Grass pollen allergoids conjugated with mannan for subcutaneous and sublingual immunotherapy: a dose-finding study

Background Polymerized allergoids conjugated with mannan represent a novel approach of allergen immunotherapy targeting dendritic cells. In this study, we aimed to determine the optimal dose of mannan-allergoid conjugates derived from grass pollen (Phleum pratense and Dactylis glomerata) administered via either the subcutaneous or sublingual route. Methods A randomized, double-blind, placebo-controlled trial with a double-dummy design was conducted, involving 162 participants across 12 centers in Spain. Subjects were randomly allocated to one of nine different treatment groups, each receiving either placebo or active treatment at doses of 500, 1,000, 3,000, or 5,000 mTU/mL over four months. Each participant received five subcutaneous (SC) doses of 0.5 mL each, every 30 days, and a daily sublingual (SL) dose of 0.2 mL. Participants who received active treatment through SC, received placebo through SL. Participants who received active treatment through SL, received placebo SC. One Group, as control, received bot SC and SL placebo. The primary efficacy outcome was the improvement in titrated nasal provocation tests (NPT) at the end of the study compared to baseline. Secondary outcomes included specific antibody (IgG4, IgE) and cellular (IL-10 producing and regulatory T cell) responses. All adverse events and side reactions were recorded and assessed. Results Post-treatment, the active groups showed improvements in NPT ranging from 33% to 53%, with the highest doses showing the greatest improvements regardless of the administration route. In comparison, the placebo group showed a 12% improvement. Significant differences over placebo were observed at doses of 3,000 mTU/mL (p=0.049 for SL, p=0.015 for SC) and 5,000 mTU/mL (p=0.011 for SL, p=0.015 for SC). A dose-dependent increase in IgG4 was observed following SC administration, and an increase in IL-10 producing cells for both routes of administration. No serious systemic or local adverse reactions were recorded, and no adrenaline was required. Conclusion Grass pollen immunotherapy with mannan-allergoid conjugates was found to be safe and efficacious in achieving the primary outcome, whether administered via the subcutaneous or sublingual routes, at doses of 3,000 and 5,000 mTU/mL. Clinical trial registration https://www.clinicaltrialsregister.eu/ctr-search (EudraCT), identifier 2014–005471–88; https://www.clinicaltrials.gov, identifier NCT02654223.


Drop-outs
The following table shows the list of abandons and the cause of these abandons.

Cell analysis 5.1. Phleum specific CD4 T cell epitopes
Experimentally verified timothy grass specific CD4 T cell epitopes targeted by allergic subjects were retrieved from the Immune Epitope Database (IEDB) [PMID: 22610854] after the following search: 1) Epitope, linear peptide; 2) Organism, Phleum pratense; 3) Host, human; 4) T cell assay with positive result, 5) Class II restriction and 6) Disease Allergy.CD-HIT [PMID: 16731699] was used to identify and cluster CD4 T cell epitopes with overlapping amino acid sequences (100 % identity threshold) (1) .Clusters were processed and CD4 T cell epitopes with ≥ 7 residue-overlaps were combined into extended peptides as described in (2) .As a result, 20 peptides bearing one or more CD4 T cell epitopes were selected for peptide synthesis (Table VIS).Peptides were synthesized by ProteoGenix (Schiltigheim, France) at ≥95% purity as confirmed by reversed-phase high-performance liquid chromatography (RP-HPLC).Lyophilized peptides were dissolved in 40% dimethyl sulfoxide, diluted in ultra-pure water to a peptide concentration of 5 mM, and stored at −80°C until use.

Determination of Phleum-specific IL-10 producing cells
In vitro expansion of Phleum-specific T cells Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient centrifugation (800g, 20 minutes) from patient's blood samples collected at baseline and at the end of the study and were kept in liquid nitrogen until processing.For the in vitro expansion of Phleum pratense-specific T lymphocytes, PBMCs from allergic patients were stimulated with the pool of Phleum peptides 10 μM (Proteogenix, France) and 10 U/ml of IL-2 (Immunotools, Germany).PBMCs were kept at 37 °C and 5% CO2 for 5 days with additional doses of the stimuli on day three of the expansion.RPMI 1640 (Gibco, NY, USA) supplemented with 5% of human serum (Gibco, NY, USA), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine (Lonza, Walkersville, USA) was used.The expanded cells were washed twice with PBS and allowed to rest for 4 hours in RPMI 1640 without human serum or stimulation prior to ELISPOT assay development.
ELISPOT assays 96-well PVDF ELISPOT plates (Mabtech, Sweden) were activated with 20 μl of ethanol for 1 minute, washed with PBS (Gibco) and coated with anti-IL-10 capture antibody (mAb 9D7; Mabtech, Sweden).Plates were incubated for 48 hours at 4 °C with the capture antibody, washed with PBS and blocked for 30 minutes with RPMI 1640 (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, NY, USA).PBMCs from allergic patients were added to the ELISPOT plates and incubated for 24 hours at 37 °C and 5% CO2.Phleum-specific IL-10 responses were detected by stimulating PBMCs with 10 μM of the Phleum peptide pool in the ELISPOT plate.Basal cytokine production in the absence of stimuli and response to phytohemagglutinin (PHA) (1 μg/ml) (Sigma-Aldrich, Germany) were evaluated as negative and positive control for cytokine production, respectively.

SFC detection
The number of spot forming cells (SFC) was detected following manufacturer's instructions (Mabtech).In brief, plates were washed with PBS and incubated for 2 hours with anti-IL-10 detection antibody (mAb 12G8-biotin; Mabtech, Sweden).Subsequently, plates were washed with PBS and Streptavidin-ALP (1:1000) (Mabtech, Sweden) was added for 1 hour at room temperature.After several washes, 100 μl of the BCIP/NBT substrate (Mabtech, Sweden) was added.The enzymatic reaction was stopped with tap water and plates were allowed to dry for at least 48 hours before being analyzed.SFC were counted with an ELISPOT reader (ImmunoSpot 5.0, CTL Analyzers, LLC, OH, USA) considering a minimum spot size of 500 μm 2 .ELISPOT assays were run in triplicate and the mean ± standard deviation of the basal control was subtracted to each value obtained.

Safety
All adverse events occurring during the trial were recorded and assessed.These events were thoroughly explored, both during scheduled visits and at any time in the subject reporting an abnormal incidence.EMA Good Pharmacovigilance Practice Guidelines (3) were followed.
The adverse events could be divided in non-drug related or drug related adverse events (also known as adverse reactions).
Adverse reactions known to be related to the intrinsic (allergenic) properties of the product were classified as: • Depending on the site of appearance: o Local: appear in the place of administration.The severity of these reactions was classified as: ▪ Mild: event that the subject easily tolerates and that causes minimal discomfort without interfering with daily activities.▪ Moderate: event that produces discomfort in a way that interferes with activities of daily living.▪ Intense or severe: event that prevents daily activities.o Systemic: appear in another part of the body different from the site of administration.The severity of these adverse reactions was graded according to the following grades: (4) ▪ Grade 0: No symptoms or nonspecific symptoms.
▪ Grade 2: Moderate systemic reaction.Usually slow onset (> 15 minutes) of generalised urticaria and/or moderate asthma (PEF < 40% reduction from baseline).Moderate limitation of the activity, with little medical intervention or hardly needing therapy.
▪ Grade 3: Severe systemic reaction.Rapid onset (< 15 minutes) of generalised urticaria, angioedema or severe asthma (PEF > 40% reduction from baseline).Severe limitation of activity that requires some type of therapy or medical assistance with possible hospitalization.
• Depending on the time of appearance: o Immediate: those that appeared within the first 30 min after administration of the treatment.o Delayed: those that appeared later than the first 30 min after administration of the treatment.
The study population received the treatment for 4 months.178 patients were screened for eligibility, of which 162 were included.In the end, 12 patients dropped and 150 completed the study.

Adverse Events (AEs)
In this study 128 events were reported, 54 were considered to be adverse drug related (related to the IMP; adverse reactions) and 74 were non-drug related adverse events (not related to IMP).
Regarding the 74 non-drug related adverse events registered in a total of 37 (22.8%)participants, 11 events were experienced in a total of 7 subjects in the placebo group.In the groups receiving subcutaneous active treatment 35 events were reported by a total of 14 subjects.On the other hand, in groups receiving sublingual active treatment, 28 non-drug related adverse events were experienced by a total of 16 participants.Of all these events, 61 were classified as mild and 13 as moderate.
In reference to the drug related adverse events (adverse reactions), 54 events were reported in a total of 22 subjects, 45 were local and 9 systemic.In the groups receiving subcutaneous active treatment 51 events were reported, of which 44 were local and 7 systemic.Of the 45 local adverse reactions, 33 were described as mild and 12 as moderate.
The most common drug related adverse events were local reactions (in the injection site), no other adverse reaction was experienced by more than 5% of the study population.

Table IS .
Other allergen sensitizations of subject, skin prick test results.
Figure 1S.Number of subjects experiencing or not improvement in the nasal provocation test in each group administered subcutaneously (Panel A) or sublingually (Panel B).

Table IIIS
. Median (Q1 and Q3) of Phleum Specific IgE values and comparative statistics P.

Table IVS .
Median (Q1 and Q3) of Ratio sIgE/IgG4 values and comparative statistics

Table VS .
Drop-outs

Table VIS .
Peptide sequences contained in Phleum peptide pool