L16 - Engineered Nanobodies to Visualize and Direct Antigen-Specific Tolerance

Introduction: : Autoimmune diseases affect ~10% of the global population and can lead to debilitating, life-threatening complications. Current therapies depend on broad immunosuppression, which increases infection risk and compromises protective immunity. There is a critical need for antigen-specific therapies that restore immune tolerance without affecting general immune function.

We present a modular immunomodulatory strategy using alpaca-derived single-domain antibody fragments (nanobodies or VHHs) that specifically target MHC class II (MHCII) molecules, enabling delivery to all professional antigen-presenting cells (APCs). Through site-specific chemo-enzymatic conjugation, we engineered VHH MHCII constructs with a range of autoantigens and immunomodulatory cargos such as dexamethasone (DEX). A single dose of a VHH MHCII conjugated to myelin oligodendrocyte glycoprotein peptide and DEX (VHH MHCII-MOG-DEX) effectively prevents and reverses disease in a mouse model of multiple sclerosis while preserving immune responses against pathogens.

This approach is broadly adaptable to other autoimmune diseases—including type 1 diabetes, rheumatoid arthritis, and allergy—by changing the antigen payload. We also developed nanobody-based PET imaging agents to monitor biodistribution and tolerogenic activity in vivo, providing a powerful platform for both therapeutic intervention and immune monitoring.

Materials and

Methods: : Nanobodies targeting MHCII were generated through alpaca immunization followed by phage display selection. Selected VHHs were engineered to include a C-terminal sortase tag, allowing site-specific modification via chemo-enzymatic conjugation. Autoantigenic peptides (e.g., MOG 35–55) and immunosuppressive drugs (e.g., dexamethasone) were conjugated to the nanobodies using optimized linkers. The resulting constructs (e.g., VHH MHCII-MOG-DEX) were purified by size-exclusion chromatography and validated biochemically.

For in vivo studies, C57BL/6 mice were induced with experimental autoimmune encephalomyelitis (EAE) using MOG 35–55 in complete Freund’s adjuvant. Mice received a single intravenous dose of nanobody conjugates either prophylactically or after symptom onset. Clinical scoring was performed daily.

Immune responses were characterized via flow cytometry to assess antigen-specific regulatory T cells (Tregs), and transcriptomic profiling was used to analyze tolerogenic signatures in lymphoid organs. For biodistribution and activity monitoring, we developed PET tracers using radiolabeled VHH MHCII and anti-idiotypic nanobodies targeting disease-relevant TCRs (e.g., 2D2 TCR specific for I-Ab-MOG). PET/CT imaging was performed in symptomatic mice to detect nanobody localization and T cell infiltration. Of note, anti-idiotypic nanobodies were isolated using phage display and validated for specificity and in vivo imaging applications.

Results, Conclusions, and Discussions:: A single dose of VHH MHCII-MOG-DEX provided durable protection against EAE when administered prophylactically and reversed established paralysis when given therapeutically. Mice maintained normal responses to unrelated pathogens, confirming that global immunity remained intact. Flow cytometry revealed an increase in MOG-specific FoxP3⁺ regulatory T cells, and transcriptomic analysis indicated induction of tolerogenic gene programs and bystander suppression. The modularity of this platform was demonstrated by swapping in other autoantigens and cargoes, showing promise for targeting additional diseases like type 1 diabetes and rheumatoid arthritis. Notably, the delivery of antigens via MHCII-targeted nanobodies enabled efficient uptake by professional APCs, which likely underlies the robust and specific tolerogenic response observed.

To monitor immune modulation in vivo, PET imaging with radiolabeled VHH MHCII showed selective accumulation in lymphoid tissues. Additionally, we developed anti-idiotypic nanobodies that bind disease-specific TCRs (e.g., 2D2 TCR), allowing us to track autoreactive T cells via ImmunoPET. In symptomatic EAE mice, these probes revealed T cell infiltration in the CNS, validating both disease presence and nanobody activity.
In conclusion, our nanobody platform offers a versatile and powerful approach for inducing antigen-specific tolerance while preserving host immunity. It also enables real-time tracking of immune modulation, making it well-suited for both therapeutic and diagnostic applications across a wide range of immune-mediated diseases. The ability to achieve lasting immune tolerance with a single dose represents a transformative step forward in the treatment of autoimmunity.

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