Peptide Immunotherapy; short but long lasting?

Opinion Statement

Allergen immunotherapy (AIT) with whole allergens or allergen extracts has been in use for more than one hundred years. It is clinically efficacious and disease-modifying. However, AIT is also associated with a significant adverse events profile, including the potential to cause severe, systemic allergic reactions. One alternative to traditional whole-antigen AIT is peptide immunotherapy, which uses small synthetic peptide immunoregulatory epitopes (SPIRE) representing T cell epitopes from the allergen of interest. Peptide immunotherapy is being developed for the treatment of allergic and autoimmune diseases where pathogenesis is T cell-dependent. Short, soluble, monomeric peptide fragments avoid the problem of IgE-mediated adverse events (since the peptides will not cross-link allergen-specific IgE on the surface of effector cells such as mast cells and basophils). However, such peptides retain the capability to induce T cell tolerance and immunoregulation. In early clinical trials, efficacy has been demonstrated months to years after the cessation of a short course of treatment, supporting the conclusion that this approach is disease-modifying, changing the natural history of the disease. The improved safety profile of short peptides allows for larger molar-equivalent doses to be administered in shorter time frames than AIT; treatment can be completed in as few as four intradermal injections, while efficacy persists for two years or more.

Introduction

Allergen immunotherapy (AIT) is clinically efficacious and disease-modifying. Studies of both subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) have demonstrated reductions in symptoms scores and medication usage (1,2). Clinical benefit persists beyond the treatment period, suggesting a resetting of the allergen-specific adaptive immune response. AIT has been shown to prevent progression of allergic rhinitis towards asthma and to reduce the risk of developing new allergic sensitizations. The mechanisms of action of this approach remain incompletely understood, but several studies have supported a role for the induction of allergen-specific T cells and B cells with immunoregulatory or suppressive characteristics such as IL-10-secreting Tr1 T cells and B10 B cells (3-6). Furthermore, clinical efficacy resulting from treatment with whole allergen molecules, or extracts, is associated with induction of allergen-specific IgG (particularly the IgG4 isotype). While absolute levels of serum allergen-specific IgG4 do not correlate with clinical efficacy, there is increasingly convincing data that high affinity allergen-specific IgG4 may better represent functional “blocking” antibody which may be capable of sequestering allergen before it reaches effector cells (7). However, the use of whole allergen proteins carries a significant risk of adverse events. Allergic adverse events result from conformational epitopes that are able to able to cross-link IgE molecules bound to the surface of effector cells (mast cells and basophils) and can cause both local and systemic allergic reactions. Adverse events lead to dose limitation and a protracted course of treatment that typically lasts about 3 years and requires monthly injections (SCIT), or daily dosing (SLIT). Lack of compliance is increasingly recognized as a major limiting factor with whole allergen approaches and is related to the onerous nature of the treatment regimen. For example, a recent European study found poor overall real-life compliance to SCIT and SLIT, with only 18% of 6486 patients reaching the minimally required duration of treatment of 3 years (SCIT, 23%; SLIT, 7%) (8••).

Numerous strategies have been developed to overcome allergic adverse events in AIT. These approaches primarily involve altering the structure of the antigen to maintain the ability to induce tolerance, but ameliorate the risk of eliciting allergic reactions. A promising approach is the use of synthetic peptide immunoregulatory epitopes (SPIREs) which are short soluble peptides containing the major T cell epitopes of clinically important allergen proteins. This therapeutic strategy delivers the relevant T cell epitopes required for tolerance induction, but does so using peptide fragments that lack the length and conformational structure that would allow them to cross-link IgE molecules on the surface of effector cells.

It is currently believed that the presentation of peptide epitopes to T cells in the absence of pro-inflammatory signals induces a state of antigen-specific immune tolerance. This manifests itself as inactivation of antigen-specific T cells (anergy), skewing of the T helper cell phenotype from Th2 to Th1 and the induction of regulatory T cells which are further able to down-regulate inflammatory immune responses through production of the tolerogenic cytokines IL-10 and/or TGF-β (3, 4).

Peptide Immunotherapy for Cat Allergy

Allergy to cat dander is relatively common; with 17% of the population of the United States yielding a positive skin prick test to cat (9). Almost 30% of allergic asthma in the US is attributable to cat sensitization (10). The application of allergen immunotherapy (AIT) has been shown to ameliorate some aspects of cat allergy (11, 12). Administration of large doses of cat dander, which contains the primary cat allergen Fel d1, results in significant reductions in skin reactivity and airway hypersensitivity in allergic patients (11-12). Despite these positive results, the use of whole allergen in AIT leads to unpredictable anaphylactic reactions in some patients (12). This is primarily due to the fact that whole Fel d1 molecules are able to cross-link IgE molecules bound to effector cells in sensitized individuals. To circumvent this problem, the regions of Fel d1 recognized by T cells of cat-allergic individuals were identified and formulated into peptide-based therapies.

Based on MHC binding studies assessing the binding affinity of candidate T cell epitope peptides (from the sequence of Fel d 1) to the 10 most common HLA-DR molecules in the human population, Worm et al., were able to define seven short peptides (13-17 amino acids; ToleroMune Cat) (13•) with moderate to high affinity binding to multiple MHC gene products expressed by more than 95% of the population. Cellular reactivity and the potential to induce basophil histamine release among the selected peptides was evaluated in cat allergic subjects. The selected peptides were formulated into an adjuvant-free therapeutic preparation and evaluated for safety and surrogate clinical efficacy, via two different administration routes, in a phase 2a clinical trial. Using an incremental dose cohort design, the authors showed that Cat-PAD was well tolerated in doses from 0.03nmol to 12nmol (intradermal administration), or 20nmol (subcutaneous administration) (13•). Based on the ability of a single administration of Cat-PAD to reduce the magnitude of the late-phase skin reaction (LPSR), 3nmol (containing approximately 35μg of Fel d 1 epitopes) and 6nmol doses and intradermal administration were selected to progress into phase 2b clinical studies.

Clinical efficacy of the 3nmol and 6nmol doses of Cat-PAD was subsequently evaluated in two separate, repeat administration, Phase 2b studies performed in an allergen exposure model (14•, 15••). Subjects in both studies were exposed to physiologically relevant airborne levels of cat allergen extract (Fel d 1 concentrations of approximately 50ng/m³) in an environmental exposure chamber (EEC). In the first of these studies, subjects with allergic rhinoconjunctivitis triggered by exposure to cats, attended the EEC for four consecutive days and were exposed to airborne cat allergen extract for three hours on each day. EEC challenges were performed at baseline and 18-22 weeks after the initiation of the study. One hundred and twenty one subjects meeting the inclusion/exclusion criteria were randomized to receive placebo or one of four Cat-PAD regimens consisting of either 3nmol or 6nmol doses administered in either four, or eight administrations over 12-14 weeks. Efficacy was assessed in terms of changes to the Total Rhinoconjuncivitis Symptom Score (TRSS) which measured eight symptom fields consisting of both nasal (sneezing, itchy nose, runny nose, nasal congestion) and ocular (itchy eyes, watery eyes sore eyes, red eyes) outcomes. Each outcome field had a score of 0-3 points with “0” representing no symptoms and “3” representing severe symptoms. A baseline TRSS score of at least 10 out of 24 (including 6 out of 12 for nasal symptoms) on at least one time point on days 3 and 4 of EEC exposure was a prerequisite for study entry. Treatment with Cat-PAD was safe and well tolerated in this study. The frequency of treatment-emergent adverse events (TEAE), related and unrelated to treatment, was generally lower in the Cat-PAD-treated arms than in placebo, although in the 6nmol group the frequency of TEAE was higher. Sub-analysis of respiratory system TEAEs showed low incidence of respiratory symptoms in all groups, with the majority of reported events occurring as a result of the EEC exposure to airborne allergen. Administration of eight intradermal injections of Cat-PAD at the 3nmol dose with two week intervals resulted in a greater improvement in symptoms scores than placebo with a mean treatment difference of 2.9 units in TRSS between 1-3 hours on Days 2-4 (pre-specified primary outcome) of the EEC challenge (14•).

More recently, Patel and colleagues compared the 3nmol and 6nmol dosing schedules in a similar study design utilizing an EEC (15••). In addition to the post-treatment efficacy outcomes measured at 18-22 weeks, efficacy was also evaluated one year after the initiation of the study, nine months after cessation of treatment. Here, as before, treatment with Cat-PAD resulted in improvement in the total rhinoconjunctivitis score (TRSS) at both the short-term (18-22 week) outcomes. Two hundred and two subjects were randomised to one of three regimens (8x3nmol 2 weeks apart; 4x6nmol 4 weeks apart with infill placebo to maintain blinding; or 8 x placebo). Of these, 174 subjects completed at least one post-treatment EEC visit and these comprised the intention to treat (ITT) population. One hundred and seventy subjects completed the first phase of the study such that they had EEC data on days 2-4 of the post treatment challenge. At 18-22 weeks the mean change in TRSS in Cat-PAD-treated groups exceed that of the placebo group (8x3nmol -5.136; 4x6nmol -5.406; placebo -2.786), with both Cat-PAD treatment arms performing similarly.

In a protocol extension, in which investigators and participating subjects remained blinded to treatment allocation, all subjects were invited to participate in a one year follow-up study. As a result, eighty-nine subjects were re-enrolled in a follow on study and returned to the EEC, at 50-54 weeks, for a further repeat of the EEC challenge of four days of allergen exposure. The aim of the extension was to assess continuing efficacy following a period of nine months without further treatment. Treatment with the 6nmol dose of Cat-PAD resulted in persistent efficacy. In the nonasthmatic population (pre-defined primary outcome measure), the 6nmol dose resulted in a decrease in mean TRSS from baseline of -6.778 points in comparison with the 3nmol dose (-3.893) and placebo (-2.908). The change in TRSS score observed with 6nmol was significantly different to both the 3nmol dose (p=0.03) and placebo (p=0.01). Similar results were obtained when a pooled analysis of asthmatic and non-asthmatic subjects was performed. Interestingly, the improvement in TRSS at the 50-54 week outcome appeared to be greater for the 6nmol group than at 18-22 weeks. Analysis of total nasal symptom scores (TNSS) in the non-asthmatic population demonstrated statistically significant decreases in mean change from baseline TNSS for the 6nmol dose (-3.435) and placebo (-1.625; p=0.02 for 6nmol vs placebo). Similarly, total ocular symptom scores (TOSS) showed a larger decrease in mean change from baseline TOSS for the 6nmol dose (-3.343) when compared to both 3nmol (-1.716; p=0.03 vs 6nmol) and placebo (-1.283; p=0.01 vs placebo) (15).

In a further extension of this study, 51 subjects were re-enrolled, with study subject and investigator blinding maintained. Subjects were returned to the EEC for a further four consecutive days of allergen exposure (102 to 106 weeks after the initiation of the study). No further treatment with Cat-PAD or placebo was administered between the initial course of treatment and the two year EEC outcome. A greater improvement was observed in TRSS in the 6nmol Cat-PAD group (mean reduction in TRSS from baseline -5.87) compared to placebo (mean reduction in TRSS from baseline -2.02), with a mean treatment effect on TRSS between the two groups of approximately 5 units at the end of fourth day of EEC challenge (16).

In a post hoc analysis of peak nasal symptoms (Total Nasal Symptom Score; TNSS), nasal symptoms were found to peak between two and three hours on each of the four EEC challenge days. Comparison of the least squares mean TNSS between groups on all 4 EEC visits demonstrated that symptoms were significantly (p<0.05) lower in the 6 nmol Cat-PAD group compared to placebo at the 2 year follow up. No significant difference was observed at two years between the 3nmol treatment group and placebo (17).

In order to examine the consistency of the treatment effect of Cat-PAD at the level of individual study subjects, a post-hoc analysis was performed to evaluate the correlation between the change from baseline in mean TRSS on days one to four at the 18-22 week outcome, versus the change at the later (50-54 week and 100-104 week) outcomes. For the 89 subjects who returned at 50-54 weeks the treatment effect for each individual correlated strongly with their response at 18-22 weeks (regression slope=0.85; correlation coefficient=0.74). Similarly, for the 51 subjects who returned for the 100-104 week outcome, the treatment effect correlated strongly (correlation coefficient=0.89); indeed the slope of the regression curve (1.36) suggested an increased treatment effect at this follow up (18).

House Dust Mite (HDM)

Based on in silico and in vitro analysis of the ability of peptide sequences derived from major house dust mite allergens to function as synthetic peptide immune-regulatory epitopes (SPIRE), seven short peptides were selected and formulated for intradermal administration. An escalating dose, phase 2a study was employed to evaluate first-into-man safety/tolerability and to identify active doses of HDM-SPIRE to be advanced into phase 2b studies (19). Subjects with a history of allergic rhinoconjunctivitis triggered by HDM were recruited to a randomized, double-blind, placebo-controlled trial of HDM SPIRE performed in an EEC challenge model with each challenge period consisting of three consecutive days with four hours of airborne allergen exposure (D. pteronyssinus extract 20-120ng/m³) (20, 21). Baseline TRSS was assessed in the EEC prior to treatment with placebo, or one of three HDM-SPIRE regimens (11 intradermal administrations of 3nmol, 11 × 6nmol, or 11 × 12nmol; all administrations at 4 week intervals). In the initial phase of the study 20 subjects were recruited per treatment arm. Subjects returned for EEC challenges at weeks 19, 36 and 49-50. At the 19 week time point, following the fourth of the eleven administrations (i.e. after 4 × 12nmol HDM-SPIRE), a pre-specified interim analysis of efficacy was performed in order to identify the most efficacious dose for further study. In the second phase of the study the 11 × 12nmol HDM-SPIRE and placebo groups were expanded to 44 subjects. The interim analysis at 19 weeks showed a mean change in TRSS of -3.97 in the 12nmol HDM-SPIRE group compared to -1.83 in the placebo group. As a result of these positive results, a 4 × 12nmol regimen was also evaluated in the second phase of the study in an expanded number of subjects (n=44). At the 49-50 week EEC visit, the 4 × 12nmol group showed a mean change in TNSS of -5.82 compared to placebo at -3.04 (p=<0.05). A subjective global assessment of symptom improvement revealed that 68% of subjects treated with 4 x 12nmol HDM-SPIRE scored “any better” compared to 44% of the placebo group (p=0.05). Treatment with the full 11 injection regimen proved no more effective than 4 administrations (20, 21).

Grass Allergy

The efficacy of Grass-SPIRE, consisting of seven immunodominant T cell epitopes from several grass species, was evaluated in an Environmental Exposure Unit (EEU) model in which Subjects were exposed for four consecutive days (3 hours per day) to mean circulating rye grass pollen counts of 3500+/- 500 grains/m³. EEU visits occurred at baseline (approximately four months prior to the grass pollen season) and approximately 25 weeks after the initiation of Grass-SPIRE therapy. TRSS was recorded at 30 minute intervals on a scale of 0-24 encompassing eight nasal and ocular symptoms fields. At the end of the final EEU visit, subjects completed a seven point global assessment of changes in symptomatology comparing the baseline and post-treatment EEU challenges. Two hundred and eight subjects were randomized to one of three Grass-SPIRE dosing regimens. Subjects treated with 8x6nmol Grass-SPIRE (two week dosing interval) showed a mean change in TRSS of -5.4 in the protocol-specified primary endpoint (all time points on days 2, 3 and 4 in participants with a mean baseline TRSS>8 points), in comparison with a mean change of -3.8 in the placebo group. The protocol-specified secondary endpoint (change in mean TRSS at EEU visit 25 weeks after randomization in subjects with baseline TRSS>12) revealed a greater treatment effect with Grass-SPIRE (-5.3 versus placebo -3.4; p=0.05) (22). Changes in nasal symptoms scores were also apparent in this study. The 8x6nmol group showed a change in mean TNSS of -2.3 versus placebo (-1.6; p<0.05). Furthermore, evaluation of total non-nasal symptom scores (TNNSS) showed a statistical trend for a greater treatment effect in the 8x6nmol group (-3.1) versus the placebo group (-2.2; p=0.08). Forty four percent of subjects in the 8x6nmol group considered themselves very much better after treatment, compared with 22% of subjects receiving placebo (p<0.01) (23).

Conclusion

Although further well designed clinical trials are required (Phase 3 with Cat-PAD is nearing completion at the time of writing this article), peptide immunotherapy is emerging as a promising treatment option for allergic diseases. By using synthetic peptide immunoregulatory epitopes (SPIRE), functional immune tolerance can be established through a combination of mechanisms including immune deviation (Th2 to Th1) and the generation of regulatory T cell that appear to exert their suppression of allergen-specific immune responses via the actions of IL-10. Growing evidence that patient compliance with both subcutaneous and sublingual allergen immunotherapy is poor, highlights the need for a rapid, safe treatment modalities that relieve symptoms for extended periods of time. The studies described here demonstrate that treatment with as few as four intradermal injections of SPIREs, can provide symptom relief that is sustained for two years. Furthermore, clinical studies to date demonstrate a favourable safety profile for this form of therapy. Further studies of peptide immunotherapy may not only produce a new class of specific disease-modifying therapeutics, but also provide fresh insight into the immunological mechanisms underlying allergy and immune tolerance.