Impact of cross-linking stoichiometry on the structure and allergenicity of glutaraldehyde-polymerized allergen extracts

Sandra Sivill¹Marcos Viñuela¹Diego García-Puentes¹Lucia Moreno-Serna²Emilio Nuñez-Borque²Rodrigo Jiménez-Saiz^2,3,4Maria Vila-Gonzalo¹Miguel Fernandez-Arquero⁵Irene Real-Arévalo¹Salvador Iborra¹Jose Luis Subiza¹Jose Fernando Cantillo^6*

• ¹Inmunotek SL, Alcala de Henares, Spain
• ²Instituto de Investigacion del Hospital de La Princesa, Madrid, Spain
• ³Universidad Francisco de Vitoria, Pozuelo de Alarcón, Spain
• ⁴McMaster University Immunology Research Centre, Hamilton, Canada
• ⁵Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos, Madrid, Spain
• ⁶Inmunotek SL, Alcalá de Henares, Spain

Introduction: Polymerized allergen extracts are widely used in allergen immunotherapy (AIT) to reduce allergenicity and enhance safety. However, the impact of cross-linking stoichiometry on the structural and allergenic properties of these polymers remains insufficiently characterized. This study systematically investigates how varying glutaraldehyde-to-protein ratios influences the resulting polymers and their potential relevance for AIT. Methods: Pollen extracts from Phleum pratense and Betula verrucosa were polymerized using five different glutaraldehyde-to-protein ratios. The resulting polymers were structurally characterized by SDS-PAGE, nuclear magnetic resonance (NMR), transmission electron microscopy (TEM) and mass spectrometry (MS). Allergenicity was evaluated by measuring IgE reactivity through ELISA, Western blotting and mast cell activation test (MAT). Immunogenicity was assessed by analyzing serum specific IgG and ex vivo lymphocyte responses in mice immunized with the least allergenic polymer formulation. Results: All cross-linking conditions produced polymers with distinct differences in size, morphology, and yield. Despite retaining similar peptide profile to native allergens, as confirmed by MS, the polymers exhibited increased stability, as shown by NMR, and significantly reduced allergenicity, accordingly to ELISA and MAT. Notably, increased polymer size and density, as determined by NMR and TEM, correlated with lower allergenicity. The most extensively cross-linked, high-density polymers, optimized for minimal allergenicity, elicited immunogenic responses comparable to those induced by native extracts when tested against unmodified allergens. Conclusion: Cross-linking stoichiometry critically shapes the structural and immunological properties of polymerized allergen extracts. Adjusting the glutaraldehyde-to-protein ratio to produce highly polymerized, dense polymers enables a well-balanced profile of safety and efficacy for use in AIT.