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1 Supplementary Online Content Scadding GW, Calderon MA, Shamji MH, et al. Effect of 2 Years of treatment with sublingual grass pollen immunotherapy on nasal response to allergen challenge at 3 years among patients with moderate-to-severe seasonal allergic rhinitis: the GRASS randomized clinical trial. JAMA. doi: /jama emethods. Supplement to the Methods efigure. Out of Season Global Evaluation (0-18) etable 1. Peak Nasal Inspiratory Flow (L/min, 0-10 Hours) Change from Pre Challenge Baseline Area Under the Curve etable 2a. Mini-Rhinoconjunctivitis Quality of Life Questionnaire (mini-rqlq) (0 6) etable 2b. Visual Analogue Scale (0 10 cm) etable 2c. Out of Season Global Evaluation (0-18) etable 3a. Return Rates of Rescue Medications etable 3b. Rescue Medication Scores etable 4a. Early Skin Response (mm) etable 4b. Late Skin Response (mm) etable 5a. Allergen-specific IgE (ku/l) etable 5b. Allergen-specific IgG4 (mga/l) etable 6. Local and Systemic Adverse Events in the First Hour After the First Tablet at Updosing Visit 1 etable 7a. Systemic Adverse Reactions in Participants on Active SCIT Injections During Study Participation (WAO Classification) etable 7b. Systemic Adverse Reactions in Participants on Active SLIT Tablets During Study Participation (WAO Classification) etable 8. Adverse Events Table ereferences This supplementary material has been provided by the authors to give readers additional information about their work.

2 Table of Contents emethods. Supplement to the Methods PARTICIPANT INCLUSION/EXCLUSION CRITERIA Inclusion Criteria Exclusion Criteria RANDOMIZATION AND BLINDING Randomization Blinding CALCULATION OF PRIMARY ENDPOINT NASAL ALLERGEN CHALLENGE Peak Nasal Inspiratory Flow Reporting of Peak Nasal Insipatory Flow INTRADERMAL ALLERGEN CHALLENGE RHINITIS ASSESSMENTS RESCUE MEDICATION SCORES SUBCUTANEOUS AND SUBLINGUAL IMMUNOTHERAPY PROTOCOLS ADVERSE EVENT REPORTING Expected Adverse Reactions Adverse Reactions to Study Medication Adverse Reactions to Study Procedures Adverse Events Not Related To Study Medication or Procedures ANALYSIS SAMPLES SAMPLE SIZE AND STUDY POWER Supplementary Figure efigure. Out of Season Global Evaluation (0-18) Supplementary Tables etable 1. Peak Nasal Inspiratory Flow (L/min, 0-10 Hours) Change from Pre Challenge Baseline Area Under the Curve etable 2a. Mini-Rhinoconjunctivitis Quality of Life Questionnaire (mini-rqlq) (0 6) etable 2b. Visual Analogue Scale (0 10 cm) etable 2c. Out of Season Global Evaluation (0-18) etable 3a. Return Rates of Rescue Medications etable 3b. Rescue Medication Scores etable 4a. Early Skin Response (mm)... 20

3 3.8 etable 4b. Late Skin Response (mm) etable 5a. Allergen-specific IgE (ku/l) etable 5b. Allergen-specific IgG4 (mga/l) etable 6. Local and Systemic Adverse Events in the First Hour After the First Tablet at Updosing Visit etable 7a. Systemic Adverse Reactions in Participants on Active SCIT Injections During Study Participation (WAO Classification) etable 7b. Systemic Adverse Reactions in Participants on Active SLIT Tablets During Study Participation (WAO Classification) etable 8. Adverse Events Table ereferences... 34

4 1. EMETHODS. SUPPLEMENT TO THE METHODS 1.1 PARTICIPANT INCLUSION/EXCLUSION CRITERIA INCLUSION CRITERIA 1. Adults age 18 to 65 years. 2. A clinical history of grass pollen-induced allergic rhinoconjunctivitis for at least 2 years with peak symptoms in May, June, or July. 3. A clinical history of moderate - severe rhinoconjunctivitis symptoms interfering with usual daily activities or with sleep as defined according to the ARIA classification of rhinitis A clinical history of rhinoconjunctivitis for at least 2 years requiring treatment with either antihistamines or nasal corticosteroids during the grass pollen season. 5. Positive skin prick test response, defined as wheal diameter greater than or equal to 3 mm, to Phleum pratense. 6. Positive specific IgE, defined as greater than or equal to IgE class 2 (0.7 ku/l), against Phleum pratense. 7. A positive response to nasal allergen challenge with Phleum pretense, defined as an increase in TNSS greater than or equal to 7 points. 8. For women of childbearing age, a willingness to use an effective form of contraception for the duration of the trial. 9. The ability to give informed consent and comply with study procedures EXCLUSION CRITERIA 1. Prebronchodilator FEV1 less than 70% of predicted value at either screening or baseline visit. 2. A clinical history of moderate - severe allergic rhinitis, according to the ARIA classification, due to tree pollen near or overlapping the grass pollen season A clinical history of persistent asthma requiring regular inhaled corticosteroids for > 4 weeks per year outside of the grass pollen season. 4. A clinical history of moderate- severe allergic rhinitis, according to the ARIA classification, caused by an allergen to which the participant is regularly exposed History of emergency visit or hospital admission for asthma in the previous 12 months. 6. History of chronic obstructive pulmonary disease. 7. History of significant recurrent acute sinusitis, defined as 2 episodes per year for the last 2 years, all of which required antibiotic treatment. 8. History of chronic sinusitis, defined as a sinus symptoms lasting greater than 12 weeks that includes 2 or more major factors or 1 major factor and 2 minor factors. Major factors are defined as facial pain or pressure, nasal obstruction or blockage, nasal discharge or purulence or discolored postnasal discharge, purulence in nasal cavity, or impaired or loss of smell. Minor factors are defined as headache, fever, halitosis, fatigue, dental pain, cough, and ear pain, pressure, or fullness. 9. History of systemic disease affecting the immune system such as autoimmune diseases, immune complex disease or immunodeficiency. 10. At randomization, current symptoms of, or treatment for, upper respiratory tract infection, acute sinusitis, acute otitis media, or other relevant infectious process; serous otitis media is not an exclusion criterion. Participants may be re-evaluated for eligibility after symptoms resolve. 11. At randomization, inflammatory conditions in the oral cavity with severe symptoms such as oral lichen planus with ulcerations or severe oral mycosis. 12. Active malignancy at randomization. 13. Any tobacco smoking within the last 6 months or a history of greater than or equal to 10 pack years. 14. Previous treatment by immunotherapy with grass pollen allergen within the previous 5 years. 15. Any history of grade 4 anaphylaxis due to any cause as defined by the WAO grading criteria for immunotherapy (see appendix 7). 16. A history of allergy to vertebrate/finned fish with, if positive, an associated elevated positive skin test (or elevated specific IgE) to vertebrate/finned fish. 17. History of bleeding disorders or treatment with anticoagulation therapy 18. History of anti-ige monoclonal antibody treatment. 19. Ongoing systemic immunosuppressive treatment. 20. History of intolerance to the study therapy, rescue medications, or their excipients. 21. For women of childbearing age a positive serum or urine pregnancy test with sensitivity of less than 50 miu/ml within 72 hours before the start of study therapy. 22. The use of any investigational drug within 30 days of the screening visit. 23. The presence of any medical condition that the investigator deems incompatible with participation in the trial.

5 1.2 RANDOMIZATION AND BLINDING RANDOMIZATION Eligible participants were randomized to one of the three treatment arms administered in a double-blind, double-dummy fashion in an approximately 1:1:1 ratio. Rho was responsible for the development, validation, and implementation of the investigational medicinal product (IMP) treatment assignment and randomization system (RhoRAND, a central automated web-based randomization system). This system ensured protection of vital study data with user authentication protocols, dedicated servers, remote network backup, and 24-hour support. Rho monitored the randomization system on a daily basis, investigated any errors, and modified the system as necessary. Rho provided a list of kit IDs and associated treatment assignments directly to the Brecon Pharmaceuticals Ltd project manager. Each participant s treatment was randomly assigned, and a randomization ID associated with that randomization was associated with the participant. Additionally, the participant was assigned a specific IMP kit, from those available at the site that corresponded to the treatment assignment. Authorized study personnel entered required data into the web-based system. The system generated the blinded randomized treatment assignment code. Each participant was assigned a kit ID from the site s inventory whose treatment matches the group to which the participant was randomized. The treatment assignment notification was sent to authorized study personnel and the investigational pharmacy via and/or fax BLINDING Blinding was maintained for all study participants throughout the treatment phase of the study and during the 1-year withdrawal phase after treatment was discontinued. Data analysis for primary and secondary endpoints were performed after the database was locked and the code was strictly maintained throughout the duration of the study for the participants and clinical and laboratory teams on site at this single center study. 1.3 CALCULATION OF PRIMARY ENDPOINT The primary endpoint was the nasal response to allergen challenge at 3 years, defined as the equally weighted average of the total nasal symptom score (TNSS/hour) measured during the early response (0-1 hour) and late response (1-10 hours) after challenge. The trapezoidal rule was used to calculate the AUCs. The formula for the AUC is = TNSS n+tnss n 1 (t 2 n t n 1 ). The linear trapezoidal method divides the entire 10 hour challenge into a series of trapezoids, finds the area of each trapezoid, and then adds them together to get the overall AUC. Using the above participant outcome data, the primary endpoint was calculated as follows: (9 + 0)( ) (4 + 9)( ) (4 + 4)( ) (4 + 3)(1 0.5) EPR(0 1 hour) = = (3 + 3)(2 1) (3 + 3)(3 2) (3 + 3)(4 3) (2 + 2)(10 9) LPR(1 10 hours) = = EPR (0 1 hour) LPR (1 10 hours) Primary Endpoint = + = =

6 The minimal clinically important difference (MCID) for TNSS was determined using the distribution-based method as there are no publications that provide an anchor-based MCID for TNSS measured during nasal allergen challenge. The MCID was calculated as 0.5*SD=1.08, which is considered to be a conservative approach. 2 Missing TNSS AUC at Year 3 was imputed using data from subjects in the same treatment arm who had available TNSS AUC values at both time t (t = Baseline, Year 1 or Year 2) and Year 3. Specifically, a linear regression line and 95% confidence bands were fit where Year 3 values are regressed on TNSS AUC values at time t (e.g. Year 1). In each arm, a missing Year 3 AUC value was imputed as the value predicted from the linear regression line. 1.4 NASAL ALLERGEN CHALLENGE Nasal allergen challenges were performed outside the pollen season (October to December) with Timothy grass pollen extract (Aquagen SQ, ALK-Abello, Denmark) with measurement of TNSS and peak nasal flow at 0, 5, 15, 30 and 60 minutes and thereafter continued hourly up to 10 hours post-challenge. Nasal challenges were performed at screening visit, at baseline (pre-treatment), at 1 and 2 years during treatment and at year 3, one year after discontinuation of treatment (efig.1). To clear the nasal cavities of preexisting secretions, nasal lavage was performed prior to challenge using 240 ml of sodium chloride and bicarbonate solution (Sinusrinse, NeilMed, USA), as described previously. 3,4 Timothy grass pollen extract (Aquagen SQ, ALK-Abello, Denmark) was reconstituted at 100,000 SQ-U/ml (equivalent to 30,000 biological units (BU)/ml or 20.2 µg/ml major allergen, Phl p 5) in albuminbased diluent (ALK-Abello), before serial dilution in normal saline to give concentrations of 10,000 BU/ml (6.7 µg/ml major allergen), 5,000 BU/ml (3.4 µg/ml) and 1,500 BU/ml (1.0 µg/ml). One hundred microlitres of solution was applied to each nostril using the Bi-dose nasal applicator device (Aptar Pharma, Germany) as previously described. 3,4 At screening, participants underwent a titrated up-dosing challenge: until reaching a total nasal symptom score (TNSS, range 0-12) 3,4 of at least 7; individuals failing to reach this score were excluded from the study. At all subsequent nasal challenge visits ie at baseline (pre-treatment) and at 1, 2 years and 3 years, participants received a single dose challenge with the lowest dose which provoked a TNSS of at least 7 at screening (efigure 1). At the baseline challenge (pre-treatment) if the single dose identified at the screening visit failed to produce a TNSS of 7 or more, the next dose up was given in addition (including a dose of 30,000 BU/ml if the single dose was 10,000 BU/ml), and TNSS was recorded following this second dose. The same two doses were then given, sequentially (irrespective of the TNSS response), at years 1, 2 and PEAK NASAL INSPIRATORY FLOW Peak nasal inspiratory flow (PNIF) has good reproducibility, is responsive to both nasal histamine application and nasal decongestants, and has been used previously in nasal allergen challenge studies. Normal ranges have been established in different populations. 5 The best of three PNIF attempts made using a modified Youlten peak flow meter (Clement Clarke, UK) plus facemask (disposable anaesthetic mask, size 5, Intersurgical Ltd, Berkshire, UK) was recorded at each symptom assessment. All measures were made whilst sniffing in from full expiration with participants standing. Additional attempts were made if the participant s technique was not optimal or in the presence of a variable nasal valve effect. The scale on the meter runs from 30 to 300 L/min. Non-movement of the recording needle, or movement without reaching 30 L/min was recorded as zero; movement of the needle past 300 L/min was recorded as 300 L/min. The minimal clinically important difference (MCID) for PNIF was determined using the distribution-based method as there are no publications that provide an anchor-based MCID for PNIF measured during a nasal allergen challenge. The MCID was calculated as 0.5*SD=33.9, which is considered to be a conservative approach REPORTING OF PEAK NASAL INSIPATORY FLOW The Peak Nasal Inspiratory Flow (PNIF) was reported as a change from the 0 minute assessment time. The 0 minute PNIF assessment was subtracted from all measurements taken after the 0 minute assessment. This delta outcome was used to calculate an area under the curve using the same methodology used for the primary outcome that is outlined in section INTRADERMAL ALLERGEN CHALLENGE One hour after nasal challenge, participants received intradermal injections of 1 BU (equivalent to 0.7ng Phl p 5) Phleum Pratense extract (Aquagen ) on the right outer forearm. Wheal responses were measured at 15 minutes, and late phase swelling at 8 hours as described. 6,7 Responses were delineated using a fine black ink ballpoint marker pen and imprinted on transparent tape and transferred onto paper for a permanent record. The size of the responses (in mm) were recorded as the mean of the longest diameter and the orthogonal perpendicular diameter.

7 1.6 RHINITIS ASSESSMENTS Rhinitis symptoms were assessed by: visual analogue scale (VAS), 8 the Mini Rhinoconjunctivitis Quality of Life Questionnaire 9 (MiniRQLQ) and Global rhinitis severity scores. VAS assessments (linear scale 0-10 cm) were self-administered by participants every week from mid-may until the end of July during years 1, 2 and 3. VAS assessment referred to overall nasal and eye symptoms. Participants were instructed to place a vertical mark on the line where they felt the severity of their symptoms were during the week prior to completing the assessment. If they placed a mark on the far left of the line, it meant they were completely symptom free and a mark at the far right of the line indicated their symptoms were as bad as they possibly could be. Higher VAS values reflect subjects who experience worse hay fever symptoms. The minimal clinically important difference for this measure within a subject is 1.0 cm. 8 The MiniRQLQ is a fully validated self-administered questionnaire with14 questions about nose/eye symptoms in five domains (activity limitation, practical problems, nose symptoms, eye symptoms and non-nose/eye symptoms). The MiniRQLQ has good reliability, cross-sectional validity, responsiveness and longitudinal validity 9 and was assessed at baseline and then weekly during the pollen season from mid-may until the end of July during years 1, 2 and 3. MiniRQLQ values range from 0-6. Higher RQLQ values reflect subjects who experience more troublesome nose, eye, and other symptoms effecting regular activities resulting in a lower quality of life. The minimal clinically important difference for this measure within a subject is MiniRQLQ and VAS data are visually represented with curves that have been smoothed using a cubic spline smoothing function. The cubic spline method uses a set of third-degree polynomials spliced together such that the resulting curve is continuous and smooth at the splices (knot points). The estimation is done by minimizing an objective function that is a combination of the sum of squares error and a penalty for curvature integrated over the curve extent. Global evaluations of rhinitis severity were assessed after the pollen season during August-October each year. The participant responded to the question How do you assess the severity of your hay fever symptoms overall during the pollen season (Tick each single symptom)? with 4 possible answers (0=none, 1=mild, 2=moderate, 3=severe) on 6 symptoms (4 nasal and 2 eye). Total scores ranged from 0 to 18. The minimal clinically important difference (MCID) for the global evaluation was determined using the distribution-based method as there are no publications that provide an anchor-based MCID for the global evaluation measured during a nasal allergen challenge. The MCID was calculated as 0.5*SD=1.4, which is considered to be a conservative approach RESCUE MEDICATION SCORES Use of the following rescue medications was assessed by asking participants to bring their used and unused medication packages to each visit, at which time any remaining pills were counted and any remaining liquid in bottles was weighed in order to determine the number of drops and sprays taken: Nasal corticosteroid (fluticasone propionate aqueous NS) Antihistamine (desloratidine) Ophthalmic antihistamine (olopatadine) Oral corticosteroids (prednisolone) A composite rescue medication score was derived using the following algorithm for each prescribed medication (or equivalent): Medication Medication score Daily score (recommended intake) Desloratadine 5mg 6 points / tablet 6 Olopatadine eye drops 1.5 points / drop 6 Fluticasone Propionate nasal spray 2 points / spray 8 Prednisolone 5mg tablet 1.6 points / tablet 16 (50mg, max) The mean composite score in each treatment group at years 1, 2, and 3 was computed and compared.

8 1.8 SUBCUTANEOUS AND SUBLINGUAL IMMUNOTHERAPY PROTOCOLS Alutard SQ Grass Pollen (SCIT) or identical matched Alutard SQ placebo were injected in hospital by the attending physician or study-nurse with close observation and monitoring of peak expiratory flow (PEF). The up-dosing phase comprised 15 injections over 8 weeks followed by monthly maintenance injections. Desloratadine 10mg tablet was given at least 15 minutes before each injection. Where appropriate, doses were adjusted following established clinical guidelines. 10,11 Injection medication was provided as a suspension in vials identified by participant s name, study and randomization numbers. Active and placebo treatments were identical in appearance. A four-eye checking policy with a doctor and nurse present at every injection was followed throughout the study. Participants remained under observation for 60 minutes following injections. Grazax (75,000 SQ-T) (SLIT) or identical matched Grazax placebo tablets were provided to each participant in blister packs for home sublingual administration for daily continuous use for two years. No up-dosing regimen was employed. The first dose was given in hospital under the supervision of the study doctor and participants remained under observation for 60 minutes. Thereafter participants were instructed to take tablets once daily immediately after opening the blister. Each tablet was placed underneath the tongue and swallowing avoided for 1 minute. Food and beverages were not allowed for at least 5 minutes. 1.9 ADVERSE EVENT REPORTING Adverse Events were reported by observing the participant, questioning the participant in an objective manner or receiving an unsolicited complaint from the participant. Any reactions occurring in the clinic due to procedures, administration of subcutaneous immunotherapy or the first administration of sublingual immunotherapy were recorded on related CRFs and entered into the electronic database. Reactions occurring outside the clinic to either subcutaneous or sublingual immunotherapy or other adverse events were assessed by study staff at clinic visits and recorded on related CRFs and entered into the electronic database EXPECTED ADVERSE REACTIONS It was anticipated that all participants would experience seasonal symptoms of itching sneezing, watery discharge, nasal congestion, and eye symptoms, with or without wheezing from April through August. These are usual symptoms of seasonal allergic rhinitis which represented their entry criteria for the study. In addition, participants had free access to anti-allergic drugs that included antihistamines, intranasal corticoid steroids, and albuterol for relief of their symptoms. This data was captured in the CRFs in the form of a standard format throughout the pollen season (i.e. Visual Analogue Scale, and Rescue Medication Score). These symptoms and requirements for rescue medications were not reported as adverse events. Additionally, there were a number of procedures throughout the study which were associated with expected symptoms. These included: o immunotherapy is associated with local swelling, redness, heat, minor itching, and discomfort. These may last up to 5 to 60 minutes. Frequently there is persistent swelling at the injection site that may persist 24 to 48 hours. As long as these symptoms were not bothersome (i.e. interfered with usual daily activities or sleep) they were not reported as adverse events. o immunotherapy involves taking a daily tablet under the tongue. This is frequently associated with oral itching and mild swelling. These symptoms may last from minutes to hours or longer in some cases. As long as these symptoms were not bothersome (i.e. interfered with usual daily activities or sleep) they were not reported as adverse events. Intradermal and skin prick tests o Intradermal and skin prick tests are associated with local redness, itching and wheal formation at 5 to 30 minutes that resolves within 1to 3 hours. This may also be associated with mild swelling which last for up to 6 to 48 hours. As long as these symptoms were not bothersome (i.e. interfered with usual daily activities or sleep) they were not reported as adverse events. Nasal allergen challenge o Nasal allergen challenge is associated with symptoms of itching, sneezing, watery discharge, nasal congestion, and eye symptoms which begin within minutes and last for 0 to 60 minutes. In some patients nasal congestion and watery discharge may persist up to 6 to 24 hours. Unless these symptoms required rescue medication they were not reported as adverse events.

9 1.9.2 ADVERSE REACTIONS TO STUDY MEDICATION The study site graded the severity of AEs for the following study medication adverse reactions according to the following criteria: All local reactions unassociated with systemic signs or symptoms will be graded according to the following criteria: o o immunotherapy (local reactions) Grade 1: Bothersome (interfering with usual daily activities or sleep) OR moderate swelling of the tongue as assessed by the study physician that is unassociated with systemic signs or symptoms. Grade 2: Bothersome, associated moderate difficulty in swallowing or breathing and/or requiring a visit to the doctor for treatment OR severe swelling of the tongue as assessed by the study physician that is unassociated with systemic signs or symptoms. Grade 3: Bothersome associated with severe difficulty in swallowing or breathing (life threatening) and/or requiring hospitalization immunotherapy (local reactions) Grade 1: Bothersome (interfering with usual daily activities or sleep) Grade 2: Bothersome requiring medication Grade 3: Bothersome requiring a visit to the study physician/emergency room for treatment All systemic reactions to subcutaneous and sublingual immunotherapy were graded according to the WAO Systemic Reaction Grading System 12 (shown below). The following modification applied for sublingual immunotherapy: Under Grade 1 (Symptoms/signs of 1 organ system present), throat clearing (itchy throat) was not considered a systemic reaction to sublingual immunotherapy. All other adverse events related to study medications not covered by the criteria above were graded according to the criteria set forth in the National Cancer Institute s Common Terminology Criteria for Adverse Events, Version 4.0 (published May 28, 2009 and updated June 14, 2010) per the protocol.

10 1.9.3 ADVERSE REACTIONS TO STUDY PROCEDURES The study site graded the severity of AEs for the following selected study procedures according to the following criteria: All local reactions to allergen skin testing and nasal allergen challenge that are to be reported as AEs and are unassociated with systemic signs or symptoms were graded according to the following criteria: o Intradermal and skin prick test procedure (local reactions) Grade 1: Bothersome (interfering with usual daily activities or sleep) Grade 2: Bothersome requiring medication Grade 3: Bothersome requiring a visit to the study physician/emergency room for treatment

11 o Nasal challenge procedure (local reactions) Grade 1: Requiring rescue medication (antihistamines, decongestants, topical steroids) Grade 2: Requiring rescue medication (oral steroids) Grade 3: Life threatening and/or requiring hospitalization (local upper airway obstruction) All systemic reactions related to intradermal or skin prick test procedure or to nasal allergen challenge procedure were graded according to the WAO Systemic Reaction Grading System. The following modification applied for nasal allergen challenges: Under Grade 1 (Symptoms/signs of 1 organ system present), rhinitis (e.g., sneezing, rhinorrhea, nasal pruritus and/or nasal congestion) as well as cough perceived to originate in the upper airway, not the lung, larynx or trachea were not considered systemic reactions to nasal allergen challenge. All other adverse events related to study procedures not covered by the criteria above were graded according to the criteria set forth in the National Cancer Institute s Common Terminology Criteria for Adverse Events, Version 4.0 (published May 28, 2009 and updated June 14, 2010) per the protocol ADVERSE EVENTS NOT RELATED TO STUDY MEDICATION OR PROCEDURES All adverse events not related to study medication or procedures were graded according to the criteria set forth in the National Cancer Institute s Common Terminology Criteria for Adverse Events, Version 4.0 (published May 28, 2009 and updated June 14, 2010) per the protocol ANALYSIS SAMPLES Intent-to-treat (ITT) sample was defined as all randomized participants. ITT participants were analyzed with the group to which they were randomized, regardless of the medication actually received. If participants drop out postrandomization, they were invited to complete study assessments throughout the duration of the trial. Modified ITT sample included all randomized participants with an evaluable endpoint. Per-protocol (PP) sample was defined as ITT sample participants who remained in the study for at least 3 years and in whom the primary endpoint was assessed. Participants in the PP sample must have been compliant with study medication, defined as taking 50% or more of their study medication for the duration of the study. Compliance with study medication was as assessed by pill count for SLIT/SLIT placebo and by observation for SCIT/SCIT placebo. Safety sample (SS) was defined as all randomized participants who received at least one dose of study medication. Participants in the safety sample were analyzed with the group according to the medication they actually received, regardless of their randomized assignment SAMPLE SIZE AND STUDY POWER The study was powered at 90% to detect a standardized mean difference between groups of approximately 40% allowing for 15% dropout. All of the sample size calculations for this trial were based on preliminary data for TNSS measured from 0 to 6 hours, whereas our primary outcome was defined as TNSS measured from 0 to 10 hours. Although data for TNSS measured from 0 to 10 hours were not yet available during the planning phases of the trial, we expected that the effect measured during the first 10 hours following the challenge was likely to be larger and/or more precise than the effect measured during the first 6 hours: hence the sample size calculations which follow were likely to be conservative. The table below provides sample size for the TNSS average of the first hour AUC and the 1-6hour AUC. Given that this study had a four-year duration, estimates of drop-out rates were critical for determining sample size. The table below shows estimates of sample sizes for both a 30% total drop-out rate (approximately 10% per year) and a less conservative 15% total drop-out rate. For the average of the first hour AUC and the 1-6hour AUC TNSS, assuming a 30% drop-out rate, a total sample size of 87 participants (29 per group) provided a power of 80% to detect a 40% difference between

12 SLIT and placebo. Assuming a 15% drop-out rate, we would have achieved 90% power to detect a 40% difference between groups with a total sample of 93 participants (31 per group). The nasal allergen challenge data presented in the table below came from unpublished data (personal communication from Miss Jackie Turner). From these data, we estimated that the average AUC of the TNSS 0 6 hours after nasal challenge in the placebo group to be 12.4 with a standard deviation of Of the 106 randomized participants, 92 completed the primary endpoint evaluation at 3 years. This represented a dropout rate of less than 5% per year, totaling 13.2% from randomization to study completion. Given the retention rate of the trial, we were adequately powered to detect a 40% difference between SLIT and placebo if that difference exists. Total Nasal Symptom Score Number of Patients needed per group Parameter Mean SD Diff Between Effect Size Groups (mean/sd) No Dropout 15% Dropout 30% Dropout 80% Power 90% Power 80% Power 90% Power 80% Power 90% Power 50% = Average of AUC 0-1hr and 1-6hr % = % =

13 2. SUPPLEMENTARY FIGURE 2.1 efigure. Out of Season Global Evaluation (0-18) Global evaluations of rhinitis severity were assessed after the pollen season during August-October each year. Scores range from 0-18 (scores of 0-3 in 6 domains). Higher scores indicate more severe rhinoconjunctivitis symptoms. Multicolored bands represent quantiles, red lines represent means, and black lines represent medians. P-values are calculated using an ANCOVA model adjusted for pre-treatment baseline values. At Year 1 for SCIT, the mean and median lines overlap and only the line representing the median is visible in the figure. The Global evaluation was analyzed in the modified ITT population comprising subjects in each group at each time point. See etable 2c for corresponding data.

14 3. SUPPLEMENTARY TABLES 3.1 etable 1. Peak Nasal Inspiratory Flow (L/min, 0-10 Hours) Change from Pre Challenge Baseline Area Under the Curve Unadjusted Model Adjusted vs vs vs Baseline n Mean % CI ( , ) ( , ) ( , ) Median Min, Max , , , 26.7 Year 1 p-value 0.02 < n Mean Mean Difference % CI (-98.32, ) ( , ) (-76.50, ) 95% CI (7.62, 66.86) (29.19, 87.42) (-41.93, 3.68) Median Min, Max , , , 34.7 Year 2 p-value 0.01 < n Mean Mean Difference % CI (-92.41, ) ( , ) (-76.66, ) 95% CI (15.83, 89.94) (30.27, 99.82) (-37.59, 15.89) Median Min, Max , , , 32.5 Year 3 p-value n Mean Mean Difference % CI ( , ) ( , ) ( , ) 95% CI (-23.64, 43.30) (-17.58, 49.98) (-32.72, 24.10) Median Min, Max , , , Note: A larger change in peak nasal inspiratory flow (PNIF) indicates a higher burden of symptoms during the nasal challenge. Change was defined relative to the 0 time point in the challenge. The minimal clinically important difference for this measure within a subject is (emethods 1.4). The p-values, mean differences, and 95% CIs reported compare the delta PNIF area under the curve (AUC) between treatment groups and were calculated using an analysis of covariance (ANCOVA) model at the 0.05 level of significance adjusted for pretreatment baseline AUC measures. The unit of measure is L/min.

15 3.2 etable 2a. Mini-Rhinoconjunctivitis Quality of Life Questionnaire (mini-rqlq) (0 6) Unadjusted Model Adjusted vs vs vs Baseline n Mean % CI (1.53, 2.32) (1.64, 2.39) (1.68, 2.50) Median Min, Max 0.1, , , 5.1 Year 1 p-value n Mean Mean Difference % CI (0.87, 1.42) (1.45, 1.93) (0.91, 1.40) 95% CI (-0.86, -0.20) (-0.86, -0.22) (-0.32, 0.35) Median Min, Max 0.0, , , 2.6 Year 2 p-value n Mean Mean Difference % CI (0.68, 1.33) (1.09, 1.55) (0.72, 1.26) 95% CI (-0.67, 0.04) (-0.70, -0.03) (-0.29, 0.44) Median Min, Max 0.0, , , 3.0 Year 3 p-value n Mean Mean Difference % CI (0.72, 1.35) (0.82, 1.38) (0.77, 1.46) 95% CI (-0.45, 0.32) (-0.46, 0.35) (-0.44, 0.42) Median Min, Max 0.0, , , 3.2 Note: Mini-Rhinoconjunctivitis quality of life questionnaire (mini-rqlq) values range from 0-6. Higher values reflect subjects who experience more troublesome nose, eye, and other symptoms effecting regular activities resulting in a lower quality of life. The minimal clinically important difference for this measure within a subject is (emethods 1.6). The p-values, mean differences, and 95% CIs reported compare the average mini-rqlq between treatment groups and were calculated using an analysis of covariance (ANCOVA) model at the 0.05 level of significance adjusted for pre-treatment baseline measures.

16 3.3 etable 2b. Visual Analogue Scale (0 10 cm) Unadjusted Model Adjusted vs vs vs Baseline n Mean % CI (2.23, 3.86) (2.08, 3.60) (2.69, 4.44) Median Min, Max 0.2, , , 8.3 Year 1 p-value n Mean Mean Difference % CI (1.82, 3.17) (2.49, 3.51) (1.76, 2.79) 95% CI (-1.35, 0.31) (-1.50, -0.05) (-0.51, 1.13) Median Min, Max 0.0, , , 5.3 Year 2 p-value n Mean Mean Difference % CI (1.29, 2.69) (1.91, 2.84) (1.41, 2.60) 95% CI (-1.24, 0.34) (-1.21, 0.31) (-0.78, 0.99) Median Min, Max 0.0, , , 6.6 Year 3 p-value n Mean Mean Difference % CI (1.32, 2.74) (1.49, 2.70) (1.40, 2.64) 95% CI (-1.00, 0.84) (-1.03, 0.74) (-0.79, 1.07) Median Min, Max 0.0, , , 5.4 Note: Visual analogue scale (VAS) values range from 0-10 cm. Higher values reflect subjects who experience worse hay fever symptoms. The minimal clinically important difference for this measure within a subject is (emethods 1.6). The p-values, mean differences, and 95% CIs reported compare the average VAS between treatment groups and were calculated using an analysis of covariance (ANCOVA) model at the 0.05 level of significance adjusted for pre-treatment baseline measures.

17 3.4 etable 2c. Out of Season Global Evaluation (0-18) Unadjusted Model Adjusted vs vs vs Baseline n Mean % CI (12.75, 14.64) (12.53, 14.35) (11.53, 13.52) Median Min, Max 5.0, , , 18.0 Year 1 p-value n Mean Mean Difference % CI (6.61, 9.64) (9.09, 11.39) (6.62, 9.38) 95% CI (-4.05, -0.60) (-3.53, -0.16) (-2.35, 1.62) Median Min, Max 0.0, , , 16.0 Year 2 p-value n Mean Mean Difference % CI (5.56, 8.83) (8.68, 11.51) (6.18, 9.19) 95% CI (-5.05, -0.81) (-4.39, -0.25) (-2.71, 1.76) Median Min, Max 0.0, , , 16.0 Year 3 p-value n Mean Mean Difference % CI (5.56, 8.71) (6.56, 9.57) (5.51, 8.94) 95% CI (-3.04, 1.07) (-2.75, 1.74) (-2.75, 1.85) Median Min, Max 0.0, , , 16.0 Note: Global evaluations of rhinitis severity were assessed after the pollen season during August-October each year. Scores range from 0-18 (scores of 0-3 in 6 domains). Higher scores indicate more severe rhinoconjunctivitis symptoms. The minimal clinically important difference for this measure within a subject is (emethods 1.6). P-values, mean differences, and 95% CIs were calculated using an ANCOVA model adjusted for baseline at the 0.05 level of significance.

18 3.5 etable 3a. Return Rates of Rescue Medications (N=36) (N=34) (N=36) Year 1 Did Not Return For Visit Did Not Return Any Medication 4 (11.8%) 2 (5.9%) 1 (2.9%) Returned Some Medication 19 (55.9%) 14 ( (41.2%) %) Returned All Medication 11 (32.4%) 18 (52.9 %) 19 (55.9%) Year 2 Did Not Return For Visit Did Not Return Any Medication 4 (12.9%) 2 (6.3%) 6 (17.6%) Returned Some Medication 11 (35.5%) 15 ( (38.2%) %) Returned All Medication 16 (51.6%) 15 (46.9 %) 15 (44.1%) Year 3 Did Not Return For Visit Did Not Return Any Medication 2 (6.5%) 2 (6.5%) 2 (6.3%) Returned Some Medication 7 (22.6%) 6 (19.4%) 9 (28.1%) Returned All Medication 22 (71.0%) 23 (74.2 %) 21 (65.6%) Note: See emethods 1.7 for additional detail about rescue medications..

19 3.6 etable 3b. Rescue Medication Scores Statistics Treatment Comparisons vs vs vs Year 1 p-value < n Mean Median Median Difference % CI (56.00,179.00) (194.00,405.00) (174.00,378.00) 95% CI ( ,-57.00) ( ,101.00) ( ,-45.00) Min, Max 1.0, , , Year 2 p-value n Mean Median Median Difference % CI (52.00,352.00) (194.00,340.00) (133.00,371.00) 95% CI ( ,1.00) ( ,120.00) ( ,11.00) Min, Max 3.0, , , Year 3 p-value n Mean Median Median Difference % CI (53.00,295.00) (179.00,424.00) (141.00,495.00) 95% CI ( ,28.00) ( ,199.00) ( ,14.00) Min, Max 4.0, , , Note: See emethods 1.7 for additional detail about rescue medications. P-values were calculated using Wilcoxon Rank Sum Tests. Median differences and 95% Cis were calculated using Hodges-Lehmann estimates.

20 3.7 etable 4a. Early Skin Response (mm) Unadjusted Model Adjusted vs vs vs Baseline n Mean % CI (19.71, 22.50) (17.75, 20.73) (17.40, 20.53) Median Min, Max 15.0, , , 31.5 Year 1 p-value 0.01 < n Mean Mean Difference % CI (14.92, 17.62) (15.75, 18.86) (11.62, 13.70) 95% CI (-3.88, -0.55) (-5.94, -2.71) (1.07, 4.24) Median Min, Max 9.8, , , 19.3 Year 2 p-value <0.001 < n Mean Mean Difference % CI (14.86, 18.01) (17.15, 20.62) (12.43, 14.21) 95% CI (-5.75, -1.72) (-6.97, -3.51) (0.41, 3.85) Median Min, Max 9.8, , , 19.0 Year 3 p-value < n Mean Mean Difference % CI (15.33, 18.05) (17.20, 19.72) (14.20, 16.62) 95% CI (-4.27, -0.94) (-4.49, -1.25) (-1.54, 1.67) Median Min, Max 10.5, , , 22.0 Note: See emethods 1.5 for additional detail about the Intradermal Allergen Challenge procedure. P-values, mean differences, and 95% CIs were calculated using an ANCOVA model adjusted for baseline at the 0.05 level of significance. The early skin response was collected 15 minutes into the 10 hour nasal allergen challenge.

21 3.8 etable 4b. Late Skin Response (mm) Unadjusted Model Adjusted vs vs vs Baseline n Mean % CI (65.87, 80.73) (63.09, 77.79) (57.39, 71.93) Median Min, Max 22.3, , , Year 1 p-value <0.001 <0.001 <0.001 n Mean Mean Difference % CI (39.31, 55.11) (62.74, 78.07) (21.50, 26.78) 95% CI (-34.30, ) (-53.24, ) (13.65, 29.81) Median Min, Max 20.5, , , 41.8 Year 2 p-value <0.001 <0.001 <0.001 n Mean Mean Difference % CI (34.65, 47.84) (67.71, 80.94) (23.19, 27.57) 95% CI (-42.52, ) (-54.46, ) (8.10, 21.78) Median Min, Max 13.0, , , 41.8 Year 3 p-value <0.001 < n Mean Mean Difference % CI (42.35, 55.99) (66.76, 81.63) (30.18, 38.02) 95% CI (-35.34, ) (-45.75, ) (5.07, 19.40) Median Min, Max 18.0, , , 58.5 Note: See emethods 1.5 for additional detail about the Intradermal Allergen Challenge procedure. P-values, mean differences, and 95% CIs were calculated using an ANCOVA model adjusted for baseline at the 0.05 level of significance. The late skin response was collected at hour 8 of the 10 hour nasal allergen challenge.

22 3.9 etable 5a. Allergen-specific IgE (ku/l) Unadjusted Model Adjusted vs vs vs Baseline n Geometric Mean % CI ( 13.52, ) ( 18.58, ) ( 14.24, ) Median Min, Max 2.58, , , 176 Year 1 p-value < <0.001 n Geometric Mean Ratio % CI ( 44.82, ) ( 17.96, 36.8 ) ( 16.86, ) 95% CI ( 2.39, 4.37 ) ( 0.95, 1.74 ) ( 1.84, 3.42 ) Median Min, Max 7.65, , , 287 Year 2 p-value <0.001 n Geometric Mean Ratio % CI ( 22.93, ) ( 20.22, 45 ) ( 9.07, ) 95% CI ( 1.02, 1.86 ) ( 0.46, 0.84 ) ( 1.63, 3.03 ) Median Min, Max 5.16, , , 297 Year 3 p-value <0.001 n Geometric Mean Ratio % CI ( 19.76, ) ( 21.18, ) ( 10.16, ) 95% CI ( 0.86, 1.58 ) ( 0.48, 0.87 ) ( 1.32, 2.45 ) Median Min, Max 3.27, , , 235 Note: P-value, group ratio, and 95% CI were calculated using a linear mixed model adjusted for baseline at the 0.05 level of significance. The summary statistics were back-transformed from log10 values. Allergen-specific IgE was analyzed in the Per Protocol Population, which included participants who were compliant with study medications, defined as taking 50% or more of their study medication for the duration of the study, and who had an assessment of the primary endpoint.

23 3.10 etable 5b. Allergen-specific IgG4 (mga/l) Unadjusted Model Adjusted vs vs vs Baseline n Geometric Mean % CI ( 0.19, 0.39 ) ( 0.16, 0.29 ) ( 0.2, 0.35 ) Median Min, Max 0.06, , , 1.27 Year 1 p-value <0.001 <0.001 <0.001 n Geometric Mean Ratio % CI ( 0.93, 2.28 ) ( 0.16, 0.29 ) ( 3.34, 8.04 ) 95% CI ( 3.77, 8.39 ) ( 13.63, ) ( 0.18, 0.42 ) Median Min, Max 0.13, , , 23.4 Year 2 p-value <0.001 <0.001 <0.001 n Geometric Mean Ratio % CI ( 1.36, 3.44 ) ( 0.18, 0.35 ) ( 11.93, ) 95% CI ( 4.83, ) ( 35.95, ) ( 0.09, 0.2 ) Median Min, Max 0.19, , , 61.9 Year 3 p-value <0.001 <0.001 <0.001 n Geometric Mean Ratio % CI ( 0.72, 1.52 ) ( 0.19, 0.34 ) ( 2.71, 7.23 ) 95% CI ( 2.31, 5.15 ) ( 9.97, 22.2 ) ( 0.15, 0.35 ) Median Min, Max 0.1, , , 15 Note: P-value, group ratio, and 95% CI were calculated using linear mixed model adjusted for baseline at the 0.05 level of significance. The summary statistics were back-transformed from log10 values. Allergen-specific IgG4 was analyzed in the Per Protocol Population, which included participants who were compliant with study medications, defined as taking 50% or more of their study medication for the duration of the study, and who had an assessment of the primary endpoint.

24 3.11 etable 6. Local and Systemic Adverse Events in the First Hour After the First Tablet at Updosing Visit 1 Description of Local Reaction to Tablet Participant # Treatment Group Symptom Location Severity Duration (minutes) Systemic Reaction to Tablet? 1 SLIT Tingling Under tongue Mild 10 No 2 SLIT Tingling Mouth Not bothersome 5 No 3 SLIT Itching Under tongue Mild No 4 SLIT Tingling Upper lip Not bothersome No 5 SLIT Swelling Itching Throat Lips Not bothersome 6 SLIT Itching Ears Not bothersome No No 7 SLIT Tingling Itching Mouth Not bothersome 60 No 8 SLIT Tingling Mouth Mild No 9 SLIT Itching Back of throat Mild 15 No 10 SCIT Tingling Throat Not bothersome No 11 SLIT Itching Tingling Mouth Throat Not bothersome No Note: These reactions were observed in 11 out of 106 participants.

25 3.12 etable 7a. Systemic Adverse Reactions in Participants on Active SCIT Injections During Study Participation (WAO Classification) Reaction Participant Grade Relationship to Relationship to # (WAO) Description of Event Active Injection Tablet Treatment Given 1 Grade 1 DELAYED GRADE 1 SYSTEMIC REACTION (ANGIOEDEMA IN LIP + FEET) Possibly Related Unrelated DESLORATADINE 2 Grade 1 MILD SNEEZING AND RUNNY NOSE (DELAYED GRADE 1 SYSTEMIC REACTION) Probably Related Unrelated Grade 2 WIDESPREAD SWELLING EXTENDING FROM AXILLA REGION OF LEFT ARM, DOWN TO UPPER PART OF LEFT THIGH, ASSOCIATED WITH ITCHY EARS (DELAYED GRADE 2 SYSTEMIC REACTION) Probably Related Unrelated DESLORATADINE Grade 3 DROWSY AND HEADACHE 10 MINS POST SCIT. DEVELOPED INTO DIZZINESS, FEELING SICK AND TIGHT CHEST 20 MINS POST SCIT. SYMPTOMS GOT WORSE WITHIN 5 MINS, PLUS SENSATION OF THROAT CLOSURE. (EARLY; WAO G3) Probably Related Unrelated ADRENALINE (1MG/ML)/ CHLORPHENIRAMINE/ HYDROCORTISONE Grade 1 RUNNY/BLOCKED NOSE AND SNEEZING (DELAYED GRADE 1 SYSTEMIC REACTION) Probably Related Unrelated NEOCLARITYN 3 Grade 1 SNIFFY AND RUNNY NOSE 30 MINS AFTER INJECTION (IMMEDIATE) Probably Related Unrelated 4 Grade 1 TEARING, SWOLLEN EYES (DELAYED) Probably Related Unrelated 5 Grade 1 GRADE 1 SYSTEMIC REACTION (DELAYED) - ITCHY NOSE AND THROAT Probably Related Unrelated Grade 1 ITCHY, FLAKY SKIN Possibly Related Unrelated Grade 2 SNEEZING, ITCHY NOSE AND EYES ON SECOND DAY OF INJECTION, RESOLVED WITHIN 24 HOURS (DELAYED GRADE 2 SYSTEMIC REACTION) Probably Related Unrelated Grade 2 ITCHY SKIN AND DIARRHOEA (DELAYED GRADE 2 SYSTEMIC REACTION) Possibly Related Unrelated 6 Grade 1 SNEEZED, ITCHY AND RUNNY NOSE 2 HOURS POST SCIT FOR FURTHER 2 HOURS (DELAYED) Probably Related Unrelated 7 Grade 2 MILD ITCHY, BLOCKED NOSE, MILD ITCHING OVER BODY, SAME DAY (DELAYED GRADE 2 SYSTEMIC REACTION) Probably Related Unrelated DESLORATADINE Grade 2 BLOCKED NOSE, ITCHY EYES AFTER 1-2 HOURS (DELAYED GRADE 2 SYSTEMIC REACTION). Probably Related Unrelated Grade 2 VERY MILD BLOCKED NOSE AND ITCHY EYES AFTER 25 MINUTES (IMMEDIATE GRADE 2 SYSTEMIC REACTION) Possibly Related Unrelated Grade 1 MILD BLOCKED NOSE 1-12 HOURS AFTER SCIT (DELAYED GRADE 1 SYSTEMIC REACTION) Possibly Related Unrelated 8 Grade 1 SNEEZING AND NASAL CONGESTION (IMMEDIATE) Probably Related Unrelated 9 Grade 1 HIVES ON BOTH WRISTS & ONE ON CHEST, ABOUT 2X2CM. (DELAYED G1 SYSTEMIC REACTION) Possibly Related Unrelated NEOCLARITYN 10 Grade 1 ITCHY NOSE AND SNEEZING AT 8 48 HOURS (DELAYED GRADE 1 SYSTEMIC REACTION) Probably Related Unrelated Grade 1 ITCHY EYES. (IMMEDIATE GRADE 1 SYSTEMIC REACTION) Probably Related Unrelated Grade 1 ITCHY EYES Probably Related Unrelated Grade 1 ITCHY EYES (IMMEDIATE GRADE 1 SYSTEMIC REACTION) Possibly Related Unrelated Grade 1 ITCHY EYES (DELAYED GRADE 1 SYSTEMIC REACTION) Possibly Related Unrelated Grade 1 MILD SNEEZING (DELAYED GRADE 1 SYSTEMIC REACTION) Possibly Related Unrelated 11 Grade 1 ITCHING SPREAD FROM INJECTION SITE TO CHEST AND OTHER ARM(DELAYED) Probably Related Unrelated 12 Grade 2 SKIN RASH ON CHIN AND BEHIND EARS; TINGLING, ITCHINESS NOSE, EARS AND THROAT; TIGHT CHEST, CLEAR ON AUSCULTATION, BLOCKED NOSE (DELAYED SYSTEMIC REACTION) Probably Related Unrelated