use.…”

“…The open-field test was performed after ten weeks of treatment, as described previously, using a square-shaped white open-field box (72 cm × 72 cm with 36 cm walls) with a center square (36 cm × 36 cm) to measure locomotion and exploration [ 18 ]. Briefly, each mouse was gently placed in the white open-field box, and their movements were recorded for 5 min.…”

“…Further, TfRMAb-TNFR fusion protein treatment resulted in low antidrug-antibody formation, which was comparable to etanercept after twelve weeks of chronic dosing, with no signs of an immune response or cerebral microhemorrhage development [ 17 ]. Furthermore, the antidrug-antibodies formed against TfRMAb-TNFR are expected to be non-neutralizing as no changes in TfRMAb-TNFR plasma concentrations or clearance were observed following chronic dosing [ 18 ]. However, the effect of initiating TfRMAb-TNFR treatment in older APP/PS1 mice when the AD pathology is full-blown, and its safety profile following chronic administration in comparison to TfRMAb alone, have not been studied and were the focus of the current investigation.…”

Modulation of hepatic amyloid precursor protein and lipoprotein receptor-related protein 1 by chronic alcohol intake: Potential link between liver steatosis and amyloid-β

“…The frozen brain tissues were mounted in Tissue-Tek OCT compound (Fisher Scientific, MA, United States), and sliced into 20 μm-thick sagittal sections at −25°C using a cryostat (Micron Instruments, CA, United States). Three sections (600 µm apart) per mouse were used for immunostaining as described below ( Ou et al, 2021 ).…”