Biological standardization and maximum tolerated dose estimation of an Alternaria alternata allergenic extract.

ABSTRACT

BACKGROUND: The manufacture of allergenic extracts from the mold Alternaria alternata is influenced by factors such as strain variability, allergenic origin, culturing conditions and extraction process, which affect the reproducibility of the preparations intended for diagnostic and therapeutic use. OBJECTIVES: To select the most adequate antigenic source of A. alternata extracts and determine its maximum tolerated dose (MTD) to be used in a subsequent immunotherapy efficacy clinical trial. METHODS: Twenty-one patients monosensitized to A. alternata were involved in a biological standardization process of A. alternata extracts. Four different mold strains were cultured and used to produce extracts by three different methods, each incorporating proteins from different origins culture filtrate, buffer extractable fraction and cellular antigens. The selected extract, characterized as in-house reference (IHR) preparation was used in a MTD finding immunotherapy study. Serum IgE, IgG, IgG1 and IgG4 specific of complete extract and purified natural and recombinant forms of Alt a 1 were determined by different EIA methods. RESULTS: Culture filtrate extract containing the allergens secreted to the spent medium was shown to be the most adequate option for establishing an IHR preparation for A. alternata extract manufacturing. A maximum dose of 1670 UBE, equivalent to 0.1 microg Alt a 1, was determined as MTD for immunotherapy. One year of administration of such a dose at monthly intervals elicited pronounced immunological changes with statistically significant decreases in IgE and increases in IgG4, both estimated with whole extract or purified Alt a 1. CONCLUSION: A high quality natural A. alternata extract has been developed and preliminarily tested to define its MTD for subsequent determination of the optimal dose in an immunotherapy efficacy clinical trial.