Peptide allergen‐specific immunotherapy for allergic airway diseases—State of the art

Allergen‐specific immunotherapy (AIT) is the only means of altering the natural immunological course of allergic diseases and achieving long‐term remission. Pharmacological measures are able to suppress the immune response and/or ameliorate the symptoms but there is a risk of relapse soon after these measures are withdrawn. Current AIT approaches depend on the administration of intact allergens, often comprising crude extracts of the allergen. We propose that the challenges arising from current approaches, including the risk of serious side‐effects, burdensome duration of treatment, poor compliance and high cost, are overcome by application of peptides based on CD4+ T cell epitopes rather than whole allergens. Here we describe evolving approaches, summarize clinical trials involving peptide AIT in allergic rhinitis and asthma, discuss the putative mechanisms involved in their action, address gaps in evidence and propose future directions for research and clinical development.


| INTRODUC TION AND A HIS TORIC AL TRIBUTE
The concept of allergen-specific immunotherapy (AIT) was described over a hundred years ago for treatment of grass polleninduced hayfever. Bostock in 1819 delineated hayfever as a seasonal illness characterized by airway catarrh, and Dunbar in 1903 associated it with "pollen toxin". 1 Dunbar described the muco-cutaneous symptoms in hayfever and that injection of "pollen toxin" in animals induced production of neutralizing antibodies. In a landmark publication in The Lancet in 1911, Noon elegantly described that prophylactic pre-seasonal inoculation of "pollen toxin" induced "active immunity" against hayfever. 1 The concept of allergen challenge, then described as "sensitiveness," and how this was altered following repeated inoculation of "pollen toxin" was ingeniously employed to obtain an objective read out of therapeutic benefit. A standardized pollen extract was manufactured by Dunbar's method that involved extraction in distilled water, by repeated thawing and freezing. 1 The potency of different pollens was compared with a conjunctival challenge procedure, and it was established that Phleum pratense was most potent among different grass species in England. Noon developed a protocol of incremental subcutaneous inoculation of "pollen toxin" at regular intervals and discovered enhancement of "resistance" as evidenced by a "conjunctival challenge". 1 Noon administered a relatively small starting dose as 1/3rd of the minimal dose that elicited a conjunctival response to challenge.
This work was carried forward by Freeman 2 who inoculated 20 patients with pollen extract and described therapeutic efficacy in 16 patients. Frankland and Augustin conducted the first "controlled" clinical trial for hayfever and seasonal asthma and showed efficacy of "crude" and "purified pollen extracts". 3 The fundamental principle underpinning AIT has not changed since it was first described in 1911, but great strides have been made with respect to standardization, purification and optimization of treatment protocols in order to maintain safety and maximize therapeutic benefit. The aim of AIT is to reduce allergic mucosal inflammation by induction of immune tolerance, thereby alleviating symptoms and improving health-related quality of life.
Administration of crude allergen extracts for AIT is associated with a risk of provoking an immediate (type-1) hypersensitivity reaction due to recognition of antigen/s or allergen by specific IgE on the surface of mast cells and basophils. Hence, efforts have focussed on development of novel approaches involving administration of modified allergens (recombinant allergens or peptides) to reduce/ circumvent IgE binding, that is reduce allergenicity but maintain immunogenicity.
An ideal AIT product should meet the following criteria: costeffective, minimal allergenicity and maximal immunogenicity and tolerogenicity, needing few doses or a short treatment course with ease of administration (preferably self-administration unsupervised), providing long-term therapeutic efficacy and importantly carrying minimal side-effects in terms of provoking local and systemic allergic reactions.

| Treatment modalities
The two main modalities currently employed world-wide for AIT in allergic rhinoconjunctivitis (ARC) include subcutaneous injection immunotherapy (SCIT) and sublingual immunotherapy (SLIT). As per current British and European guidelines, these treatments are offered to patients with moderate-severe ARC that is either unresponsive or partially responsive to standard pharmacotherapy. 4,5 An important recent development has been the recommendation by the European Academy of Allergy and Clinical Immunology (EAACI) and Global Initiative for Asthma (GINA 2020; https://ginas thma.org/wpconte nt/uploa ds/2020/06/GINA-2020-report_20_06_04-1-wms. pdf) regarding use of AIT in house dust mite-driven allergic asthma. 6 HDM SLIT tablets have been recommended for adults as an "add-on" or adjunct therapy in those with controlled or partially controlled disease 6 with an aim to reduce acute attacks. HDM SLIT drops have been similarly recommended for children with well-controlled disease in order to reduce medication use and better control of asthma symptoms. Similarly, HDM SCIT has been recommended for adults with well-controlled disease for better symptomatic control and reduction of medication usage. 6 It has been acknowledged by the authors of EAACI guidelines that the quality of evidence, however, is fairly limited at present with respect to use of AIT to treat chronic asthma. Well-designed studies are needed to delineate the role for AIT in the context of mild-moderate asthma endotypes 7 (type-2 "high" and non-type-2 or type-2 "low") based on predominant underlying inflammatory pathways, although it is likely to be more effective in type-2 "high."

| Safety aspects
Safety remains an important concern, as uncontrolled asthma is an important risk factor for fatal and near fatal systemic allergic reactions to AIT. Hence, patient selection is paramount, and AIT is restricted to specialist use only in a secondary care setting. As regards UK practice, use of AIT in ARC is a relative contraindication in patients with concomitant mild-moderate well-controlled asthma as per The British Society for Allergy and Clinical Immunology (BSACI) guidelines and an absolute contraindication in those with uncontrolled, severe or brittle asthma. 5 Furthermore, AIT is not recommended for the treatment of chronic asthma per se in the UK practice.
Several clinical trials with different AIT products have highlighted safety and efficacy (short-term and long-term) of SCIT and SLIT, particularly for grass pollen, birch pollen, ragweed and house dust mite. [8][9][10][11][12] They involve administration of a standardized intact whole allergen extract, although modified allergens (allergoids and polymerized extracts) have also been shown to be safe and effective. SCIT formulations include aqueous and depot (aluminium hydroxide or tyrosine to enhance immunogenicity) preparations. SLIT is available as dissolving tablets or drops. Systematic review and meta-analysis of SCIT and SLIT for ARC have shown that they have a modest/moderate therapeutic benefit. 11,12 Whilst mild oropharyngeal reactions are relatively frequent and commonly associated with SLIT, systemic reactions and anaphylaxis are very rare. 4,5 SCIT is associated with the risk of systemic reactions and anaphylaxis, and rarely fatal anaphylaxis could occur. 4,5,[13][14][15] The SCIT national surveillance project from North America reported 7 fatalities between 2004 and 2017 out of 54.7 million injection visits. SCIT-associated systemic reactions occurred in 0.1% of injection visits, involving varying severity with greater risks associated with accelerated (rush and ultra-rush) protocols. 15 Hence, preference is to employ a conventional up dosing protocol involving 12 weekly visits, particularly in the UK, where most whole allergen extracts for SCIT are currently unlicensed. 5

| Practical aspects
There are recognized logistic issues with SCIT and SLIT. 4,5 SCIT should be administered under specialist supervision and in secondary care with access to critical care should anaphylaxis occur. Hence, there are overhead expenses attached to administration of SCIT.
It involves considerable time commitment for patients with multiple visits and with a waiting time for an hour following each injection. SLIT, on the other hand, is initiated in hospital, and patients are trained for self-administration. Standard recommended duration of both treatments is 3 years. Those failing to show therapeutic response can be identified early and treatment may be withdrawn.
Compliance to treatment has been highlighted as a concern in a reallife setting. A study from The Netherlands in 6486 patients (2789 SCIT and 3690 SLIT) 16 reported an overall 18% compliance with respect to completion of the 3-year course of standard treatment (median 1.7 and 0.6 years, respectively, for SCIT and SLIT). One of the predictors for discontinuation was the prescriber, with patients more likely to persist with treatment with a general practitioner than with a specialist.

| Other AIT modalities
Other approaches that have shown some promise include AIT with adjuvants (TLR-4 agonist, monophosphoryl lipid A; TLR-9 agonist, bacterial DNA oligonucleotides containing a CpG motif), combination of AIT with omalizumab, recombinant Bet v 1, recombinant B cell epitope-based vaccine, comprising a recombinant hybrid grass allergen mix conjugated with a hepatitis B domain surface. [17][18][19][20][21][22][23] Other routes of administration have also been attempted including intradermal, epicutaneous and intralymphatic [24][25][26] routes. There have been several studies during the last 2 decades investigating efficacy and safety of short and long contiguous overlapping peptides (COPs) targeting dominant T cell epitopes of major allergens as an alternative to employing intact or whole allergens for AIT.
Long COPs represent fragments of overlapping peptides covering the entire sequence of respective allergen, thereby preserving the relevant peptides for T cell recognition, but carrying an advantage of lacking conformation of the whole molecule to prevent IgE binding on the surface of mast cells of basophils. Similarly, short peptides, generally comprising single T cell epitopes, are usually 8-20 amino acids. Selection of the correct sequence and optimizing the length of peptide is critical to safety, success and cost of peptide AIT.
The main aim of this review is to critically appraise AIT involving peptide-based treatment for ARC. We will review clinical trials that have investigated the therapeutic efficacy and safety of peptide AIT for ARC, challenges associated with this modality, and describe putative mechanisms and future directions.

| R ATI ONALE B EHIND PEP TIDE IMMUNOTHER APY
Peptide AIT in allergic airway disease rests on two fundamental principles, 27,28 (a) a pivotal role for T cells in orchestrating chronic allergic mucosal inflammation in ARC and asthma and (b) circumventing IgEmediated recognition and cross-linking with allergen on the surface of mast cells and basophils, the mechanism underpinning systemic type-1 hypersensitivity reactions to whole allergen AIT.
Predefined and well-mapped short peptides (SPs) or long COPs of major allergens representing dominant T cell epitopes, with the ability to bind to a vast array of HLA class II alleles, can be administered at regular intervals, either intradermally or subcutaneously to induce allergen-specific T cell tolerance. 27,28 This concept has been also been tested with some success in the context of autoimmune diseases where there are recognized target antigens such as in autoimmune liver diseases, rheumatoid arthritis, type-I diabetes, multiple sclerosis and Graves' disease. [28][29][30][31][32][33][34] Targeting pathogenic T cells in organ-specific autoimmune diseases with T cell epitopes induces antigen-specific regulatory T cells and this strategy is under evaluation in clinical trials.
From a logistical and cost viewpoint, short peptides can be manufactured in a standardized fashion with low production costs and are relatively stable in a lyophilized form at room temperature. 35 Also, cost of production of peptide-based therapeutics is low since mass production of high purity product is achievable. Therefore, as soon as development costs are recovered, we predict that treatments for common allergic disorders will be inexpensive.
Efficacy was measured using a standardized clinical scoring system of combined nasal/bronchial and ocular symptom and medication scores, night-time nasal symptom scores, health-related quality of life scores, lung function (in some cases also airway hyperresponsiveness), early-and late-phase skin test response, measurement of biomarkers such as allergen-specific IgE (sIgE), allergen-specific IgG 4 and facilitated IgE binding assays.

| Therapeutic response to peptide AIT
Peptide AIT has been shown to improve clinical responses to allergen challenge either following a natural exposure or during/after a controlled exposure in an Environmental Exposure Chamber (EEC) alongside alteration in immunological parameters with a significant increase in sIgG 4 and reduction in sIgE/IgG 4 ratio. Recent studies have also shown a sustained clinical benefit at years 1-3 following a relatively short course of therapy, alongside persistent immunological signals as described above, albeit with a downward trend from immediate post-treatment metrics. 37,40 Ellis et al 39 reported a phenomenon of a bell-shaped pharmacological response rather than a linear dose-response association with grass pollen peptides, highlighting the importance of dose titration in early clinical trials to inform robust design of phase III trials and beyond. Whilst a cumulative dose of 48 nmol of grass peptides (4 or 8 injections) induced a significant therapeutic benefit, a similar (or expected dose-response benefit) response was not detected with a higher dose of 96 nmol. This is in contrast to SCIT clinical trials involving an alum preparation of Phleum pratense which showed a linear dose-response relationship, that is a maintenance dose of 100,000 SQ. U was superior to 10,000 SQ. U of whole allergen extract in terms of therapeutic efficacy and associated with more frequent local and systemic treatment-emergent allergic reactions. 8

| Safety aspects
Whilst recent studies with grass 39,41,42 and cat 55 peptides have been shown to be relatively safe with respect to grade 3 or 4 systemic reactions, as per the World Allergy Organization (WAO) grading system, others, particularly those involving grass pollen, 38 cat 47,52 and birch pollen 36 have reported relatively more severe early and delayed (>3 h) systemic allergic reactions. Specifically, lower airway symptoms with a decline in FEV 1 (>30%) have been reported, and this has been attributed to a MHC restricted allergen-specific T cell activation. 57 It has been suggested that the late asthmatic response to peptides improves with repeated dosing, 52 but this needs confirmation. Local injection site reactions have been less frequent and relatively less severe.

| Putting evidence into context
In summary, peptide AIT is a promising option as an immunomodulatory and disease modifying treatment for ARC and asthma, but further work is clearly required in well-characterized patients to demonstrate its long-term efficacy and safety. Current recommendations with whole allergen extracts of SLIT and SCIT are that 3 years of treatment is recommended to achieve long-term efficacy. 4,5 Whilst there is preliminary and promising evidence that a short course of peptide AIT for 4-6 weeks confers sustained 37,40,48 clinical benefit at years 1-3, without additional treatment, further studies are clearly warranted to determine long-term efficacy with optimization of dosing regimens, route of administration (intradermal vs subcutaneous), duration of therapy and safety specifically in asthma. Further research is also needed in children in order to determine the role for peptide AIT not only in established allergic airway disease, but also with respect to prevention of asthma and newer sensitizations as reported with whole allergen AIT. 58 Further research to investigate the efficacy and safety of peptide AIT in house dust mite-related ARC and asthma is also needed. Importantly, it would be crucial to have a proportionate representation of Black, Asian and Minority Ethnic population in peptide AIT clinical trials to be able to maintain credibility and generalizability of findings at a global level. Table 1 summarizes key studies in peptide AIT undertaken during the last 3 decades and includes details regarding study design, efficacy and safety. Notably, AIT with Fel d 1-derived peptides showed a stable treatment effect when the clinical response was measured employing an EEC. The significance of this effect however was challenged in a phase 3 study (ClinicalTrials.gov Identifier: NCT01620762; unpublished) in which primary outcome was measured by a combined score [CS =Total Rhinoconjunctivitis Symptom Score (TRSS) + Rescue Medication Score (RMS)] involving natural cat allergen exposure over a 3-week period as opposed to challenge in an EEC, and no significant differences were detected between the treatment and placebo groups. The reason for this study not reaching its desired primary end-point is not clear.
It is plausible that differences in study methodology, specifically method of cat allergen challenge (natural exposure vs EEC) and psychosomatic factors might potentially impact in cat allergy clinical trials. This needs due consideration in future peptide AIT clinical trials which should include a combined clinical cum biomarker approach, as successfully shown in recent studies involving grass pollen peptide AIT.

| Lessons regarding induction of immune tolerance from whole allergen AIT studies
The fundamental question relating to effective AIT is the type of immune modulation required to produce effective tolerance towards the allergen. On the one hand, there is evidence that administration of conventional whole allergen-based immunotherapy leads to generation of allergen-specific blocking antibodies, that is IgG 2 and especially IgG 4 isotypes that compete for binding antigen and block cross-linking of IgE on mast cells. For example, Zhao and colleagues have shown that the IgE-blocking factor induced by AIT correlates with IgG 4 antibodies and a decrease of symptoms in HDM-allergic children. 59 Furthermore, Shamji and colleagues have shown that grass pollen SCIT leads to serum IgG 4 inhibitory antibodies that prevent IgE facilitated allergen binding by B cells and hence presentation to T cells. 60 On the other hand, there is evidence that AIT leads to the deletion, induction of immunological paralysis (anergy) or immune modulation (alteration of cytokine secretion with respect to dampening of Th2 and skewing towards a Th1 or Tr1 phenotype) of allergen-specific T cells. 61 The key question is which form and mode of delivery of the antigenic component of an allergen is safe and effective in

| Peptides vs whole allergen
The advantage of using peptides rather than intact antigens is that peptides generally do not fold into the conformation found in the native antigen. As a result, it is most unlikely that a peptide would cross-link surface-bound IgE antibodies even if the peptide retained a low affinity B cell epitope. Furthermore, this is even less likely with short linear peptides that generally form a random coil state.
The aims for a safe, effective and durable impact of AIT are to: 1. Induce immunological tolerance by administering a preparation that limits the risk of cross-linking IgE and hence causing The only way to prevent IgE binding to a desensitizing agent is to disrupt the conformation of the allergen and its associated B cell epitopes. This can be achieved using allergen fragments generated by enzymatic digestion of the allergen or synthesis of either COPs 36,37,62 or the design of short synthetic peptides (SPs) representing dominant T cell epitopes of the allergen. 63 Here we will discuss the pros and cons of the latter two approaches.

| COPs vs SPs
COPs contain all of the CD4 + T cell epitopes within the specific al-  48 Campbell and colleagues confirmed that this approach generates IL-10 + regulatory T cells capable of "linked" suppression of the response to distinct T cell epitopes within the same allergen. 66 Similar observations have been made in SP-AIT studies of bee venom, 67 grass allergen 39,40 and peanut allergy. 68 The induction of IL-10-secreting T cells in these SP-AIT studies is important since these Tr1 cells are known to promote IgG 4 production. Therefore, the induction of blocking antibodies, the aim of active immunization with whole allergens or COPs, can also be achieved by tolerance induction with SPs.

| Experience from peptide antigen immunotherapy in autoimmune diseases
The clinical trials summarized above are reminiscent of similar stud- Importantly, no unexpected safety signals arose from administration of these SPs. It is interesting to note that a short course of treatment with SPs in ARC has been shown to induce long-term suppression of symptoms, 37,40 whereas the use of SPs produces only short-term benefit in autoimmune disease. 32,69 We propose that this is due to the continued exposure of allergic patients to strong antigens and the generation of memory B cells producing blocking antibodies.
Effective use of SPs for autoimmune diseases will require repeated administration to maintain suppression of the relatively weak response to their self-antigens.

| Mechanisms associated with peptide antigen immunotherapy
The Wraith laboratory has used experimental models to reveal the detailed mechanism of antigen-specific immunotherapy with SPs ( Figure 1). Not all T cell epitopes induce tolerance and it transpires that peptides must bind directly to MHC II on antigen-presenting cells (APCs) to induce tolerance. 70 Recent work has shown that these antigen processing independent T cell epitopes (apitopes)

| FUTURE DE VELOPMENT OF PEP TIDE IMMUNOTHER APY FOR TRE ATMENT OF ALLERGY AND A S THMA
The time required to achieve effective tolerance by AIT for allergic airway diseases is currently months to years. Based on initial results of peptide therapy, however, it appears that tolerance to allergens is achievable with a short course of peptide AIT. Even so, there are likely to be combination therapies that could enhance AIT without disrupting tolerance induction. Whilst the use of steroids can inhibit T cell activation, previous studies have shown that combined dexamethasone and vitamin D3 promotes Tr1 cell differentiation. 79 In addition, antibodies targeting effector arms of the allergic response, including anti-IgE or anti-IL-5, could be given whilst concurrently inducing tolerance by peptide therapy.
Furthermore, it may now be possible to reduce levels of plasma cells using anti-CD38 antibodies, as tested recently in systemic lupus erythematosus. 80 As mentioned above, COPs and SPs depend on fundamentally different mechanisms of active immunization versus tolerization, respectively. It is possible that the effect of COPs could be improved by combination with virosomes. Anergis, the company producing COPs, has announced improved efficacy of their treatment when combined with virosomes from Mymetics, in an unpublished pre-clinical study.
A number of groups are investigating different ways to deliver SPs for treatment of autoimmune diseases. 81 These include presentation by tolerogenic DCs, generated in vitro and then transferred back to the patient; delivery on red blood cells or nanoparticles designed to target the tolerogenic environment of the liver; combination with immunosuppressive drugs in nanoparticles or delivery on MHC coated nanoparticles. Although each of these approaches is tried and tested in pre-clinical models of autoimmune disease, none have yet been designed to treat allergic diseases. Any one of these approaches could improve on currently available approaches.
At present, we know little about the mechanism of action (MoA) of novel AIT approaches being test in clinical trials. We need to define the optimal conditions for induction of tolerance, immune correlates of effective desensitization, kinetics of tolerance, longevity of disease suppression and molecular basis of effective immunotherapy. We now have the tools allowing us to focus on antigen-specific cells isolated from patients 82 using peptide-MHC multimers and high-throughput transcriptional profiling to define MoA. These tools enable us to address the critical questions listed above. Furthermore, we must use precision medicine approaches to define why each patient does or does not respond to treatment and hence allow better patient stratification.
In conclusion, we are now beginning to understand the MoA of conventional and novel approaches to AIT. We propose that peptide AIT will greatly enhance the safety and ultimately compliance levels in those treated and hence improve management of this rapidly increasing group of diseases. A number of distinct approaches are in the pipeline and when proven to be both safe and effective these will greatly improve the armamentarium of the allergist.