Engineered Nanobodies as Tools to Visualize and Direct Antigen-Specific Tolerance 3116

Description Current treatments for autoimmune disorders often involve broad immunosuppression. We present a strategy to modulate immune responses to specific antigens while preserving overall immunity. We employed alpaca-derived single-domain antibody fragments (nanobodies/VHHs) targeting major histocompatibility complex class II (MHCII) and engaging all antigen-presenting cells. We developed chemo-enzymatic methods for site-specific VHH modification at the C-terminus, enabling attachment of autoantigens and anti-inflammatory drugs like dexamethasone (DEX). A single dose of VHH MHCII conjugated with myelin oligodendrocyte glycoprotein (MOG) peptides and DEX (VHH MHCII-MOG-DEX) provided lasting protection against experimental autoimmune encephalitis (EAE), a multiple sclerosis model, reversing paralysis without compromising immunity. Flow cytometry and transcriptomic analyses revealed increased antigen-specific regulatory T cells and bystander suppression. This approach can be adapted for other autoimmune diseases (type I diabetes and rheumatoid arthritis) and allergic asthma, by altering the autoantigens. All these benefits are achieved with a single dose. We also developed anti-idiotype VHHs against anti-MOG TCRs and used it as imaging agents in immuno-PET to detect infiltrating T cells in the spinal cord of symptomatic EAE mice, tracking tolerance efficacy. These engineered nanobodies offer versatile tools for targeted immune modulation and real-time immune response tracking. Funding Sources This study was supported by National Multiple Sclerosis Foundation Career Transition Award (N.P.).