Engineered Cytokines Targeting Lipid-Laden Macrophages

PROJECT SUMMARY/ABSTRACT Cytokine therapies have the potential to revolutionize treatment for immunologic diseases but are limited by their poor pharmacokinetic profiles, off-target effects, and pleiotropic nature. Engineered cytokine platforms, on the other hand, have the potential to target specific tissue environments and cell types to provide local

immunomodulation with minimal side effects. Since macrophages play a central role in many immune-mediated diseases and can be polarized toward anti-inflammatory or pro-inflammatory phenotypes, they are promising targets for cytokine therapies. In my preliminary work, I developed a platform technology to target metabolically

dysfunctional macrophages in the context of atherosclerosis, a paradigm chronic inflammatory disease with high prevalence. Since macrophages that comprise atherosclerotic plaques engulf large amounts of low-density lipoprotein (LDL) and become pro-inflammatory lipid-laden “foam cells,” I engineered a protein fusion in which

one side is an antibody fragment (Fab) that binds to LDL and the other side is the anti-inflammatory cytokine IL- 10. I have shown that Fab-IL-10 constructs attach to LDL upon i.v. injection in hypercholesterolemic mice, hitchhike a ride to inflamed regions, preferentially target macrophages, and successfully reduce inflammation.

This proposal aims to elucidate the molecular mechanisms of action whereby inflammation is locally suppressed in atherosclerosis (Aim 1), engineer additional functionalities into the construct (Aim 2), and determine its generalizability to other cytokine payloads (Aim 3). In Aim 1, we will primarily use in vitro models to dissect the

roles of different scavenger receptors involved in Fab-IL-10 binding and uptake and characterize the resulting phenotype and transcriptome of Fab-IL-10-treated lipid-laden macrophages. We will also perform single cell RNA sequencing on plaque-resident macrophages in an experimental mouse model of atherosclerosis to determine

the effects of treatment with Fab-IL-10 in vivo. In Aim 2, we will engineer and evaluate an LDL-binding full antibody-IL-10 construct with enhanced avidity due to multiple binding regions, extended half-life due to neonatal Fc receptor-mediated recycling, and higher potency due to an extra copy of IL-10 per construct. In Aim 3, we will

evaluate the generalizability of this platform to other payloads while also uncovering biological insights on the effects of less well-studied cytokines in atherosclerosis (i.e., IL-19 and IL-33). With data generated from this proposal, we will apply to multiple R01-level grants to expand this platform to target additional disease models

that are partially regulated by lipid-laden macrophages including non-alcoholic fatty liver disease and certain solid cancers. This research proposal combined with my individualized career development plan will enable me to expand my scientific and professional skillsets and will enable my seamless transition to research

independence as a future tenure-track assistant professor.