Allergen Release Profiles of Fast-Dissolving Freeze-Dried Orodispersible Sublingual Allergy Immunotherapy Tablets.

Background: Sublingual allergy immunotherapy tablets (SLIT-tablets) provide a well-tolerated and clinically efficacious treatment for allergic disease such as allergic rhinitis and allergic asthma. In SLIT, uptake of allergen by immune-competent cells in the oral mucosa activates the immune system and leads to tolerance toward the sensitizing allergen. The ability to deliver the full allergen content into solution within the recommended sublingual holding time is therefore an essential quality of SLIT-tablets that must be supported by the tablet formulation for all relevant allergen sources. SLIT-tablets based on a fast-dissolving orodispersible freeze-dried formulation (Zydis) are currently available for 5 of the most prevalent allergens: tree (birch and related species from the birch-homologous group), grass, ragweed, Japanese cedar, and house dust mite. Objectives: The purpose of this study was to examine the allergen release properties of three freeze-dried SLIT-tablets containing tree, ragweed, and Japanese cedar extracts, respectively. The correlation between SLIT-tablet allergen release and the level of allergen-specific T-cell activation was examined for the tree SLIT-tablet. Methods: Allergen release kinetics and tablet disintegration times for the 3 freeze-dried SLIT-tablets were examined. For all 3 tablets, the magnitude of solubilized major allergen relative to time in solution was compared to external controls to achieve a measure of the total allergen release. Additional assessments of allergen release occurring after the initial timepoint (15 or 30 seconds in solution) were done independently of external controls by linear regression analyses. For the tree SLIT-tablet, the immunological potency of the released major allergen was assessed at each experimental timepoint by a Bet v-specific T-cell activation assay. Results: All 3 SLIT-tablets disintegrated within 1 second after contact with assay buffer without any detectible residue. Complete release of major allergens (Bet v 1, Amb a 1, and Cry j 1, respectively) was seen at the earliest experimental time points (15 or 30 seconds). For the tree SLIT-tablet, full T-cell activation was achieved at 30 seconds (earliest experimental time point). Conclusions: The freeze-dried SLIT-tablet formulation consistently provides rapid and complete release of allergen from a wide range of species in a standardized in vitro assay. Full release of the SLIT-tablet allergen content within the sublingual holding time is a prerequisite for maximal exposure of allergens to the sublingual mucosa immune system. The freeze-dried SLIT-tablet formulation examined here supports short sublingual holding times and furthermore offers a convenient administration form of allergy immunotherapy.

State of the Art: Development of a Sublingual Allergy Immunotherapy Tablet for Allergic Rhinitis in Japan.

Allergic rhinitis (AR) caused by house dust mite (HDM) and Japanese cedar pollen (JCP) represents a significant, expanding health problem in Japan. Allergic symptoms often have a severe impact on the QOL such as sleep disturbance and reduced school and work performance. In addition to the classical symptoms, AR is known to be a risk factor for the development of allergic asthma, a potentially life-threatening condition. Allergy immunotherapy (AIT) is a well-documented, safe, effective treatment option for respiratory allergic disease. It has been demonstrated that AIT can provide relief from clinical symptoms and that AIT has the potential to provide long-term post-treatment effect. Although the mechanism of AIT is not fully understood, it can actively modulate protective allergen-reactive pathways of the immune system and alter the natural course of disease. Unlike pharmacotherapy, AIT addresses the basic immunological mechanisms that are responsible for the development and persistence of allergic conditions. Currently two main routes of AIT administration are commonly available, subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). Both SCIT and SLIT are clinically effective, and SLIT is particularly well tolerated, with a lower risk of systemic allergic reactions compared with SCIT. To date, SLIT tablets have been developed for a range of different allergies including HDM and JCP and are the best-documented AIT treatment form. Here we introduce the current status of development of a SLIT tablet in Japan for AR, examine the clinical aspects and mechanism of action of AIT, and discuss the future directions of SLIT.

Allergen Stability and Immunological Reactivity during Co-dissolution and Incubation of House Dust Mite and Japanese Cedar Pollen SLIT-Tablets.

Japanese allergic subjects are commonly sensitized to both house dust mite (HDM) and Japanese cedar pollen (JCP) and combined treatment with sublingual immunotherapy (SLIT) tablets is desirable. However, mixing extracts of two non-homologous allergens may compromise allergen stability and affect the clinical outcome. Therefore, we investigated the stability of major allergens and total allergenic reactivity of HDM and JCP SLIT-tablets following dissolution in human saliva or artificial gastric juice. Two fast-dissolving freeze-dried SLIT-tablets were completely dissolved and incubated at 37 °C. Major allergen concentrations and total allergenic reactivity were measured. After mixing and co-incubation of HDM and JCP SLIT tablets in human saliva for 10 min at 37°C, there were no statistically significant changes in major allergen concentrations. In addition, no loss of allergenic reactivity of the mixed two SLIT-tablet solutions was seen. In contrast, complete loss of allergenic reactivity and detectable major allergen concentrations occurred when the two SLIT-tablets were dissolved and incubated in artificial gastric juice. These results demonstrate that HDM or JCP major allergens and the total allergenic reactivity of both SLIT-tablets measured here remain intact after dissolution and co-incubation in human saliva, supporting the possibility of a dual HDM and JCP SLIT-tablet administration regimen if clinically indicated. The complete loss of allergenic reactivity after incubation in artificial gastric juice can furthermore be taken to indicate that the immunological activity of the allergen extracts contained in the two SLIT-tablets is likely to be lost or severely compromised upon swallowing.

A birch sublingual allergy immunotherapy tablet reduces rhinoconjunctivitis symptoms when exposed to birch and oak and induces IgG4 to allergens from all trees in the birch homologous group.

BACKGROUND: This randomized, double-blind trial was conducted to determine the optimal dose for clinical efficacy of the SQ tree SLIT-tablet. An environmental exposure chamber (EEC) was used to reduce variability of allergen exposure and allow investigation of symptom reduction towards different species from the birch homologous group in separate EEC sessions. METHODS: Eligible subjects (N = 219) were randomized to receive treatment with placebo or the SQ tree SLIT-tablet (2, 7, or 12 DU) for 24 weeks. EEC pollen challenges were conducted outside the birch pollen season and included four birch and two oak EEC sessions. The primary efficacy endpoint was the average allergic rhinoconjunctivitis (ARC) total symptom score (TSS) after 24 weeks of treatment. RESULTS: There was a statistically significantly lower TSS during the 24-week birch EEC session for 7 DU and 12 DU compared to placebo with relative differences of 24% (P = 0.03) and 25% (P = 0.02). For the 24-week oak EEC session, there was a statistically significant difference for 12 DU (24%, P = 0.03). IgE and IgG4 measurements supported these findings and demonstrated cross-reactivity to all other species within the birch homologous group. Treatment was well-tolerated with the most frequently reported adverse reactions being the local reactions in the oral cavity of mild-to-moderate severity. CONCLUSION: This trial demonstrates that the SQ tree SLIT-tablet reduce ARC symptoms triggered by birch or oak pollen. The optimal dose for further development was 12 DU. Clinical and immunological findings suggest that the tablet may be used to treat allergies to all species within the birch homologous group.

Establishment of enzyme-linked immunosorbent facilitated antigen binding as a biomarker assay for Japanese cedar pollen allergy immunotherapy.

BACKGROUND: Clinical efficacy of allergen-specific Immunotherapy (AIT) towards Japanese cedar (JC) pollen allergy is firmly established but JC pollen-specific biomarker assays are lacking. Treatment-related increase of allergen-specific antibodies is a robust biomarker of successful AIT. Allergen-specific non-IgE antibodies are believed to reduce the effects of allergen exposure by competing with IgE for allergen binding, and in-vitro assays quantifying the effects of AIT-induced IgE-blocking antibodies are advantageous. A cell-free enzyme-linked immunosorbent facilitated antigen binding (ELIFAB) assay of JC pollen was established. METHODS: Serum IgE-allergen complexes were captured by immobilized recombinant CD23, and allergen-IgE-CD23 complexes were detected by a biotin-conjugated anti-human IgE antibody. Sera from JC pollen-allergic subjects without or with subcutaneous immunotherapy (SCIT) with JC pollen extract were used (n = 11/group). RESULTS: Optimal assay conditions were established at 20 µg/mL CD23 and 0.3 µg/mL JC pollen extract, and the dependency on CD23 and IgE was verified. The data show that the JC pollen ELIFAB assay is fit for purpose and demonstrates that the IgE-blocking activity is significantly increased in the JC pollen SCIT group compared with the non-treated group. CONCLUSION: The JC pollen ELIFAB assay represents a simple, cell-free biomarker assay for monitoring the development of IgE-blocking antibody activity during JC pollen AIT.

The Effective Allergenic Reactivity of House Dust Mite Sublingual Immunotherapy Tablets Is Determined by Tablet Formulation.

House dust mite (HDM) sublingual immunotherapy (SLIT) in the form of SLIT-tablets is now an established treatment option for HDM allergy and HDM-induced allergic asthma. In SLIT-tablet immunotherapy allergen extracts are formulated as dry tablets and administered under the tongue where it must be solubilized in saliva in order to be able to interact with the immune system of the sublingual mucosa. Solubilization of the extract must occur within a short time span of about one minute after administration, determined by the sublingual holding time recommended by the manufacturer. Currently, two types of HDM SLIT-tablets are available. Both tablet types contain natural HDM extracts from two common HDM species as the active ingredient, but differ with regard to formulation as one tablet type is based on a freeze-dried tablet formulation while the other is based on a compressed formulation. HDM extracts contain a number of major and minor allergens, which in combination provide the allergenic activity that drives the immunological response and in turn the clinical efficacy of the tablets. Here, a biologically relevant human immunoglobulin E (IgE)-based assay is used to compare the ability of the two HDM SLIT-tablet types to deliver HDM allergenic reactivity from the dry tablet into soluble form. The experiments demonstrate that the freeze-dried formulation delivers HDM allergenic activity into solution faster and more efficiently than the compressed formulation.

Bioavailability of House Dust Mite Allergens in Sublingual Allergy Tablets Is Highly Dependent on the Formulation.

BACKGROUND: In sublingual immunotherapy (SLIT), the immune system is addressed by solubilized allergen that interacts with immunocompetent cells of the oral mucosa, the efficiency of which is governed by 2 main factors of SLIT allergen bioavailability: the allergen concentration and the mucosal contact time. Recently, 3 house dust mite (HDM) SLIT tablets were developed that differ with regard to allergen content, nominal strength (maintenance doses: 6 SQ-HDM/10,000 Japanese Allergen Units [JAU], 12 SQ-HDM/ 20,000 JAU, and 300 IR/57,000 JAU), and formulation (freeze-dried/compressed). Here, the importance of the SLIT tablet formulation for HDM major allergen bioavailability is examined. METHODS: The HDM major allergen content, tablet disintegration times, and allergen release kinetics were determined. Dissolution kinetics (allergen concentration vs. time) of Der f 1, Der p 1, and Der 2 were measured. Area under the curve (AUC) was used as a surrogate parameter for allergen bioavailability. RESULTS: The release of HDM major allergens from the freeze-dried tablets was complete after 30 s, while only partial release was achieved with the compressed tablets, even after prolonged dissolution. At 1 min, i.e., the recommended sublingual holding time for the freeze-dried tablets, the allergen bioavailability (AUC) of the compressed 300 IR/57,000 JAU tablet was 4.7-fold (Der f 1), 10.8-fold (Der p 1), and 23.6-fold (Der 2) lower than that of the freeze-dried 12 SQ-HDM/20,000 JAU tablet and similar to (Der f 1) and 5.3-fold (Der p 1) and 12.5-fold (Der 2) lower than that of the freeze-dried 6 SQ-HDM/10,000 JAU tablet. CONCLUSIONS: SLIT tablet allergen bioavailability depends highly on the tablet formulation. Only the fast-dissolving freeze-dried tablets provide maximal delivery of soluble allergens and achieve allergen concentrations that reflect the nominal tablet strengths within the recommended sublingual holding time.

Major allergen content consistency of SQ house dust mite sublingual immunotherapy tablets and relevance across geographic regions.

BACKGROUND: Consistency in composition and potency, particularly regarding major allergens, is crucial for the quality of extracts for allergen immunotherapy. OBJECTIVE: To characterize the major allergen composition of house dust mite (HDM) extracts commercially available in the United States and the SQ HDM sublingual immunotherapy (SLIT) tablet, and to relate the composition to patient sensitization patterns. METHODS: Der 1/Der 2 ratios were determined in 10,000- and 30,000-AU/mL HDM extracts from 5 US companies and the SQ HDM SLIT-tablet. Allergen content was analyzed by enzyme-linked immunosorbent assay and compared with an in-house reference. Sensitivity toward Der p 1, Der p 2, and Der p 10 was determined in serum from randomly selected subgroups of 220 individuals from North American and European SQ HDM SLIT-tablet trials. RESULTS: Mean Der 1/Der 2 ratios in US HDM extracts ranged from 0.4 to 20.5. For the SQ HDM SLIT-tablet (20 batches), variability did not exceed 12% regarding content of Der f 1 (SD, 11.9%; 95% confidence interval [CI], 0.94-1.06), Der p 1 (SD, 6.1%; 95% CI, 0.97-1.03), and combined Der 2 allergen (SD, 6.4%; 95% CI, 0.97-1.03), indicating a consistent Der 1/Der 2 ratio. High allergen sensitivity frequencies toward Der p 1 and Der p 2 were observed regardless of geographic region. Efficacy of the SQ HDM SLIT-tablet has been demonstrated in 5 clinical trials. CONCLUSION: The SQ HDM SLIT-tablet has efficacy potential for a broad range of patients because it includes a consistent 1:1 ratio of the 2 major HDM allergens to which individuals were most frequently sensitized across geographic regions. Efficacy has been demonstrated.

Effect of Polarization on Airway Epithelial Conditioning of Monocyte-Derived Dendritic Cells.

Airway epithelial cells (AECs) form polarized barriers that interact with inhaled allergens and are involved in immune homeostasis. We examined how monocyte-derived dendritic cells (MDDCs) are affected by contact with the airway epithelium. In traditional setups, bronchial epithelial cell lines were allowed to polarize on filter inserts, and MDDCs were allowed to adhere to the epithelial basal side. In an optimized setup, the cell application was reversed, and the culture conditions were modified to preserve cellular polarization and integrity. These two parameters were crucial for the MDDCs' immunoregulatory properties; thus, previous observations obtained using traditional setups should be considered with caution. Using the optimized setup, AEC conditioning of MDDCs led to increased expression of programmed death 1 ligand 1, immunoglobulin-like transcript 3, CD40, CD80, and CD23. This increased expression was accompanied by decreased secretion of monocyte chemotactic protein 1 and eotaxin and donor-variable effects on IL-12 and IL-10 secretion. Conditioning varied between maturation states and depended partly on direct contact between AECs and MDDCs. The setup allowed MDDCs on the basal side of the epithelium to sample allergens administered to the apical side. Allergen uptake depended on polarization and the nature of the allergen. AEC conditioning led to decreased birch allergen-specific proliferation of autologous T cells and a trend toward decreased secretion of the Th2-specific cytokines IL-5 and IL-13. In conclusion, we determined that AEC conditioning favoring cellular integrity leads to a tolerogenic MDDC phenotype, which is likely to be important in regulating immune responses against commonly inhaled allergens.

Short ragweeds is highly cross-reactive with other ragweeds.

BACKGROUND: The most widespread ragweed (Ambrosia) species in North America are short ragweed (Ambrosia artemisiifolia; Amb a), giant ragweed (Ambrosia trifida; Amb t), and western ragweed (Ambrosia psilostachya; Amb p). Varied geographic distributions of ragweed species raise questions regarding the need for ragweed species-specific allergen immunotherapy. OBJECTIVE: To determine allergenic cross-reactivity among ragweed species by immunologic analyses of sera from subjects allergic to ragweed from North America and Europe. METHODS: Sera were collected from 452 subjects allergic to ragweed who participated in Amb a sublingual immunotherapy tablet clinical trials. All subjects had positive skin prick test and serum IgE against Amb a. Ragweed-specific IgE (pre treatment) and IgG4 (post treatment) were measured by ImmunoCAP. IgE inhibition studies among Amb a, Amb t, and Amb p were conducted. Using pooled sera from another ragweed-allergic population, IgE inhibition studies of 7 less widespread Ambrosia species also were conducted. RESULTS: A strong correlation between Amb a vs Amb p and Amb t serum IgE levels was observed. In the vast majority of pretreatment sera, Amb a inhibited Amb a, Amb p, and Amb t IgE reactivity by more than 90%. Strong correlations were observed between Amb a vs Amb p and Amb t post-treatment IgG4 levels. In pooled sera, Amb a extract inhibited the binding of serum IgE to all 10 ragweed species by 98%-100%. CONCLUSION: In a population of subjects allergic to Amb a, substantial allergenic cross-reactivity among Amb a, Amb p, and Amb t was demonstrated. These in vitro data suggest that an Amb a-based single-species ragweed allergen immunotherapy may be therapeutically active in patients exposed to diverse ragweed pollens. TRIAL REGISTRY: Clinicaltrials.gov, NCT00770315, NCT00783198, and NCT00330083.

Epitope grafting, re-creating a conformational Bet v 1 antibody epitope on the surface of the homologous apple allergen Mal d 1.

Birch-allergic patients often experience oral allergy syndrome upon ingestion of vegetables and fruits, most prominently apple, that is caused by antibody cross-reactivity of the IgE antibodies in patients to proteins sharing molecular surface structures with the major birch pollen group 1 allergen from Betula verrucosa (Bet v 1). Still, to what extent two molecular surfaces need to be similar for clinically relevant antibody cross-reactivity to occur is unknown. Here, we describe the grafting of a defined conformational antibody epitope from Bet v 1 onto the surface of the homologous apple allergen Malus domestica group 1 (Mal d 1). Engineering of the epitope was accomplished by genetic engineering substituting amino acid residues in Mal d 1 differing between Bet v 1 and Mal d 1 within the epitope defined by the mAb BV16. The kinetic parameters characterizing the antibody binding interaction to Bet v 1 and to the mutated Mal d 1 variant, respectively, were assessed by Biacore experiments demonstrating indistinguishable binding kinetics. This demonstrates that a conformational epitope defined by a high affinity antibody-allergen interaction can successfully be grafted onto a homologous scaffold molecule without loss of epitope functionality. Furthermore, we show that increasing surface similarity to Bet v 1 of Mal d 1 variants by substitution of 6-8 residues increased the ability to trigger basophil histamine release with blood from birch-allergic patients not responding to natural Mal d 1. Conversely, reducing surface similarity to Bet v 1 of a Mal d 1 variant by substitution of three residues abolished histamine release in one patient reacting to Mal d 1.

Isoallergen variations contribute to the overall complexity of effector cell degranulation: effect mediated through differentiated IgE affinity.

Most allergens exist in several variants (isoallergens), each of which may be recognized differently by patient IgE. We have previously shown that several properties of the IgE repertoire, including IgE affinity and IgE clonality, are important factors determining degranulation responses of effector cells involved in type I allergic reactions. However, less is known about how the repertoire of naturally occurring isoallergens may affect this response. Thus, in this study, we investigated how individual rIgE Ab clones derived from a human subject are able to distinguish among variants of Der p 2 isoallergens and assessed the impact on basophil degranulation. Biacore analyses showed that individual rIgE clones cloned from an individual allergic to house dust mites recognized Der p 2 with binding affinities varying up to 100-fold between different Der p 2 isoforms. In a well-defined biological system consisting of human basophils sensitized with low rIgE clonality, degranulation responses were directly related to rIgE affinity toward particular rDer p 2 isoallergens. However, basophils sensitized with polyclonal patients' sera showed no differences in degranulation responses toward the different rDer p 2 isoallergens. In conclusion, our study shows that individual IgE Abs are able to bind single allergens with a broad range of affinities due to natural isoallergen variations, contributing further to the overall complexity of IgE-allergen interactions at the effector cell surface, which is, however, blurred by the polyclonal nature of patients' IgE repertoires.

Facilitated antigen presentation and its inhibition by blocking IgG antibodies depends on IgE repertoire complexity.

BACKGROUND: The antibody repertoires of allergic subjects are characterized by the presence of allergen-specific IgE antibodies. We have previously shown that the composition of the IgE repertoire is critical for allergen-mediated activation of human effector cells. Activation of CD4(+) T cells in allergic subjects is highly potentiated by the process of facilitated antigen presentation (FAP), in which allergen in complex with IgE is taken up by B cells through the low-affinity IgE receptor CD23 and presented to T cells. OBJECTIVE: We sought to investigate the influence of IgE repertoire complexity on the formation of IgE/allergen/CD23 complexes on B cells and subsequent T-cell activation. METHODS: Using defined allergen-specific recombinant IgE and IgG antibodies, we investigated the influence of individual IgE affinity, IgE clonality, specific IgE concentration, and the ratio between IgE specificities on IgE/allergen/CD23 complex formation in vitro. RESULTS: Although IgE affinity is an important factor, IgE clonality seems to be governing complex formation, especially with medium- and low-affinity IgE antibodies. We demonstrate that differences in allergen-specific IgE affinity correlate with the efficiency of subsequent T-cell activation. In addition, we show that the complexity of an IgE repertoire also affects the ability of allergen-specific IgG antibodies to block FAP. CONCLUSION: The composition of allergen-specific IgE repertoires in individual patients, especially with respect to IgE clonality, might play an important role in the manifestation of allergic disease not only for the immediate allergic reaction through activation of basophils and mast cells but also for the exacerbation of allergic inflammation through recurring activation of allergen-specific T cells by FAP.

Correlation between sensitization to house dust mite major allergens, age, and symptoms in Japanese house dust mite allergic subjects.

Der p 23 has recently been recognized as a new house dust mite (HDM) major allergen that may be linked to the development of asthma in HDM allergic patients. This study aimed to investigate the frequency of sensitization to HDM major allergen components including Der p 23 and to examine the correlation between HDM-sensitization and AR symptom score in Japanese HDM allergic rhinitis (AR) patients without allergic asthma. Serum samples (n = 120) collected from Japanese HDM AR patients (12 to 64 years) without asthma were assessed for allergen-specific IgE (s-IgE) by ImmunoCAP (Dermatophagoides pteronyssinus (D. pteronyssinus; Der p) extract, Der p 23) or immunosolid-phase allergen chip (Der p 1, Der p 2). Japanese HDM AR patients without asthma showed a high prevalence of allergic sensitization to the HDM major allergens Der p 1 (94.2%), Der p 2 (97.5%) and Der p 23 (71.7%). No difference in the prevalence was detected for Der p 1 and Der p 2 s-IgE among three age groups. However, the prevalence of Der p 23 s-IgE was significantly higher in the younger group compared to the elderly group. No significant correlation was found between AR symptom scores and concentration of s-IgE towards Der p extract and any of the three HDM major allergens. Although the prevalence of sensitization towards D. pteronyssinus major allergens is high in Japanese AR patients without asthma, there was no correlation between allergen specific IgE including IgE towards Der p 23 and AR symptom in this population.

Phl p 5 resorption in human oral mucosa leads to dose-dependent and time-dependent allergen binding by oral mucosal Langerhans cells, attenuates their maturation, and enhances their migratory and TGF-beta1 and IL-10-producing properties.

BACKGROUND: Sublingual immunotherapy (SLIT) is safe and effective as treatment of allergic rhinitis and mild asthma. Oral mucosal Langerhans cells (oLCs) play a central role. However, little is known about allergen binding by oLCs during mucosal allergen resorption and its impact on oLC functions. OBJECTIVE: Binding of Phl p 5 to oLCs was studied in a standardized ex vivo model to investigate mechanisms important for SLIT. METHODS: Human oral mucosal biopsies were incubated with the grass pollen allergen Phl p 5. Migration, binding of Phl p 5, phenotype and cytokine production, and T-cell priming of Phl p 5-binding oLCs were analyzed. RESULTS: Significant uptake required more than 5 minutes, and dose-dependent binding of Phl p 5 to oLCs was saturated at 100 microg/mL Phl p 5. Furthermore, Phl p 5 significantly increased the migratory capacity of oLCs but attenuated their maturation and strongly promoted the release of TGF-beta1 and IL-10 by oLCs themselves as well as by cocultured T cells. CONCLUSION: Oral mucosal Langerhans cells bind Phlp5 in a dose-dependent and time-dependent manner, leading to an increased production of tolerogenic cytokines and an enhanced migratory capacity but decelerated maturation of oLCs.

Reduced anaphylactic potential of denatured pullulan-conjugated Cry j 1.

Japanese Cedar (JC) pollinosis is the most common seasonal allergic rhinitis in Japan. Throughout the JC pollen season, patients suffer from the allergic symptoms, resulting in a reduction of quality of life. Allergy immunotherapy (AIT) is an established treatment option for a wide range of allergens that unlike symptomatic treatments (e.g. antihistamines) may provide sustained immune tolerance. However, AIT, especially subcutaneous immunotherapy (SCIT) has a fatal anaphylaxis risk due to the use of crude allergen extracts. Consequently, development of allergen derivatives with substantially reduced anaphylactic potential is desirable. An allergen derivative that showed reduced IgE-binding and anaphylactic potential was developed through conjugation of native Cry j 1 (n Cry j 1), a major JC allergen, to the polysaccharide pullulan followed by chemical but non-covalent denaturation. The resulting Cry j 1 allergen derivative, Dn p-Cry j 1, showed reduced IgE-binding and IgE-mediated effector cell activation in vitro using an ELISA competition assay and a mast cell activation model (EXiLE). Reduced anaphylactic potential of Dn p-Cry j 1 in vivo was demonstrated using the rat passive cutaneous anaphylaxis (PCA) assay. The difference in anaphylactic potential of Dn p-Cry j 1 compared to n Cry j 1 in wild-type rats was of the same magnitude as the difference seen in the anaphylaxis reactions obtained with n Cry j 1 in wild-type rats and mast-cell deficient rats, indicating a dramatic reduction in anaphylactic potential of Dn p-Cry j 1. These results indicate that Dn p-Cry j 1 is a promising candidate for next-generation JC AIT.