IL-10 as a predictive biomarker of responsiveness to house dust mite allergen immunotherapy

Abstract

The present invention concerns the use of IL-10 as a biological marker for predicting the responsiveness of a house dust mite allergic patient to house dust mite allergen immunotherapy.

Claims

1. A method for immunotherapy of house dust mite allergy in a patient comprising: (i) selecting a patient for immunotherapy by implementing a method comprising: a) measuring abundance of IL-10 mRNA in a biological sample from said patient, b) comparing said abundance of IL-10 mRNA with a control, c) identifying said patient as likely to be a responder to immunotherapy when (c1) the abundance of IL-10 mRNA in the patient sample is equal to or greater than the level of expression in a control derived from a responder subject, or group of responder subjects, known to respond to said immunotherapy; or (c2) the abundance of IL-10 mRNA in the patient sample is greater than the level of expression in a control derived from a non-responder subject, or group of non-responder subjects; or (c3) the abundance of IL-10 mRNA in the patient sample is greater than the level of expression in a control derived from a randomly selected group of subjects; and wherein said biological sample is taken before the commencement of immunotherapy, and wherein said immunotherapy comprises administration of a house dust mite allergen to said patient in order to treat house dust mite allergy, and (ii) administering said house dust mite allergen to said patient identified as likely to be a responder to said immunotherapy.

2. The method according to claim 1, wherein a responder demonstrates at least 10% improvement in clinical symptoms of house dust mite allergy and a non-responder demonstrates less than 10% improvement in clinical symptoms of house dust mite allergy.

3. The method according to claim 1, wherein said IL-10 mRNA consists of the sequence SEQ ID NO: 1.

4. The method according to claim 1, wherein the immunotherapy comprises administration of said house dust mite allergen to a mucosal surface, or administration of allergen via a subcutaneous, transdermal, intralymphatic or epicutaneous route.

Description

FIGURES

(1) FIG. 1. Higher IL-10 mRNA levels in whole blood cells from HDM allergic patients responding to AIT. Expression of the IL-10 gene (mean±SEM) was assessed at baseline (before treatment—V2) by qPCR in whole blood cells from patients of the active and placebo groups (ARs, n=38; ANRs, n=37; PRs, n=18; and PNRs, n=59) (** P<0.01, Mann-Whitney tests). A threshold of 51.18% AASS improvement after treatment (improvement of AASS between 44-52 weeks of treatment and baseline—V17/V2) was applied to distinguish responders from non-responders.

(2) FIG. 2. IL-10 mRNA levels in whole blood cells from HDM allergic patients correlate with AIT clinical efficacy.

(3) Expression of the IL-10 gene (mean±SEM) was assessed at baseline (before treatment—V2) by qPCR in whole blood cells from patients of the active and placebo groups (Actifs=75, Placebo=77). Spearman correlations at an individual patient level of IL-10 gene levels by plotting copy numbers of IL-10 mRNA per million copies of β-actin mRNA against percentage improvements in AASS (improvement of AASS between 44-52 weeks of treatment and baseline—V17/V2) in patients from the active (upper panel) and placebo (lower panel) groups (** p<0.01, ns=non-significant; Spearman test).

(4) FIG. 3: Selection of IL-10 high patients improves the difference in clinical scores between active and placebo patients. Average Adjusted Symptom Scores (AASS; mean±SD) are presented after 12 months (weeks 44-52) of immunotherapy for 152 patients (75 receiving the active treatment and 77 receiving placebo) without using predictive biomarker criteria (upper panel). AASS are presented after 12 months (weeks 44-52) of immunotherapy for 114 patients exhibiting high levels of IL-10 mRNA (52 receiving the active treatment and 62 receiving placebo) (lower panel). (**** P<0.0001, Mann-Whitney tests).

(5) FIG. 4: ROC curves of IL-10 mRNA abundance. ROC curves of IL-10 mRNA abundance in active group with % AASS improvement as a cut off of 16.54% (A), 25% (B) and 51.18% (C) to define responder patients and in which the controls are defined as non-responder patients (i.e. patients with a percentage of improvement in AASS lower than a threshold) (AUC: area under the ROC curve).

EXAMPLES

(6) In order to identify biological parameters allowing distinguishing house dust mite allergic patients, before they initiate allergen immunotherapy (AIT), who will subsequently benefit from AIT from those who are non-responders, experiments have been conducted on biological samples from patients undergoing a phase 2/3 clinical trial with house dust mite SLIT tablet.

Example 1: Study Design for Evaluation of House Dust Mite SLIT Tablet Efficacy and Safety in a Large Scale One-Year Study in Patients with Allergic Rhinitis

(7) Clinical Trial Design:

(8) A randomized, double-blind, placebo-controlled study has been conducted to evaluate the efficacy and safety of house dust mite (HDM) tablet in adolescent and adult patients with HDM allergic rhinitis, with or without intermittent asthma. Subjects were randomized 1:1:1 to placebo or active treatment with HDM extracts at doses (expressed in Index of Reactivity [IR], the in-house standardization unit) of 300 IR or 500 IR and treated for 52 weeks.

(9) Study Population:

(10) The full analysis set included 927 subjects, encompassing patients receiving either 300 IR (N=315), 500 IR (N=296) and placebo (N=316) tablets.

(11) Participants:

(12) The main inclusion criteria were males or females; ages between 12 and 64 years; a clinical history of HDM-induced allergic rhinitis at least for 2 years; positive HDM-specific serum IgE and positive nasal provocation test.

(13) Study Treatment and Other Medications:

(14) Two doses of HDM tablets, 300 IR and 500 IR, were administered in the active treatment groups. Active tablets contain a 1:1 mixture of standardized extracts of D. pteronyssinus and D. farinae. Placebo tablets were administered to the placebo group.

(15) Subjects were instructed to use RM (Rescue Medication) if symptoms were intolerable or interfered with daily activities, and in a step-wise manner, at step 1: oral or ophthalmic antihistamine, step 2: nasal corticosteroid, step 3: oral or ophthalmic antihistamine with nasal corticosteroid.

(16) Assessments:

(17) Subjects recorded their daily nasal symptoms (0-4 for sneezing, rhinorrhea and nasal congestion, and 0-3 for nasal pruritus, total 0-15 point scale), ocular symptoms (0-4 for itchy eyes and watering eyes), interference with daily activities (0-4) and RM use, for the 2-weeks interim evaluation periods (week 8-10, 16-18, 24-26, 32-34 and 40-42) and the end of treatment evaluation period (week 44-52, primary evaluation period). Rescue medication score (RMS; range, 0-2) was assigned as 1 if a subject took oral and/or anti-histamine (Step 1), and 2 if a subject took nasal corticosteroid (Step 2). If a subject took both anti-histamine and nasal corticosteroid (Step 3), RMS of 2 was assigned.

(18) Outcomes:

(19) The primary efficacy endpoint was the AASS in the last last weeks of the 52-week treatment period (weeks 44-52) (Grouin et al. Clin Exp Allergy. 2011; 41:1282-8). The AASS is the average for each patient of the Adjusted Symptom Score (ASS) which is derived as follows. If a subject did not take RM on the day or previous day, the ASS equaled to the Rhinitis Total Symptom Score (RTSS). If a subject took RM on the day, the ASS on the day was equal to the RTSS of the day or the ASS of the previous day, whichever was higher, and the ASS of the next day was the RTSS on the next day, or the ASS of the day before, whichever was higher. The RTSS (range, 0-15) was the sum of the 4 rhinitis symptom scores. The Combined Score (CS; range, 0-2.875) is derived as (RTSS/4+RMS)/2.

Example 2: Identification of IL-10 as a Biomarker of AIT Efficacy

(20) In the context of the clinical trial described in example 1, ancillary biomarker studies were conducted in HDM allergic patients receiving daily either 300 IR HDM tablets (n=75) or a placebo (n=77). In this study clinical responses were evaluated in each individual patient as percentages of improvement in Average Adjusted Symptom Score (AASS) which adjusts symptom scores for the use of rescue medication.

(21) Methods

(22) Patient Subgroups

(23) The median percentages of AASS improvement in the active group after treatment was also used as a threshold to identify clinical responders, with patients exhibiting a ≥51.18% AASS improvement considered as responders, and those with a lower improvement as non-responders. Based on this threshold, 4 patient subgroups were defined, including active responders (ARs, n=38), active non-responders (ANRs, n=37), placebo responders (PRs, n=18), and placebo non-responders (PNRs, n=59).

(24) Sample Collection

(25) Whole blood cells were collected at baseline (Visit 2; V2) and after 2 (V6) and 12 months (V17) of AIT by using PAXgene blood RNA tubes (Qiagen), and kept frozen at −20° C. until use. All biological analyses were performed on samples coded to the operators.

(26) RNA Isolation and Quantitative Real-Time PCR Analyses

(27) Total RNAs, including miRNAs, were extracted with the nucleic acid purification kit (PAXgene Blood miRNA Kit) as per the manufacturer's instructions. cDNAs were synthesized using the SuperScript® VILOTM cDNA Synthesis kit (Applied Biosystems, Les Ulis, France) as per the manufacturer's instructions. The expression of IL-10 gene (IL-10-Hs00174066_m1) was assessed and compared with endogenous β-actin (Hs99999903_m1) as a control by quantitative PCR (qPCR) on a 7900HT Real-Time PCR system (Applied Biosystems) according to the manufacturer's instructions. The relative amounts of IL-10 mRNAs corresponding to the target gene in each sample were calculated in comparison with the calibrator gene, i.e. endogenous β-actin, using the following equation: ΔCt=(Ct IL-10-Ct β-actin). For graph analyses, the formula (2.sup.−ΔCt)×10.sup.6 was used in order to determine copy numbers of IL-10 mRNA per million copies of β-actin mRNA.

(28) Statistical Analyses

(29) Statistical differences between groups and correlation analyses were performed by using Mann-Whitney and Spearman tests, respectively, with P values <0.05 considered significant.

(30) Results

(31) At Baseline, IL-10 mRNA Levels are Higher in Whole Blood Cells of Clinical Responders than in Non-Responders and Significantly Correlate with Clinical Benefit within Patients from the Active Group

(32) To evaluate IL-10 as a predictive marker of clinical responses to AIT, we measured the expression of IL-10 by qPCR in whole blood cells collected before AIT (V2) from 152 HDM allergic patients receiving either a 300 IR HDM (n=75) or placebo (n=77) tablet sublingually, once a day, for 12 months. Patients were stratified in 4 subgroups (cf. Methods), including active responders (ARs; n=38), active non-responders (ANRs; n=37), placebo responders (PRs; n=18), and placebo non-responders (PNRs; n=59). As shown in FIG. 1, IL-10 mRNA levels were significantly (p<0.01) higher in ARs than in ANRs. No such differences were observed in the placebo group between PRs and PNRs and there was no potential bias since there was no differences between the active and the placebo groups (FIG. 1).

(33) When plotted against percentages of AASS improvement (V17/V2) for each individual patient, levels of IL-10 mRNA were significantly correlated (with a Spearman correlation R=0.3511, p=0.002) with clinical benefit within patients from the active group (FIG. 2, upper panel), whereas no such a correlation was observed in placebo-treated patients (FIG. 2, lower panel).

(34) Selection of IL-10 High Patients Improves the Difference in Clinical Scores Between Active and Placebo Patients.

(35) To assess the advantage of selecting HDM allergic patients eligible to AIT based on IL-10 mRNA levels in whole blood cells, we selected for further analyses 75% (114 out of 152) of HDM allergic patients exhibiting the highest levels of IL-10 mRNA, defined as IL-10.sup.high patients based on copy numbers of IL-10 mRNA per million copies of β-actin mRNA ≥94.455. By contrast, 25% (38 out of 152) of patient exhibiting the lowest levels of IL-10 mRNA (IL-10.sup.low patients) were withdrawn from analyses. As shown in FIG. 3, after 12-month AIT (weeks 44-52), differences in AASS in IL-10.sup.high patients receiving the active treatment versus placebo were much higher (lower panel; −34.5%) when compared to differences in AASS in the whole cohort of unselected patients (upper panel; −26.0%).

(36) The present study thus clearly establishes that the IL-10 gene is more expressed in whole blood cells from ARs relative to ANRs. These results provide for the use of IL-10 levels, in particular IL-10 mRNA levels, before AIT as a criterion to select patients more likely to benefit for treatment. As such, IL-10 represents a biomarker for predicting AIT efficacy.

(37) The pertinence of the IL-10 mRNA was further assessed by a receiver operating characteristic (ROC) analysis. The ROC curve of IL-10 mRNA copies levels of 75 active patients divided in 2 subgroups based on a threshold percentage of improvement in AASS of 16.54% is shown in FIG. 4A. The AUC was of 0.6911 (with p-value of 0.03682).

(38) The ROC curve of IL-10 mRNA copies levels of 75 active patients divided in 2 subgroups based on a threshold percentage of improvement in AASS of 25% is shown in FIG. 4B. The AUC was of 0.7242 (with p-value of 0.004345).

(39) The ROC curve of IL-10 mRNA copies levels of 75 active patients divided in 2 subgroups based on a threshold percentage of improvement in AASS of 51.18% is shown in FIG. 4C. The AUC was of 0.7116 (with p-value of 0.001627).

(40) In these ROC curves, controls are defined as non-responder patients (i.e. patients with a percentage of improvement in AASS lower than the percentage of AASS improvement thresholds defined above).

(41) These latter ROC curves in which the controls are defined as non-responder patients were associated with the following data on sensitivity and specificity.

(42) TABLE-US-00001 TABLE 1 Threshold at 16.54% improvement in AASS (IL-10 mRNA copies per million β-actin mRNA copies) Cutoff IL-10 Sensitivity % Specificity % >47.10 98.41 0 >51.00 98.41 8.333 >53.15 96.83 16.67 >55.05 95.24 16.67 >57.35 93.65 16.67 >63.40 92.06 16.67 >69.40 90.48 16.67 >70.60 90.48 25 >71.35 88.89 25 >72.60 87.3 25 >75.15 85.71 25 >77.00 85.71 33.33 >77.80 84.13 33.33 >79.40 82.54 33.33 >80.80 82.54 41.67 >81.65 80.95 41.67 >82.50 79.37 41.67 >86.70 77.78 41.67 >90.90 76.19 41.67 >91.85 76.19 50 >93.15 74.6 50 >97.40 73.02 50 >102.0 71.43 50 >105.5 68.25 50 >111.0 66.67 50 >114.5 66.67 58.33 >119.0 63.49 58.33 >124.0 61.9 58.33 >126.5 60.32 58.33 >128.5 58.73 58.33 >129.5 57.14 58.33 >131.0 55.56 66.67 >133.5 53.97 75 >137.0 52.38 75 >139.5 50.79 75 >144.0 50.79 83.33 >150.0 49.21 83.33 >152.5 47.62 83.33 >156.5 46.03 83.33 >160.5 46.03 91.67 >164.0 44.44 91.67 >171.5 42.86 91.67 >178.5 41.27 91.67 >182.5 39.68 91.67 >191.0 38.1 91.67 >199.0 36.51 91.67 >205.5 34.92 91.67 >215.5 31.75 91.67 >221.5 28.57 91.67 >226.0 25.4 91.67 >242.0 23.81 91.67 >255.5 23.81 100 >258.0 22.22 100 >262.5 20.63 100 >272.5 19.05 100 >282.0 17.46 100 >286.0 15.87 100 >291.0 14.29 100 >311.5 12.7 100 >331.5 11.11 100 >356.0 9.524 100 >417.0 7.937 100 >457.5 6.349 100 >463.5 4.762 100 >485.0 3.175 100 >543.5 1.587 100

(43) TABLE-US-00002 TABLE 2 Threshold at 25% improvement in AASS (IL-10 mRNA copies per million β-actin mRNA copies) Cutoff IL-10 Sensitivity % Specificity % >47.10 100 5.556 >51.00 100 11.11 >53.15 98.25 16.67 >55.05 96.49 16.67 >57.35 94.74 16.67 >63.40 92.98 16.67 >69.40 91.23 16.67 >70.60 91.23 22.22 >71.35 91.23 27.78 >72.60 89.47 27.78 >75.15 87.72 27.78 >77.00 87.72 33.33 >77.80 85.96 33.33 >79.40 84.21 33.33 >80.80 84.21 38.89 >81.65 82.46 38.89 >82.50 82.46 44.44 >86.70 80.7 44.44 >90.90 78.95 44.44 >91.85 78.95 50 >93.15 77.19 50 >97.40 77.19 55.56 >102.0 75.44 55.56 >105.5 73.68 61.11 >111.0 71.93 61.11 >114.5 71.93 66.67 >119.0 68.42 66.67 >124.0 66.67 66.67 >126.5 64.91 66.67 >128.5 63.16 66.67 >129.5 61.4 66.67 >131.0 59.65 72.22 >133.5 57.89 77.78 >137.0 56.14 77.78 >139.5 54.39 77.78 >144.0 54.39 83.33 >150.0 52.63 83.33 >152.5 50.88 83.33 >156.5 49.12 83.33 >160.5 49.12 88.89 >164.0 47.37 88.89 >171.5 45.61 88.89 >178.5 43.86 88.89 >182.5 42.11 88.89 >191.0 40.35 88.89 >199.0 38.6 88.89 >205.5 36.84 88.89 >215.5 33.33 88.89 >221.5 29.82 88.89 >226.0 26.32 88.89 >242.0 24.56 88.89 >255.5 24.56 94.44 >258.0 22.81 94.44 >262.5 21.05 94.44 >272.5 19.3 94.44 >282.0 17.54 94.44 >286.0 17.54 100 >291.0 15.79 100 >311.5 14.04 100 >331.5 12.28 100 >356.0 10.53 100 >417.0 8.772 100 >457.5 7.018 100 >463.5 5.263 100 >485.0 3.509 100 >543.5 1.754 100

(44) TABLE-US-00003 TABLE 3 Threshold at 51.18% improvement in AASS (IL-10 mRNA copies per million β-actin mRNA copies) Cutoff Sensitivity % Specificity % >47.10 100 2.703 >51.00 100 5.405 >53.15 100 10.81 >55.05 97.37 10.81 >57.35 97.37 13.51 >63.40 97.37 16.22 >69.40 97.37 18.92 >70.60 97.37 21.62 >71.35 97.37 24.32 >72.60 97.37 27.03 >75.15 94.74 27.03 >77.00 94.74 29.73 >77.80 92.11 29.73 >79.40 92.11 32.43 >80.80 92.11 35.14 >81.65 89.47 35.14 >82.50 89.47 37.84 >86.70 86.84 37.84 >90.90 84.21 37.84 >91.85 84.21 40.54 >93.15 81.58 40.54 >97.40 81.58 43.24 >102.0 78.95 43.24 >105.5 78.95 48.65 >111.0 76.32 48.65 >114.5 76.32 51.35 >119.0 73.68 54.05 >124.0 73.68 56.76 >126.5 73.68 59.46 >128.5 71.05 59.46 >129.5 71.05 62.16 >131.0 68.42 64.86 >133.5 65.79 67.57 >137.0 65.79 70.27 >139.5 63.16 70.27 >144.0 63.16 72.97 >150.0 60.53 72.97 >152.5 57.89 72.97 >156.5 55.26 72.97 >160.5 55.26 75.68 >164.0 52.63 75.68 >171.5 50 75.68 >178.5 50 78.38 >182.5 50 81.08 >191.0 47.37 81.08 >199.0 44.74 81.08 >205.5 42.11 81.08 >215.5 39.47 83.78 >221.5 36.84 86.49 >226.0 31.58 86.49 >242.0 28.95 86.49 >255.5 28.95 89.19 >258.0 26.32 89.19 >262.5 23.68 89.19 >272.5 23.68 91.89 >282.0 21.05 91.89 >286.0 21.05 94.59 >291.0 18.42 94.59 >311.5 15.79 94.59 >331.5 13.16 94.59 >356.0 10.53 94.59 >417.0 7.895 94.59 >457.5 7.895 97.3 >463.5 5.263 97.3 >485.0 5.263 100 >543.5 2.632 100

(45) These results confirm that IL-10 mRNA is useful to discriminate clinical responders from non-responders before AIT.

Example 3—Expression of the IL-10 in Fresh PBMCs

(46) Expression of IL-10 gene can be assessed in human PBMCs by PCR.

(47) Total RNAs were isolated from PBMCs using silica-based spin columns. Complementary DNAs were then synthesized using a reverse transcriptase enzyme and random hexamer primers. Quantification was finally carried out by quantitative real-time PCR using the TaqMan technology with primers and probes specific for the IL-10 gene. Relative gene expression was calculated using the ΔΔCt method.

Example 4—Expression of IL-10 in Restimulated PBMCs

(48) Expression of IL-10 gene can be assessed in restimulated human PBMCs by PCR

(49) PBMCs were restimulated in vitro with relevant allergens under controlled culture conditions. Total RNAs were subsequently isolated using a spin column-based method followed by a reverse transcription step to generate complementary DNAs. The IL-10 gene expression was further quantified by real-time PCR using specific primers and a Taqman probe. Results were expressed as relative quantity and evaluated using the ΔΔCt method.

Example 5—Abundance of IL-10 in Serum

(50) Measure of IL-10 polypeptide abundance in serum with aptamers

(51) Nucleic acid aptamers specific for IL-10 were used to quantify IL-10 concentration in serum using a DNA quantification technique such as microarrays. The quantification was performed following the subsequent method:

(52) (i) Aptamer reagents attached to streptavidin beads bind to serum proteins

(53) (ii) The proteins that are bound to their specific aptamer reagents are then biotinylated

(54) (iii) The aptamer-protein complexes are released by photocleaving the linker, and non-specific aptamer-protein complexes are separated

(55) (iv) Biotinylated proteins are bound to a second streptavidin bead

(56) (v) Bound aptamer reagents are removed from their protein targets

(57) (vi) Aptamer reagents are collected and denatured

(58) (vii) Aptamer reagents are measured using standard DNA analysis techniques like microarrays.