V44 - Long-Lived CNS-Targeted Antibody Cytokine Fusion Proteins for the Treatment of Neuroinflammation

Introduction: : Neurological disorders affect ~15% of the global population, with the number of chronic neurodegenerative patients expected to double over the next 20 years. Treating these diseases poses many significant challenges, including efficiently delivering therapeutics across the blood-brain barrier (BBB) and retaining them in the central nervous system (CNS) for long periods of time. Additionally, finding a suitable biologic applicable to multiple neuroinflammatory diseases is very attractive. Regulatory T cells (Tregs) have been shown to ameliorate neuroinflammation in many diseases and can be expanded with various cytokines. However, these proteins have low BBB permeability and short half-lives. In our study, we utilize a bispecific antibody shuttle composed of an anti-TfR (transferrin receptor) scFv to cross the BBB, an IgG targeting an extracellular antigen with little to no internalization to depot the protein in the CNS, and an anti-inflammatory cytokine as the therapeutic for long-lived CNS Treg expansion.

Materials and

Methods: : Proteins were expressed in HEK 293 cells and purified using Protein A beads, characterized by SDS-PAGE and SEC, and 2-step purified by SEC to >90% monomer if necessary. Cytokine function was verified in fusion protein form using an activity assay in vitro. For pharmacokinetic studies, protein was radiolabeled by conjugating radioisotope I-125 to quantitively track concentration in each tissue and injected retro-orbitally in female C57BL/6 mice. For pharmacodynamic studies, immune cells in the brain, spinal cord, and spleen were isolated, stained for relevant populations, and analyzed using spectral flow cytometry.

Results, Conclusions, and Discussions:: However, the anchoring version had a Cmax of 22 nM in the brain, with concentrations of >22 nM for 84 days. We also wanted to understand the impact antibody format had on Treg induction in the brain. Three formats were tested: anchoring TfR shuttle, non-anchoring TfR shuttle, and anchoring IgG without shuttle. We observed that our anchoring TfR shuttle increased the percentage of Tregs in the brain by 10.8 ± 2.6 percentage points (SE of difference) compared to vehicle control (adjusted p-value: 0.0007), as shown in the figure. Importantly, we also show that this format and dose had higher Treg percentages and absolute cell counts in the brain compared to all other formats after two weeks without expansion of Tregs in the periphery.
We demonstrate that our anchoring TfR shuttle can be retained in the CNS for months and can selectively expand Tregs in the brain for at least two weeks with a cytokine payload. We also show that having both a shuttling component to cross the BBB and a targeting component to depot in the CNS are essential for prolonged and selective Treg expansion in the brain. We can further optimize our delivery platform by testing different CNS anchoring targets and BBB transport pathways, as well as attaching different drug cargo. Our findings show potential for using our engineered CNS-anchoring antibody cytokine fusion proteins to be neuroprotective in various neurodegenerative diseases such as Alzheimer's, multiple sclerosis, and ALS.

Acknowledgements and/or References (Optional)::