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Immunological Parameters for Assessing the In Vitro Safety and Efficacy of Allergoid Mixtures for Immunotherapy

Calzada D1, Parody N1, Beitia JM2, Dominguez-Ortega J3, Gonzalez-de-Olano D4, Renshaw-Calderón A5, Osuna C1, Moya R1, Carnés J1

1R&D Unit (Allergy & Immunology), LETI Pharma, Madrid, Spain
2Hospital Universitario de Guadalajara, Madrid, Spain
3Department of Allergy, Hospital Universitario La Paz, Institute for Health Research (IDIPAZ), Madrid, Spain
4Hospital Ramón y Cajal, Madrid, Spain
5Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Campus de Cantoblanco, Madrid, Spain

J Investig Allergol Clin Immunol 2025; Vol. 35(6)
doi: 10.18176/jiaci.1061

Background: Most patients with respiratory allergy are polyallergic. Combining different allergen extracts in the same allergen-specific immunotherapy is a common practice. However, it should be justified.
Objective: To analyze the stability, safety, and immune response of allergen extract mixtures from nonhomologous groups.
Methods: We analyzed 2 depigmented-polymerized mixture extracts (DPmixEs): cat dander grass pollen and Alternaria alternata–grass pollen. The stability of the mixtures was investigated by studying proteolysis and degradation effects. The allergenicity and humoral and cellular immune responses of DPmixEs were also evaluated using various technical approaches, including the Bradford assay, enzyme-linked immunosorbent assay, rabbit immunization, peripheral blood mononuclear cell culture, and flow cytometry. The results were compared with those of individual depigmented-polymerized extracts (DPEs) and native mixture extracts (NmixEs).
Results: The proteolytic activity of DPmixEs was lower than that of NmixEs. The protein content of DPmixEs remained stable for 18 months, whereas that of NmixEs decreased significantly during the first month. The allergenicity of DPmixEs was similar to that of DPEs and lower than that of NmixEs. Regarding the immune response, DPmixEs induced functional specific IgG antibodies in rabbits and blocked sIgEallergen binding. Moreover, DPmixEs induced IL-10 secretion in peripheral blood mononuclear cells from polyallergic patients, improving the Treg/TH2 cell balance.
Conclusion: These findings support the use of DPmixE as a promising formulation for allergen immunotherapy, combining stability, reduced enzymatic activity, and enhanced immunological stimulation, while preserving in vitro safety and efficacy comparable to separated depigmented-polymerized extracts.

Key words: Allergen-specific immunotherapy, Polyallergy, Tolerogenic response, Multiallergen treatment, Allergoid, Immunomodulation, Mechanisms of action