Engineered Nanobodies to Direct and Visualize Durable Tolerance 2260248

Abstract Introduction Current treatments for autoimmunity and allergy manage symptoms rather than cure disease. We propose a curative nanobody-based approach that selectively silences pathogenic responses to specific antigens/allergens while preserving protective immunity, i.e., durable antigen-specific immune tolerance. We also developed nanobody tools to map antigen processing and T cell responses in vivo, enabling precise visualization and quantification of tolerance induction at the molecular and cellular levels. Methods We developed an engineering-based solution using alpaca-derived nanobodies (VHHs) that recognize all MHC class II molecules (VHH MHCII). We covalently conjugated disease- or allergen-specific peptides-derived from myelin, insulin, and citrullinated collagen, as well as the model antigen ovalbumin (OVA) and house dust mite allergen to VHH MHCII together with the anti-inflammatory drug dexamethasone (VHH MHCII-antigen-DEX). Results A single dose of these trimodal conjugates induced durable, antigen-specific tolerance by promoting deletion, anergy, and regulatory differentiation of antigen-specific T cells. VHH MHCII-Myelin-DEX reversed experimental autoimmune encephalomyelitis, VHH MHCII-collagen-DEX halted arthritis progression, and intranasal delivery of VHH MHCII-OVA-DEX reduced OVA-specific IgG/IgE, mast cell activation, and airway inflammation. Conclusion This approach imposed long-lasting tolerance in prophylactic and therapeutic settings while maintaining systemic heterotypic immunity. Complemented by newly developed anti-idiotypic nanobodies specific for myelin- and OVA-specific TCRs, functionalized for fluorescence and positron emission tomography (PET) imaging, we achieved real-time visualization of antigen processing and tolerance induction. Together, these tools reveal how APC networks orchestrate tolerance and establish a modular platform for antigen- and allergen-specific immunotherapy. The available human-specific version of VHH MHCII enables translation to a clinical setting. Funding Source Boston Children’s Hospital Faculty Development and Pilot Grants, NIH R24OD035556, NIH R01AI182177, NIH R01CA255216, Breakthrough T1D Career Development Award, Lupus Research Alliance Innovative Award, Charles H. Hood Foundation Child Health Grant, Rheumatology Research Foundation Innovative Award, National Multiple Sclerosis Foundation Career Transition Fellowship, and American Lung Association & American Academy of Allergy, Asthma, and Immunology (AAAAI) Allergic Respiratory Disease Research Award Topic Categories Therapeutic Approaches to Autoimmunity (THER)