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Keywords:

  • allergen content;
  • assay standardization;
  • dose–response;
  • regulatory guidelines;
  • specific immunotherapy

Abstract

  1. Top of page
  2. Abstract
  3. Methodology
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References

To cite this article: Calderón MA, Larenas D, Kleine-Tebbe J, Jacobsen L, Passalacqua G, Eng PA, Varga EM, Valovirta E, Moreno C, Malling HJ, Alvarez-Cuesta E, Durham S, Demoly P. European Academy of Allergy and Clinical Immunology task force report on ‘dose–response relationship in allergen-specific immunotherapy’. Allergy 2011; 66: 1345–1359.

Abstract

Background:  For a century, allergen-specific immunotherapy (SIT) has proven to be an effective treatment for allergic rhinitis, asthma, and insect sting allergy. However, as allergen doses are frequently adapted to the individual patient, there are few data on dose-response relationship in SIT. Allergen products for SIT are being increasingly required to conform to regulatory requirements for human medicines, which include the need to demonstrate dose-dependent effects.

Methods:  This report, produced by a Task Force of the EAACI Immunotherapy Interest Group, evaluates the currently available data on dose-response relationships in SIT and aims to provide recommendations for the design of future studies.

Results:  Fifteen dose-ranging studies fulfilled the inclusion criteria and twelve reported a dose-response relationship for clinical efficacy. Several studies also reported a dose-response relationship for immunological and safety endpoints. Due to the use of different reference materials and methodologies for the determination of allergen content, variations in study design, and choice of endpoints, no comparisons could be made between studies and, as a consequence, no general dosing recommendations can be made.

Conclusion:  Despite recently introduced guidelines on the standardization of allergen preparations and study design, the Task Force identified a need for universally accepted standards for the measurement of allergen content in SIT preparations, dosing protocols, and selection of clinical endpoints to enable dose-response effects to be compared across studies.

Allergen-specific immunotherapy (SIT) is used in the treatment of allergic rhinitis, asthma, and insect sting allergy. The two currently validated administration routes for SIT in adults and children are subcutaneous (SCIT) and sublingual (SLIT). Although the clinical efficacy of SIT has been demonstrated by multiple studies and systematic reviews, a correlation between the allergen dose and the clinical outcome measures has not been clearly established. Presently, SIT dose regimens used in clinical practice vary widely among countries and clinics, although some suggestions on the optimal dose have been made in a World Health Organization position paper (1). Moreover, no standards for the measurement of allergen content are universally accepted, with manufacturers using in-house reference materials and their own units to express potency (2–6), which may hamper analysis of the few available dose–response studies.

As allergen extracts are usually prepared from natural biological sources, standardization procedures are of crucial importance. The CREATE project (Development of Certified Reference Materials for Allergenic Products and Validation of Methods for their Quantification) has recently worked toward the development of certified reference materials of purified natural or recombinant allergens and the validation of enzyme-linked immunosorbent assays (ELISAs) for the quantification of major allergens (7). Additional sources of variability that further hamper a proper dose–response analysis are the large differences in the qualitative composition of the marketed products and the presence of different adjuvants and their quantities. Finally, the efficacy of an allergen extract may also depend on factors that influence bioavailability, for example, the volume in which the allergen is dissolved and for sublingual immunotherapy (SLIT), the form of administration, i.e., drops vs tablets. As a result, direct comparisons cannot be made between studies using products from different manufacturers to establish a dose–response relationship for a particular allergen extract.

Studies are available, however, in which several doses of the same extract are compared. Endpoints used in these different SIT dose–response studies vary widely and include clinical efficacy, changes in markers of immune response, number and severity of adverse events (AEs), and surrogate endpoints, such as titrated skin prick tests, nasal and bronchial challenge, and measurements of blood and intranasal cytokines. Recently, the European Medicines Agency (EMA) produced a guideline for standardization of clinical trials with SIT in allergic disease that includes some recommendations for the selection of endpoints (8). However, many SIT studies were designed or published before these recommendations, making it difficult to directly compare findings. Moreover, a change of dose can be accomplished by changing volume or concentration, or both, with or without concomitant change in the adjuvant dose (both in concentration and in volume). In most of the studies, these dosing details were not provided and thus it is not possible to assign any observed variations in effect to a single factor, such as the allergen dose. Thus, even if standardized allergens and validated ELISAs become available, it would be impossible to make cross-comparison between studies.

One of the basic principles in empirical SIT has always been that doses are adapted to the individual patient, with the highest tolerated dose for each individual considered to be the most effective. Dose–response data that could enable the physician to rapidly determine the dose with the most favorable risk-to-benefit ratio are scarce. Allergen products for SIT are being increasingly required to conform to regulatory requirements for human medicines and the recently introduced EMA guidelines on the clinical development of products for SIT (8) state that after establishing a tolerated dose range, studies should be performed to establish a dose–response relationship for clinical efficacy. These new requirements for dose–response data further underline the need to adopt international standards for quantification and investigation of allergen products for SIT, to enable harmonization of clinical trial data. Such a process would require the introduction of standard reference materials and assays for allergen products, validation of clinical endpoints, and the adoption of consistent guidelines on the conduct of clinical studies in SIT by regulatory authorities worldwide. It should be borne in mind, however, that it is intrinsically difficult with SIT to build classical dose–response curves, because the clinical effects of the treatment appear within weeks or months, and no objective marker that could replace the traditional symptom score is available.

A serious attempt at harmonizing potency and characteristics of allergen extracts has already been made in the 1970–1980s. An international team of experts created certified reference material (CRM) of five allergens, for manufacturers to use as a reference for their products (9–13). However, as the use of these CRMs was optional and their quality variable, being extracts of natural allergen sources, their use never became established.

This European Academy of Allergy and Clinical Immunology (EAACI) Immunotherapy Interest Group Task Force Report evaluates the currently available data on dose–response relationship in SIT and is aimed at professionals performing SIT or involved in the design of clinical studies.

Methodology

  1. Top of page
  2. Abstract
  3. Methodology
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References

The Task Force carried out a search of PubMed; the Cochrane Ear, Nose and Throat Disorders Group Trials Register and the Cochrane Central Register of Controlled Trials, which includes EMBASE, CINAHL, IndMed, PakMediNet, CAB Abstracts, Web of Science, BIOSIS Previews, mRCT (Current Controlled Trials), ICTRP (International Clinical Trials Registry Platform); and Google using the keywords: allergen immunotherapy, allergen subcutaneous immunotherapy, allergen SLIT, immunotherapy AND rhinitis, immunotherapy AND asthma, immunotherapy AND venom. The date of the most recent search was May 19, 2011. The searches identified a total of 1739 references. Phase I studies were excluded, and search results were assessed to identify trials of SIT that tested multiple allergen doses. Studies were selected for evaluation if the following information was provided:

  •  Route of allergen administration (subcutaneous or sublingual);
  •  allergen used; and
    • clinical or surrogate endpoints or safety outcomes.
    Additional data were included, when available:
  •  Presence and/or dilution of an adjuvant;
  •  major allergen content in manufacturers units;
  •  treatment period;
  •  duration of up-dosing period; and
  •  duration of maintenance period.

Because there are no European or international standards for measurement of allergen content, and considerable heterogeneity between study endpoints, only the outcomes of individual dose–response studies were analyzed and no attempt was made to compare data from different studies. When information was not provided on the assay or standards used to determine the major allergen content of the extracts, these data were requested from the manufacturer.

The Task Force assessed the quality of the studies and assigned a score according to the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) scoring system (14), Delphi list (15), and Consolidated Standards of Reporting Trials (CONSORT) grading (16).

Results

  1. Top of page
  2. Abstract
  3. Methodology
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References

Study design and quantification of allergen content

Analysis of the search results identified fifteen studies that used different doses of allergen extracts (Table 1). Three were non-randomized and open (17–19), one was randomized and double blind for the dosing groups, including an open control group (20). Two studies were randomized and double blind for the treatment groups, but had no control group (21, 22), and the remaining nine trials were randomized, double-blind, placebo controlled (23–31). Two large trials (23, 31) produced several companion papers describing patient subgroups or immunological data (32–35). The studies including allergen, dose, main endpoints, and results are summarized in Table 1.

Table 1.   Summary of the selected SCIT and SLIT dose-ranging studies
AuthorsAllergenRDB PCAge rangeDurationMaintenance doses and enrolled patients per group*Clinical outcomesSurrogate endpoints (clinical and immunological)Main results
  1. RDB PC, randomized double-blind placebo controlled; NR, not reported; AEs, adverse events; NPT, nasal provocation test; PEF, peak expiratory flow; QoL, quality of life; SS, symptom score; SCIT, subcutaneous immunotherapy; SLIT, sublingual immunotherapy; [UPWARDS ARROW], increase; [DOWNWARDS ARROW], decrease.

  2. Clinical endpoints include global evaluations, SS, QoL assessments, medication scores. Clinical surrogate endpoints include SPT, skin LPR, CPT, bronchial and nasal challenge tests.

  3. *Monthly maintenance dose if not otherwise specified.

  4. †Indicates the primary outcome, when stated.

SCIT studies in respiratory allergies
 Johnstone et al. (27)Multiple mixedYes2–15 years4 yearsControls (n = 42) 1:10 000 000 w/v (n = 49) 1:5000 w/v (n = 39) 1:250 w/v (n = 43)Days with no wheezing No asthma in the last year New allergies Exercise-induced asthmaNoDays with no wheezing on exertion and during upper respiratory illness and patients without asthma attacks significantly [UPWARDS ARROW] in the two higher dose groups. No patient of the two higher dose groups developed new allergies
 Franklin et al. (22)Ragweed plus mix of other allergensNoNROpen dose-escalation 1 year+Trial 4 months1:1000 w/v (n = 13) 1:50 w/v (n = 12)Severity of symptoms†NoHigher dose significantly better than lower dose
 Creticos et al. (21)RagweedNoAdults18 months13 pts progressively on 0.6, 12.4, 24.8 μg Amb a 1Rhinitis symptoms-medications during seasonsNasal-specific challenge at each dose† Specific IgGThe nasal reactivity (mediators) decreased at the two higher doses of 12.4 and 24.8 μg as compared with pretreatment challenge Progressive increase in specific IgG [DOWNWARDS ARROW] symptom and medication score in all patients vs baseline season
 Haugaard et al. (20)HDMNo10–64 years2 yearsControl (n = 19) 0.7 μg Der p 1(n = 19) 7 μg Der p 1(n = 20) 21 μg Der p 1 (n = 16)Peak expiratory flow (PEF) and medication score AEsBronchial-specific reactivity† Conjunctival-specific reactivity[DOWNWARDS ARROW] of allergen-specific bronchial reactivity and conjunctival reactivity vs controls with no difference among the doses at 1 and 2 years Significant increase in AEs (local and systemic) with increasing maintenance doses [DOWNWARDS ARROW] of medication and PEF score in all three active groups compared to pretreatment, no change in control group
 Olaguibel et al. (18)HDMNo6–50 years18 monthsMax tolerated dose: <4 μg Der p 1 (n = 22) 4–16 μg Der p 1 (n = 15)Asthma symptoms and medications† AEsEye-specific reactivity Skin prick tests Specific IgE, IgG, IgG1, IgG4No difference between doses in all parameters except for specific IgG, IgG1 and IgG4 and skin reactivity (in favor of the high dose) Increased frequency of systemic AEs after administration of higher dose
 Ewbank et al. (25)CatYesAdults5 weeksPlacebo (n = 7) 0.6 μg Fel d 1 (n = 7) 3.0 μg Fel d 1 (n = 7) 15 μg Fel d 1 (n = 7)NoTitrated skin prick tests Specific IgE, IgG4 Cytokines (flow cytometry)Progressive [DOWNWARDS ARROW] in skin reactivity across doses. Significant overall dose-dependent effect for increase in sIgG4 and decrease in CD4 + /IL-4 +  peripheral blood mononuclear cells (PBMCs)
 Nanda et al. (29)CatYesAdults1 yearPlacebo (n = 7) 0.6 μg Fel d 1 (n = 6) 3.0 μg Fel d 1 (n = 6) 15 μg Fel d 1 (n = 7)NoTitrated-specific nasal challenge and skin prick tests† Specific IgE, IgG4 Cytokines at 5 wks and 1 yearSignificant [DOWNWARDS ARROW] in nasal and skin reactivity at 5 wks and 1 year with the highest dose Significant dose effect across doses Specific IgG4 significantly [UPWARDS ARROW] at 5 wks and 1 year in the medium- and high-dose group No change in cytokines
 Lent et al. (28)DogYesAdults5 weeksPlacebo (n = 7) 0.6 μg Can f 1 (n = 6) 3 μg Can f 1 (n = 7) 15 μg Can f 1 (n = 7)NoTitrated skin prick test Nasal challenge Specific IgE, IgG4 Lymphocyte proliferation, TGFβ and TNFαOverall dose-dependent [DOWNWARDS ARROW] in skin prick test and late cutaneous response vs baseline No change in nasal reactivity Specific IgG4 significantly [UPWARDS ARROW] in low and high dose Dose-dependent [UPWARDS ARROW] in TGFβ and [DOWNWARDS ARROW] in TNFα No change in specific IgE
 Frew et al. (26)GrassYes18–60 years6.4 months (3–8 months)Placebo (n = 103) 2 μg Phl p 5 (n = 104) 20 μg Phl p 5 (n = 203)Symptom/medication scores whole season† and peak season AEsQuality of life (QoL) Bad days (VAS)Symptom and medication scores: no difference between doses in whole season, significant difference in the peak season QoL: significant difference between doses Bad days (VAS): no difference between doses Overall more AEs in the high-dose group
SLIT studies in respiratory allergies
 Durham et al. (24)GrassYes18–65 years18 weeksPlacebo (n = 286) 0.5 μg Phl p 5/d (n = 136) 5 μg Phl p 5/d (n = 139) 15 μg Phl p 5/d (n = 294)Rhinoconjunctivitis SS and medication use† QoL % well days AEsSpecific IgE, IgG4Symptom score [DOWNWARDS ARROW] in the 15 μg dose, significant [DOWNWARDS ARROW] of medication use in the 15 μg group QoL and % of well days significantly increased in the 15 μg dose group compared to placebo, no difference between the two lower doses and placebo Dose- and time-dependent increase in specific IgG4 Increased frequency of mild AEs between 0.5 and 5.0 μg dose group but not between 5.0 and 15 μg
 Valovirta et al. (31)  Savolainen et al. (35)  Nieminen et al. (33)Pollen mix: Birch, alder, hazelYes5–15 years18 monthsPlacebo (n = 33) 3.6 μg Bet v 1/wk (n = 33) 30 μg Bet v 1/wk (n = 32)Symptom and medication score†Conjunctival provocation test Methacholine bronchial provocation test Late phase skin reaction IL-5, IL-10, IL-17, IL-18 mRNA, FOXP3 mRNA[DOWNWARDS ARROW] symptoms with both doses Medications [DOWNWARDS ARROW] with higher dose, no overall difference between the two doses No significant change in the other parameters [UPWARDS ARROW] in IL-18 and FOXP3 mRNA expression in high-dose group only
 Didier et al. (23)  Didier et al. (32)GrassYes18–45 years4–6 monthsPlacebo (n = 156) 100 IR/d (n = 157) 300 IR/d (n = 155) 500 IR/d (n = 160)Rhinoconjunctivitis SS† % days with rescue medication % of symptom free days QoL AEsSpecific IgE, IgG4Significant reduction in symptom scores vs placebo only with the 300 and 500IR Significant reduction of days without symptoms, days without medications Significant improvement of QoL Dose-dependent increase in specific IgG4 No dose-dependent differences in frequency of AEs
 Skoner et al. (30)RagweedYes18–50 years18 weeksPlacebo (n = 40) 4.8 μg Amb a 1/d (n = 39) 48 μg Amb a 1/d (n = 36)Average daily SS† Medication score NPT AEsSpecific IgE, IgG4No significant reduction in symptom and medication scores compared to placebo in both dose groups In an analysis of covariance correcting for preseasonal symptoms, both symptom and medication scores significantly [DOWNWARDS ARROW] in the high-dose group only vs placebo [UPWARDS ARROW] in IgG4 with both doses Equal safety
SCIT studies in insect sting anaphylaxis
 Golden et al. (17)Bee Yellow jacket HornetNoNR20 weeks50 μg (n = 23) raw venom 100 μg-1978 (n = 22) 100 μg-1976 (n = 20)Sting challenge†Specific IgGBetter protection at challenge with the 100 μg dose Higher concentrations of specific IgG with the 100 μg dose
 Rueff et al.(19)Bee Yellow jacketNo19–72 yearsNRIncrease from 100 to 150, 200 or even 250 μgSting challenge†No8 of 10 patients still reacting at challenge when treated with 100 μg were fully protected when treated with 150 μg maintenance dose and 28 of 30 patients were fully protected at 200 μg maintenance dose and 1 patients at 250 μg

Until present, no placebo-controlled prospective dose–response studies have been published in venom immunotherapy (VIT), because of the potentially life-threatening nature of an anaphylactic reaction to a sting in patients treated with placebo. One VIT study reported a dose-dependent increase in venom-specific serum IgG and greater clinical efficacy with a 100 μg monthly venom maintenance dose as compared with a 50 μg maintenance dose. Insufficient protection to a sting challenge, while on 100 μg monthly venom maintenance dose, could be overcome by increasing the maintenance dose to either 150 or 200 μg, especially in patients with elevated baseline tryptase levels or mastocytosis (19).

The dosing data for the selected studies are presented in Table 2. Of the fifteen studies, the two earliest, both published in the 1960s, provided no information on the source of allergens or if the allergen content was quantified (22, 27). For both VIT studies, only the quantity of venom in micrograms is mentioned, as is customary in VIT, without providing information on the content of the major allergen (17, 19). Five studies used investigators’ in-house assays to determine the allergen content of commercially obtained allergen preparations (17, 18, 21, 30, 32), with only one study citing a published assay method (18). In the remaining seven studies, the allergen content was determined using the manufacturers’ in-house assay (20, 24–26, 28, 29, 31). None of the published reports of these studies provided information on the source of antibodies or reference material used to quantify the major allergens.

Table 2.   Dosing data of the evaluated individual studies
  1. DBPC, double-blind placebo-controlled; GRADE, Grading of Recommendations, Assessment, Development, and Evaluations.

  2. *Delphi score was calculated from 11 criteria from the Delphi list for assessing the quality of randomized controlled trials (15). A higher score indicates a higher quality of study.

SCIT studies in respiratory allergies
Johnston 1961, DBPC RCT
 Antigen mixture (=all inhalant allergens patients were allergic to, no details)
  Low dose: 10−7 = 1 : 10 000 000 wt/v
  High dose: 1 : 5000 wt/v
  Highest tolerated dose: maximum 0.5 ml of 1 : 250 wt/v
  Manufacturer: not specified
  Volume: 0.5 ml all groups
  Adjuvant: none
 Up-dosing: 1 : 10 000 000 w/v group, 5 weeks; 1 : 5000 w/v group, 9 weeks; 1 : 250 w/v group, NA
 Frequency at maintenance: 28 days
 Duration: 4 years
 GRADE scoring: 2, low quality of evidence
 Delphi score*: 6.5
Franklin 1967, double-blind randomized continuation of already installed high-dose ragweed pollen immunotherapy
 Antigen mixture (no details) with aqueous ragweed pollen extract in two doses
  Low dose: mean 0.3 ml of a ‘concentration’ of 1 : 1000
  High dose: mean 0.3 ml of a ‘concentration’ of 1 : 50
  Manufacturer: not specified
  Volume: variable as tolerated: low-dose range 0.1–0.4 ml (mean 0.3 ml); high-dose range 0.07–0.5 ml (mean 0.3 ml)
  Adjuvant: none
 Frequency at maintenance: every 2–3 weeks
 Duration: duration trial immunotherapy 3 months, but before that approximately 6-month dose-escalation immunotherapy with several pollens, including ragweed pollen
 Duration: 4 years
 GRADE scoring: 2, low quality of evidence
 Delphi score: 8.5
Creticos 1989, randomized double blind with no control group
 Ragweed concentrations: dose escalation. Evaluation of the patients when 0.6 μg, 12.4 mg and 24.8 μg Amb a 1 dose was reached.
  (description μg)
  Major allergen content determined by radial immunodiffusion
  Volume: NA
  Adjuvant: none
 Frequency at maintenance: 4 weeks
 Duration: 0.6 μg was reached at 4–5 months, 12.4 μg at 6–10 months, 24.8 μg at 12–18 months
 GRADE scoring: 2, low
 Delphi score: 5
Haugaard 1993, randomized, double blind in active group with open control group (controls received no injections)
 D pteronyssinus 10 000, 100 000 and 300 000 SQ-U and open control group
  100 000 SQ-U contained 23 000 UI Der p 1 (7 μg/ml) and 34,500 UI Der p 2; quantified by
  Immunoelectrophoresis; whole mite culture
  Volume: 1 ml all groups
  Adjuvant: aluminum hydroxide, same dose all 3 dosing groups
 Frequency at maintenance: 6–8 weeks
 Duration: 24 months
 GRADE scoring: 2, low quality of evidence
 Delphi score: 8
Olaguibel 1997, open dose titration trial, nonrandomized
 D pteronyssinus: maximum tolerated dose
  Pangramin® (ALK-Abelló)
  (100 BU = 40 μg Der p 1, 20 μg Der p 2. Der p 1/2 quantified by monoclonal antibody enzyme immunoassays ELISA in solid phase, whole mite culture dialyzed to remove particles smaller than 5 kDa (55)
  Volume: depending on dose 0.4–0.8 ml (lower dose had a higher volume as it was taken from the diluted vial)
  Adjuvant: aluminum hydroxide (no data on concentration)
 Frequency at maintenance: 4 weeks
 Duration build-up: 3–4 months
 Total duration: approximately 15 months
 GRADE scoring: 0, very low quality of evidence
 Delphi score: 7
Ewbank 2003, DBPC RCT
 Cat hair and dander extract (ALK-Abelló, Wallingford, CT, USA) high dose 2344 BAU, medium 469 BAU, low 94 BAU, placebo
  (10 000 BAU = 64 μg Fel d 1. Groups 15, 3, 0.6 μg Fel d 1)
  Assay: in-house assay ALK. Placebo caramelized sugar + histamine
  Volume: 0.5 ml
  Adjuvant: none
 Frequency at maintenance: weekly (only 1 dose given)
 Duration up-dosing: 4 weeks (cluster)
 Duration: 5 weeks (4 run-in, 1 maintenance week)
 GRADE scoring: 2, low quality of evidence
 Delphi score: 10
Nanda 2004, DBPC RCT
 Cat hair and dander extract (ALK Austin, TX, USA) high dose 2344 BAU, medium 469 BAU, low 94 BAU, placebo
  (10 000 BAU = 64 μg Fel d 1. Groups 15, 3, 0.6 μg Fel d 1)
  Assay: in-house assay ALK. Placebo caramelized sugar + histamine
  Volume: 0.5 ml
  Adjuvant: none
 Frequency at maintenance: monthly
 Duration up-dosing: 4 weeks (cluster)
 Duration: 12–13 months (4 weeks run-in, 1 year maintenance)
 GRADE scoring: 3, medium quality of evidence
 Delphi score: 9
Lent 2006, DBPC RCT
 AP dog extract (Hollister Stier Laboratories) high dose calculated to deliver 15 μg, intermediate 3 μg, low 0.6 μg Can f 1 and placebo
  (1 : 100 wt/vol extract of AP dog (Hollister-Stier Laboratories, Spokane, WA, USA), contained approximately 161 μg/ml
  Can f 1 (information provided by the extract manufacturer)
  Assay: in-house assay Hollister-Stier. Placebo caramelized sugar + histamine
  Volume: 0.5 ml
  Adjuvant: none
 Frequency at maintenance: monthly
 Duration up-dosing: 4 weeks (cluster)
 Duration: 5 weeks (4 weeks run-in, 1 dose maintenance)
 GRADE scoring: 2, low quality of evidence
 Delphi score: 9
Frew 2006, DBPC RCT
 Phleum pratense: High dose 100 000 SQ-U, low dose 10 000 SQ-U, placebo
  Alutard® (ALK-Abelló, Hørsholm, Denmark)
  (100 000 SQ-U = 20 μg Phl p 5. Quantification method not mentioned; probably rocket immunoelectrophoresis or radio immunodiffusion (56)
  Volume: 100 000 SQ-U/ml
  Adjuvant: aluminum hydroxide (<100 000 SQ doses used were diluted with regard to allergen as well as ALOH)
 Frequency at maintenance: 6 weeks (±2 weeks)
 Duration build-up: 8 weeks
 Total duration: up-dosing 8 weeks, average maintenance 6.4 months (range 3–8 months)
 GRADE scoring: 4, high quality of evidence
 Delphi score: 9.5
SLIT studies in respiratory allergies
Durham 2006, DBPC RCT
 Phleum pratense pollen: high dose 75 000 SQ-T, Median dose 25 000 SQ-T, low dose 2500 SQ-T, placebo
  Grazax® (ALK-Abelló, A/S, Denmark)
  (75 000 SQ-T = 15 μg Phl p 5. Quantification method not mentioned; probably rocket immunoelectrophoresis or radio immunodiffusion (56)
  Volume: oro-dispensable, fast-dissolving tablet
  Adjuvant: none
 Frequency at maintenance: daily
 Total monthly dose: high-dose group 2 250 000; medium 750 000; low 75 000 SQ-T
 Duration build-up: none
 Total duration: 18 weeks (preco-seasonal, starting approximately 2 months preseasonal)
 GRADE scoring: 4, high quality of evidence
 Delphi score: 11
Valovirta 2006, DBPC RCT
 Tree pollen extract B. verrucosa (birch), C. avellana (hazel) and A. glutinosa (alder):
  High dose 40 000 SQ-U, low dose 4800 SQ-U, placebo
  1 000 000 SQ-U contains 150 μg (Bet v 1/Aln g 1/Cor a 1) (ALK-Abelló, A/S, Denmark)
  Quantification method not mentioned; probably rocket immunoelectrophoresis or radio immunodiffusion (56)
  Volume: 400 μl. For high and low doses same volume
  Adjuvant: none
 Frequency at maintenance: 5 days a week
 Total monthly dose: not mentioned (weekly cumulative dose: 3.6 μg, 30 μg SLIT 5 times per week)
 Duration build-up: 5 weeks
 Total duration: 18 months
 GRADE scoring: 3, medium quality of evidence
 Delphi score: 11
Didier 2007, DBPC RCT
 5-grass pollen tablet Oralair® (orchard, meadow, perennial rye, sweet vernal, and timothy grasses) (Stallergènes SA, Antony, France)
  High dose 500 IR, medium dose 300 IR, low dose 100 IR, placebo
  300 IR contains 25 μg group 5 major grass allergen (Stallergènes)
  Quantification method: ELISA sandwich using two in-house monoclonal antibodies with rPhl p 1 and 5 Biomay (Vienna, Austria) reference material (57)
  Volume: tablet
  Adjuvant: none
 Frequency at maintenance: daily
 Total monthly dose: high 15 000 IR, medium 9000 IR, low 3000 IR
 Duration build-up: 5 days
 Total duration: 6 months (preco-seasonal, starting 4 months preseasonal)
 GRADE scoring: 4, high quality of evidence
 Delphi score: 8
Skoner 2010, DBPC RCT
 Glycerinated short ragweed pollen extract (Greer Laboratories, Inc, Lenoir, NC, USA)
  High dose 48 μg Amb a 1 or maximum tolerated dose (mean: 30.5 μg), medium dose 4.8 μg Amb a 1 or maximum tolerated dose (mean: 3.21 μg), placebo
  Quantification method: radial immunodiffusion assay with FDA references. (Manufacturer’s information)
  Volume: 50–140 μl
  Adjuvant: none
 Frequency at maintenance: daily
 Total monthly dose: (mean) low: 96.3 μg, high: 915 μg Amb a 1
 Duration build-up: 4 doses on 1 day
 Total duration: 17 ± 3 weeks (preco-seasonal, starting 8–10 weeks preseasonal)
 GRADE scoring: 3, medium quality of evidence
 Delphi score: 10
SCIT studies in insect sting anaphylaxis
Golden 1981, Open study, historic controls
 Yellow jacket, yellow hornet, white-faced hornet, honeybee or mixed vespid venom (Pharmacia Diagnostics, Piscataway, NY, USA)
  High dose 100 μg, low dose 50 μg venom
  Quantification method: not stated
  Volume: 1 ml (high dose), 0.5 ml (low dose)
  Adjuvant: none
 Frequency at maintenance: monthly
 Total monthly dose: 50 or 100 μg
 Duration build-up: 6 weeks
 Total duration: 16 weeks (historic controls 20 weeks), before sting challenge
 GRADE scoring: 3, medium quality of evidence
 Delphi score: 4
Rueff 2001, Open study, retrospective
 Lyophilized honeybee venom (A. mellifera) or Vespula species (Reless; ALK-SCHERAX, Hamburg, Germany)
  Maintenance 100 μg every 4 weeks, but dose increased to 150, 200, and 250 after nontolerated sting challenge
 Quantification method: not stated
  Volume: not stated
  Adjuvant: none
 Frequency at maintenance: monthly
 Total monthly dose: 100, 150, 200, 250 or 400 μg venom
 Duration build-up: normal protocol 18 weeks (conventional)
 Total duration: 10–28 months before sting challenge
 GRADE scoring: 3, medium quality of evidence
 Delphi score: 3

For the studies that cited allergen content determined by manufacturers’ in-house assays, the Task Force requested detailed information on the type of immunoassay used, sources of antibodies, source of reference material used in the preparation of standard curves, and any published data on the assay used from the manufacturers. Where available, this information is presented in Table 2.

Study evaluation according to GRADE and CONSORT and Delphi list

The Task Force evaluated the quality of evidence of studies using the GRADE scoring system (14) and Delphi list (15). In addition, the group evaluated studies for compliance with eight selected recommendations for reporting of randomized controlled trials (RCTs) outlined in the CONSORT statement (36), see Table 3. The results of these evaluations are presented in Tables 2 and 3.

Table 3.   Methodological characteristics of the evaluated studies
AuthorCONSORT flow chartDrop outs placebo/active/active (%)ITT OR per protocolAdequate blindingMethod of randomization reportedFlaw in methodPower analysisDelphi score*GRADE score
SpecifiedUsed
  1. A, active group; ITT, intention to treat; PP, per protocol; LOCF, last observation carried forward; NA1, not applicable since the first CONSORT statement was issued in 1996 (16); NA2, not applicable because the study was not randomized; P, placebo; SCIT, subcutaneous immunotherapy; SLIT, sublingual immunotherapy; CONSORT, Consolidated Standards of Reporting Trials; GRADE, Grading of Recommendations, Assessment, Development, and Evaluations.

  2. *Delphi score was calculated from 11 criteria from the Delphi list for assessing the quality of randomized controlled trials (15). A higher score indicates a higher quality of study.

  3. Post hoc analysis of co-variance: correct for differences of baseline symptoms.

SCIT studies in respiratory allergies
 Johnstone et al. (27)NA1       6.52
 Franklin et al. (22)NA1       8.52
 Creticos et al. (21)NA1       52
 Haugaard et al. (20)NA1       82
 Olaguibel et al. (18)NA1       70
 Ewbank et al. (25)No, but no dropouts0/0/0 ITT, no dropoutsYesNoOnly challenge test and immunologyNo102
 Nanda et al. (29)No14/0/0/14 PPYesNoOnly challenge test and immunologyNo93
 Lent et al. (28)Yes, in words0/14/0/0 Almost ITT (only 1 drop-out)YesNoOnly challenge test and immunologyNo92
 Frew et al. (26)Yes12/16/17NoPPYesIncomplete Yes9.54
SLIT studies in respiratory allergies
 Durham et al. (24)Yes13/10/10/15ITTAll randomized patientsYesYesNo calculation of differences between active groupsYes114
 Valovirta et al. (31)Yes18/3/22ITTPatients excluded with evaluable clinical dataYesNoPost hoc sign level changed from 1.5 to >5%Yes113
 Didier et al. (23)Yes6/11/14/12ITTITT analysis excluding drop outs with no efficacy data and LOCFYesIncomplete Yes84
 Skoner et al. (30)No10/13/8NoPP Missing data excludedYesYesBelow calculated number of patients to treat and†Yes103
SCIT studies in insect sting anaphylaxis
 Golden et al. (17)NA2NANA NANA  43
 Rueff et al. (19)NA2      No33
  •  Three studies reporting a dose–response relationship for clinical endpoints had a GRADE score of 4, indicating a high quality of evidence (23, 24, 26). Two of these studies also showed a dose–response relationship for immunological endpoints (23, 24) and one for safety endpoints (26).
  •  One study that did not show a dose–response relationship for any of the main endpoints had a GRADE score of 3, indicating a medium quality of evidence (30).
  •  The eight RCTs (23–26, 28–31) published after the first CONSORT statement was issued complied with at least three of the eight selected CONSORT recommendations.

Study outcomes

Thirteen studies (out of 15) reported a dose–response relationship for clinical efficacy (17, 19–29, 31), eight of these also reported a dose–response effect for immunological endpoints (17, 21, 23–25, 28, 29, 31) and two reported a dose–response effect for safety and tolerability scores (20, 26). One study reported a dose–response relationship for immunological endpoints and safety scores, but not for clinical endpoints (18). One study reported no significant dose–response relationship for any of the efficacy endpoints, but a dose–response relationship was observed for safety scores (30). In total, nine studies (out of ten) reported a dose–response relationship for immunological endpoints (17, 18, 21, 23–25, 28, 29, 31) and five (of seven) for safety endpoints (18, 20, 24, 26, 31).

A few SCIT studies reported the use of an adjuvant (18, 20, 22); however, information on whether the adjuvant concentration remained constant or whether this changed along with the administration volume and allergen dose was not reported in any publication. As far as data obtained on request from the manufacturers showed, the concentration of the adjuvant was always changed together with the allergen.

Two double-blind placebo-controlled (DBPC) RCTs were performed in pediatric populations, one SCIT study in children with perennial asthma reported a dose–response effect for clinical efficacy and development of new allergies (27), and a SLIT study in children with allergic rhinoconjunctivitis with/without seasonal asthma showed a dose–response relationship for both clinical and immunological endpoints (31).

Discussion

  1. Top of page
  2. Abstract
  3. Methodology
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References

Positive outcomes establish a dose–response relationship

  •  Several studies demonstrated a clear dose–response relationship in SIT, showing that the effect increases with the dose.
  •  A dose–response relationship in SIT efficacy was shown for both clinical (13 of 15 studies) and immunological (9 of 10 studies) endpoints.
  •  The concept of dose-dependent SIT effects is valid for subcutaneous and sublingual routes of administration. Three of the four SLIT studies and 10 of the 11 SCIT studies showed dose-dependent effects for clinical efficacy.
  •  In SLIT studies that reported AEs as a clinical endpoint, a dose-dependent increase in efficacy was not associated with an increase in AEs, possibly reflecting the different sites of action within the immune system. In contrast, SCIT studies reporting AEs showed increased frequencies of AEs at higher allergen doses.

Potential sources of bias to consider

Heterogeneity of effects and lack of clinical data

  •  The considerable heterogeneity in dose-dependent effects between the reviewed studies, reflects variations in study design, selection of patients and endpoints, allergen sources, product standardization, units, and other parameters. Consequently, comparisons between dose–response studies are problematic, and for this reason, studies are presented individually. Results of dose–response studies are clearly linked to a particular product and protocol, route of application, subject age, disease manifestation, and severity.
  •  At present, no dose–response data exist that enable comparison between SIT protocols. Therefore, it remains unclear how the mode of dose escalation and maintenance (co-seasonal vs perennial vs preseasonal) affects the dose dependency.
  •  The studies identified were mostly carried out before publication of the World Allergy Organization (WAO) and EMA guidelines for standardization of clinical trials with SIT (8, 37).

Methodological issues

  •  Not all studies were DBPC (9/15).
  •  Many studies were carried out in small populations.
  •  Many of the studies did not class endpoints as primary and secondary; therefore, we evaluated all measurements to determine whether any were dose related.
  •  Not all studies had high GRADE or high CONSORT scores. Of interest, 1/11 SCIT studies and 4/4 SLIT studies report whether a power analysis was performed.
  •  Published SIT studies that demonstrate dose-dependent effects of SIT compared with placebo, use superiority testing. Significant differences between doses would clearly confirm dose-dependent effects. However, this would necessitate large sample sizes and introduce multiplicity-testing issues. Therefore, differences between a limited number of doses, rather than based on predefined dose–response models, should be sufficient to demonstrate a dose–response effect in SIT (38). An alternative approach would be to use a regression framework to describe a dose–response relationship; however, although this would necessitate fewer patients per dose, many doses would be needed.

Unresolved biological issues

  •  Until present, the units used to quantify allergen preparations in Europe were based on in-house reference materials and protocols for extract characterization and standardization. Moreover, measurements of major allergen content are obtained by in-house assays, and limited information is publicly available on details, such as assay type, reference materials, antibodies, and protocols. Therefore, values from different laboratories or allergen manufacturers cannot be compared.
  •  A consortium of independent research laboratories, allergen manufacturers and the Paul-Ehrlich-Institute (6, 39) has carefully selected reference materials and searched for robust assays to detect major allergens in complex allergen preparations. Recombinant major birch pollen (rBet v 1a) and major timothy grass pollen allergen (rPhl p 5a) are promising candidates for potential reference materials, which will lead to improved standardization of birch and Phleum pratense pollen extracts.
  •  Immunoassays for the detection of allergen-specific IgG4, which are commonly used to follow SIT-induced immune responses during dose-finding studies, are not standardized. These immunoassays are based on in-house references, and their values cannot be compared between studies. Future assays should employ single (major) allergens, calibrated to validated reference materials.

Unresolved dosing problems

  •  The relationship between allergen dose and adjuvant concentration is a subject of investigation and debate. In multi-dose SCIT studies, information about the amount of adjuvant, i.e., alum, in relation to allergen dose, should be considered. An extra group might be added, in which the adjuvant concentration is varied, but in human studies the practicalities of realizing this option are limited. According to the data obtained from the manufacturers on request, the adjuvant was diluted down when lower doses were produced for the trials. Recently, animal studies have suggested that the alum dose in relation to the allergen dose is relevant for promoting a protective immune response during SCIT (40–42). Allergen-specific IgG4 measurements after short-term SCIT suggest an important role of adjuvant/allergen ratio also in humans (43, 44).
  •  Animal studies during preclinical development may be useful for investigating the relationship between allergen dose and adjuvant concentration. This relationship might vary depending on the allergen source as well as on the nature of the adjuvant. It does not seem feasible to test several allergen/adjuvant ratios in human studies.
  •  The relationship between frequency of administration and peak dose of sublingual allergen application and their contribution to the immunological effect of SLIT has sparked many discussions. Preclinical animal studies indicate that the frequency of administration, even with constant weekly cumulative dose, is of significant importance for the resulting effect, favoring a daily regimen for SLIT (45).
  •  The dose–response relationship and, consequently, the optimal dose have been established with a robust methodology only for grass allergens in SLIT tablets (23, 24). Dose-ranging studies with adequate methodology are lacking for the remaining relevant allergens (e.g., mite, birch, parietaria, and ragweed).
  •  For studies investigating the dose dependency of safety, an universal grading system is needed.

General methodological and regulatory issues

Potential role of surrogate endpoints

  •  Significant differences between SIT doses measured using surrogate endpoints are not necessarily clinically relevant. Therefore, the choice of endpoint should enable estimation of the clinical benefit of any dose–response effect. Combined symptom and rescue medication scores, proposed in the EMA guidelines (8), would enable such an estimation. Because of the difficulties in designing SIT dose-finding studies of sufficient power, clinical surrogate parameters could be a substitute for clinical endpoints; however, these require validation.
  •  Clinical surrogate endpoints for SIT dose-finding studies include challenge models (conjunctival, nasal, or bronchial provocation tests), allergen exposure (chamber) models, and patient-reported outcomes (e.g., quality of life). SIT dose-finding studies should, nevertheless, include clinical endpoints, such as symptom severity after allergen challenge.
  •  Laboratory parameters, such as allergen-specific IgG or IgG4 levels, T-cell reactivity, or cytokine production are not necessarily correlated with the clinical outcome. The in vitro parameter that best correlates with clinical efficacy in respiratory allergen SIT is FAB analysis (Facilitated Antigen binding) (46, 47). Even so, although laboratory tests can provide supportive information for dose-finding studies, they cannot be used to determine a suitable therapeutic dose.

Concepts of SIT dosing

  •  In general, pharmacokinetic and pharmacodynamic principles cannot simply be adapted to SIT dose-finding studies (48). SIT is only administered to allergic subjects and not to healthy volunteers, and still very little is known about bioavailability after subcutaneous or sublingual application of allergen preparations (49). Numerous variables potentially influence the efficacy of SCIT (volume of injection, allergen dose, modification of the allergen preparation, nature and concentration of the adjuvant) as well as SLIT dosing (aqueous or solid state, volume of solution, potential adjuvant, peak dose, contact time with the mucosa).
  •  Dose-finding studies can be designed to define a recommended maximum tolerated dose in allergic individuals. Recent safety studies of SLIT have indicated a logarithmic distribution, which is linked to a continuous increase in both frequency and severity of AEs (50). The concept of maximum tolerated dose requires guidelines on how the frequency and severity of local and systemic AEs should be taken into account (51).
  •  Unlike IgE response, which has a logarithmic curve and broad dose–response curve (e.g., titrated skin tests, challenge tests, basophil studies), the dose–response relationship in SIT might follow different rules – some studies report a rather steep relationship between the allergen dose and the clinical effects in SIT. Comparisons of immunological and clinical dose–response curves obtained in parallel would be helpful to investigate the relationship between these endpoints during dose–response studies.
  •  At present, definitions such as ‘low dose’ or ‘high dose’ are meaningless, unless these are defined relative to a common standard. Designation of lower and higher doses is only relevant for identical allergen preparations used within a given study.

Framework of present regulations on allergen products in Europe

If a medicinal product (i.e., allergen preparation for SIT) is to be developed for two or more European countries, an application for marketing authorization must be submitted via the Mutual Recognition Procedure or the Decentralized Procedure. A marketing authorization granted by the Centralized Procedure is valid in the whole European Community and is mandatory for recombinant or biotechnologically generated products. Three guidelines of the EMA have a potential to shape the future development of allergen preparations for SIT (8, 52–54). Parallel to the European initiatives, the Paul-Ehrlich-Institute has recently targeted ‘Named-Patient products’ used for SIT in Europe. So far, no dose–response studies have been requested for these already marketed allergen preparations. New products, however, will be required to show a tolerated dose range and follow the recently introduced EMA guidelines for further development and registration (8).

Future implications

  •  New clinical trials in children and adults will be necessary for registration of new and currently available products for SIT. These will include dose-finding studies for safety as well as for efficacy endpoints, according to the EMA guidelines outlined above.
  •  Novel SIT preparations and possibly currently available products that lack dose–response data will be tested for safety and efficacy in dose-escalation schemes. This will produce a large pool of data that will help to define the dose–response relationship in SIT and mark a major advance in the understanding of the treatment of allergic conditions. The Task Force strongly supports this for the most prevalent allergens.
  •  Improved characterization and standardization of allergen products for SIT is to be expected. As a small-scale follow-up to the CREATE project, validation of allergen reference materials in Europe will continue through the Biological Standardization Program (BSP090) of the European Directorate for the Quality of Medicines & HealthCare in conjunction with the European Pharmacopoeia.
  •  Vigorous testing by reference laboratories and a ring test trial should prove the suitability of the recombinant birch and grass pollen allergens rBet v 1 and rPhl p 5a and the robustness of the appropriate antibody-based immunoassays for their detection. The same approach is foreseeable for other recombinant major allergens (i.e., from house dust mite). After validation, these materials could become biological reference preparations and establish the basis for future in-house reference preparations for allergen manufactures and regulatory authorities. Only then would labeling of allergen products, including their major allergen content in mass units, become a possibility.
  •  The recombinant venom allergens Api m1, Ves v5 and Ves v1 are already available for use in diagnosis. Future use of recombinant venom allergens for VIT will also necessitate that these conform to guidelines for standardization of allergen preparations.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Methodology
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References

The Task Force was financed by the EAACI. The authors thank EAACI for their financial support in the development of this Task Force report. Special thanks to Dr Domingo Barber, ALK-Abelló, Madrid, Spain, for his academic input and encouragement and also to Dr Claude Andre, Stallergenes, France, for his participation and valuable discussion. We acknowledge the editorial support of Dr Ron Hogg, OmniScience SA, who was financed by a grant from EAACI.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Methodology
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References

MC has acted as a consultant for ALK-Abelló and a speaker for ALK-Abelló, Allergy Therapeutics (UK) Ltd, MSD and GSK. He has appeared on advisory boards for Allergy Therapeutics (UK) Ltd and ActoGeniX, received honoraria from ALK-Abelló, Allergy Therapeutics (UK) Ltd, Schering-Plough, and MSD, and research grants from ALK-Abelló and GSK. DL has received lecture fees from ALK, Schering Plough, MSD and FDA Allergenic (Brasil) and travel grants from ALK and Stallergènes. JKT has received lecture fees from Allergopharma, ALK-Abelló, Bencard, HAL Allergy, Leti, Novartis, Roxall Medizin/Dr. Beckmann Pharma, and Stallergènes, and consultancy fees from ALK-Abelló, Bencard, HAL Allergy, and Novartis, and research and safety board honoraria from Allergopharma, ALK-Abelló, HAL Allergy, Leti and Merck/Shering-Plough. LJ has acted as consultant for the European Allergen Manufacturers Group. GP has received lecture fees from ALK Abelló, Abbott, Almirall, AstraZeneca, GSK, Lofarma, Menarini, MSD, Phadia, Schering Plough, and Stallergènes. PAE has received lecture fees from ALK, Allergopharma, Astra, Glaxo, MSD, Stallergènes, and Phadia, and consultancy fees from ALK, Allergopharma, MSD and Novartis. EMV declares no conflict of interest. EV has received lecture fees from ALK, GSK, MSD, Schering-Plough, AllergoPharma and Nycomed, and consultancy fees from ALK, Nycomed, Schering-Plough and MSD. CM declares no conflict of interest. HJM has received research support from, and served on the advisory boards of, ALK-Abelló and Stallergenes, and received speaker fees from ALK-Abelló, Stallergenes, and Allergopharma. EAC declares no conflict of interest. SD has received research funding via Imperial College from ALK-Abelló and Novartis, lecture fees from ALK-Abelló and consultancy fees from ALK-Abelló, Circassia and Merck. PD has received lecture fees from ALK, GSK, MSD, Schering-Plough, Stallergènes, and Thérabel, and consultancy fees from ALK, BioXtract, Crucell, Schering-Plough, Stallergènes, and Thérabel.

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  6. Acknowledgements
  7. Conflict of interest
  8. References
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