New product development with the innovative biomolecular sublingual immunotherapy formulations for the management of allergic rhinitis
Medical and Scientific Department, Stallergenes, Milan, Italy; Interdepartmental Centre of Bioclimatology, University of Florence, Florence, Italy; Allergy and Clinical Immunology Section, Azienda Sanitaria di Prato, Prato, Italy; Experimental Allergy Unit, IDI-IRCCS, Rome, Italy; Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy; Allergy/Pulmonary Rehabilitation, ICP Hospital, Milan, Italy; 7Department of Pediatrics, Allergy Unit, University of Messina, Messina, Italy
Abstract: The molecular allergy technique, currently defined as component-resolved diagnosis, significantly improved the diagnosis of allergy, allowing for differentiation between molecules actually responsible for clinical symptoms (genuine sensitizers) and those simply cross-reacting or shared by several sources (panallergens), thus influencing the appropriate management of a patient's allergy. This also concerns allergen immunotherapy (AIT), which may be prescribed more precisely based on the component-resolved diagnosis results. However, the advance in diagnosis needs to be mirrored in AIT. According to consensus documents and to expectations of specialists, therapy should be based on standardized extracts containing measured amounts of the clinically relevant molecules, ie, the major allergens. The new generation of extracts for sublingual immunotherapy fulfills these requirements and are thus defined as biomolecular (BM). BM refers to natural extracts with a defined content of major allergens in micrograms. All Staloral BM products are indicated for the treatment of allergic rhinitis with or without asthma. The effectiveness of AIT is related to its ability to modify the immunological response of allergic subjects. The 5-grass and house dust mite extracts were evaluated addressing the T helper 1, T helper 2, and T helper 3 cells by polymerase chain reaction array on mRNA extracted from Waldeyer's ring tissue (adenoids). Sublingual immunotherapy with a defined content of major allergens in micrograms induced a strong downregulation of genes involved in T helper 2 and T helper 1 activation and function, allowing the definition of the immunologic effect as "bio-homeostatic". This clinical and immunological model must be implemented with respect to other allergens, thus expanding the application of a treatment with a unique disease-modifying capacity.
Keywords: allergen immunotherapy, allergy, component resolved diagnosis, major allergens, allergen molecules
Corrigendum for this paper has been published
Introduction
The birth of molecular allergology was a major advance in the diagnosis of hypersensitivity diseases. In fact, the technique of component resolved diagnosis, measuring the specific IgE antibodies to individual allergen molecules (natural or recombinant), rather than to the entire sources containing them, provides more precise data for the diagnosis.1 In particular, separating the molecules actually responsible for clinical symptoms (genuine sensitizers) from those simply cross-reacting or shared by several sources (panallergens) allows for the identification of causative allergens in polysensitized patients and, hence, appropriate management of the patient’s allergy.2 This is true for food allergy3 and is of paramount importance with respect to respiratory allergy. Actually, more refined and precise pollen maps were recently obtained by using molecular techniques in place of the common measurement of pollen grains.
As far as the prescription of allergen immunotherapy (AIT) is concerned, Sastre et al demonstrated that the use of molecular diagnosis substantially changed the previous decision, which was based on conventional diagnosis.5 This observation was confirmed also in children with pollen allergy.6 However, the concern is the choice of the allergen source to be used (birch pollen, grass pollen, olive pollen, etc) but not the molecular composition of the extract. In fact, the possibility of performing a tailored AIT, that is, based on products containing a composition of recombinant allergens reproducing the patient’s individual sensitization profile, has long been envisioned.7 However, considering the large number of different sensitization profiles, the commercial availability of these products is unlikely, due to the high cost required to register each product. Notably, a controlled trial on patients with rhinoconjunctivitis induced by birch pollen showed that the clinical efficacy of two preparations containing the recombinant or natural major allergen Bet v 1 from birch pollen was similar to that of a high-quality, licensed birch pollen extract.8 Indeed, this shows a noninferiority of the molecular preparations versus high-quality conventional extracts, and makes apparent that when the diagnosis is sound and the AIT product fulfills the quality standards, the results are clinically comparable to molecular preparations.
The importance of titrating the major allergen content in micrograms (μg) was recently supported by the opinions of allergy specialists. In a recent survey, specialists evaluated the importance of the different issues with respect to AIT, ranking high the documentation of the content of the major allergens in μg, along with the level of evidence-based validation of efficacy and safety, and the standardization of the products.9 Actually, consensus documents on AIT view the documentation on the content of major allergens in μg as a need to be met by producers.10,11 This is also a requirement by the European Medicine Agency guidelines for the production and quality of AIT preparations.12
Here, we review the background and the characteristics of Staloral biomolecular (BM) products for AIT, based on defined contents in μg of major allergens.
Production technology
Each allergen source contains a large number of components, some of them eliciting a specific IgE response. The frequency of IgE binding in a population of patients allergic to a given source, higher or lower than 50%, defines major allergens and minor allergens, respectively.13 For decades, allergen extracts for AIT were measured by their protein content, the protein nitrogen unit being the most used quantification unit; but, in the 1980s, the development of international standards redefined biological potency units.14 Among these units, the histamine equivalent in prick testing, the biological units, and the bioequivalent allergen units were the most used. Table 1 shows the characteristics of such units.15 In 2008, the CREATE (Certified REference Materials for Allergenic Products and Validation of MeThods for their Quantification) project, involving research laboratories, allergen manufacturers, clinical researchers, and biotech companies from European Union member states, was launched with the aim to develop certified reference materials for allergen extracts and to validate the enzyme-linked immunosorbent assays (ELISA) for the measurement of major allergens.16
Table 1 Biological potency units for allergen extracts
Abbreviations: BAU, bioequivalent allergen unit; BU, biological unit; HEP, histamine equivalent in prick testing.
The measurement in μg of the content in major allergens of allergenic products is currently considered mandatory in the certification of their quality.11,15 Every manufacturer of allergen extracts has its in-house reference standard measuring the allergenic potency. For extracts from Stallergenes (Antony, France), the potency is measured by the index of reactivity (IR) per milliliter, with 100 IR/mL corresponding to the allergenic potency of an extract eliciting a mean wheal diameter of 7 mm by skin prick tests performed in patients allergic to the specific allergen source.17 The product Staloral300, obtained from the same source material used for BM products but titrated only in IR, was demonstrated to be efficacious by 20 double-blind, placebo-controlled trials on patients allergic to pollens and dust mites.18 The quantification of allergens is generally made by antibody-based techniques such as radioimmunoassay, ELISA, radial immunodiffusion, and rocket immunoelectrophoresis (RIE), but due to the variations of measurement depending on the technique used, the modern approach used in production to quantify the relevant allergens is also based on mass spectrometry (MS).19 MS is an analytical technique that produces spectra of the masses of the atoms or molecules comprising a sample of material, which was applied since 2002 in the study of allergens.20 The use of these techniques, ie, the quantification in μg with the respective measurement in 100 IR, led to the documentation of the correspondence between IR and μg for the different allergens (Table 2). The use of MS has overcome the limitation of antibody-based immunoassays, which, because of the variability of the natural allergen structure, often resulted in excess of specificity and, consequently, in an underestimated allergen concentration. Instead, MS is able to detect the peptides that are conserved in all the different isoforms, allowing the proper quantification of allergens naturally occurring in different isoforms, such as the group 1 allergens from grass pollen or Der p 2 from Dermatophagoides pteronyssinus.21 Another prerequisite for quality is the stability of the product, which is essential in maintaining allergenic potency. High temperature is the most critical factor affecting stability. The Staloral extracts for AIT from Stallergenes were tested for temperature during transport in the warm season, as occurs when the product is sent from the manufacturer to a patient’s house, using a thermal-insulating packaging. ELISA-inhibition tests of extracts subjected to temperatures up to 45°C showed that an immunologic activity higher than 75% of that measured in the production phase was