Anti-allergen monoclonal antibodies for the treatment of allergies

To tackle the growing allergy epidemic, novel therapeutic approaches are urgently needed. One promising avenue is the development of anti-allergen antibody therapies. This passive immunization approach stands out from traditional allergen immunotherapy by not exposing patients to the allergen but also by potentially treating patients who are less responsive or even unresponsive to allergen immunotherapies and providing immediate protection. After a review of historical and recently published literature on the topic of anti-allergen antibodies, the status quo and recent advancements are presented. Anti-allergen antibody therapies in the context of immunological changes that occur during allergen immunotherapy are also discussed. While the protective role of anti-allergen antibodies was recognized decades ago, the advancement of antibody discovery technologies has fueled the field, and now different anti-allergen antibody therapies are approaching clinical use. These developments have also broadened our understanding of the allergens and allergenic epitopes responsible in different allergic diseases, and also the role played by immunoglobulins in shaping the immune system. The development of anti-allergen antibody therapies offers great potential for the treatment of allergies. Of interest, the efficacy of passive immunization approaches will likely extend beyond allergen neutralization as in the presence of the allergen they might contribute to long-lasting disease modification.


Introduction
With the prevalence of allergies increasing worldwide, there is an urgent need to find novel therapies for allergic patients.Over the last few decades, allergen immunotherapy (AIT) has been an important treatment option in both food and environmental allergies and is the only disease-modifying therapy available to date.However, limitations remain in terms of AIT efficacy, safety, and treatment regimen, which entails that important segments of the allergic patient population do not have access to effective therapies.Thus, the administration of allergen-specific neutralizing antibodies provides a novel, promising treatment for allergies.

Protective role of anti-allergen IgG in allergic disease
One of the main immunological changes during allergen immunotherapy, observed over 50 years ago, is an increase in allergen-specific IgGs, followed by a decrease in allergen-specific IgE levels [1].Elevated levels of anti-allergen IgGs have also been discovered in beekeepers, a population repetitively exposed to the bee venom allergen, albeit protected from venom allergic reactions [2].Based on these observations it has been postulated that allergen-specific IgGs compete with IgEs for binding to the allergen, thus, preventing allergen-mediated cross-linking of allergen-specific IgE bound on basophils and mast cells and hence cell degranulation.One of the first studies to confirm mini-review the protective role of anti-allergen IgGs showed that blood transfusion or sera injections from patients successfully desensitized from hay fever to allergic patients were able to transfer protection [3].Similarly, the plasma of hyperimmune beekeepers allowed bee venom allergic patients to tolerate 1.5-5 times the venom dose that had previously elicited an allergic response [4].While these studies have proven the protective role of anti-allergen immunoglobulins, it was later shown that anti-allergen IgG antibody levels, even though consistently increased in patients undergoing AIT, do not always correlate with successful desensitization [5].It has been speculated that this is due to the different epitope specificity of IgG induced by allergen immunotherapy in successfully desensitized individuals compared to nonresponders.Indeed, functional IgE competition with IgGs induced during AIT has been shown to better correlate with successful desensitization [6].These studies concluded that the formation of blocking antibodies that neutralize the allergen, thereby preventing allergen binding to IgE on allergic effector cells, are crucial for successful disease modification towards allergen tolerance/unresponsiveness and in raising the threshold for allergen reactivity.In line with this, monoclonal antibodies (mAbs) cloned from peanut-allergic patients who showed sustained unresponsiveness after withdrawal from AIT were shown to target slightly different epitopes on the major peanut allergen Ara h 2 than mAbs from patients who showed only transient desensitization [7,8].Immunoglobulin responses are one of the many different immunological changes that occur during immunotherapy, which also include modulation of specific T-cell subsets.For example, in peanut allergic individuals, successful AIT was associated with suppression of T helper cells 2A (TH2A), a subset of T helper cells uniquely found in allergic individuals [9,10].Interestingly T-follicular-cell subsets, important to induce IgE class switch, were shown to be more resistant to changes induced by AIT.These data highlight the challenges of inducing durable immune tolerance that blocks pathogenic IgE formation and increases protective IgGs.In conclusion, IgG/IgE ratios and the increase of allergen-neutralizing IgGs remain the best serological predictors of protection from allergic disease.Thus, the clinical use of anti-allergen neutralizing IgG antibodies has great potential and rationale for being developed as novel therapies.

Advancements in antibody discovery technologies
Early attempts to use anti-allergen antibodies, or allergen passive immunization, were, however, limited in their clinical applicability.Indeed, passive transfer of plasma encounters the same limitations observed for the use of hyperimmune sera in infectious diseases, as seen recently in the treatment of COVID-19: product variability, logistic hurdles, and even more impor-tantly low antigen-specific antibody titers important for effective, lasting antigen neutralization [11].Nevertheless, technological progress in the last decade created the right environment for the development of passive immunotherapies against allergens in a novel, targeted, and controlled way.Particularly important was the booming field of antibody discovery technologies that enabled the efficient isolation, cloning, and expression of allergen-specific antibody libraries [12].
Different technologies have been applied to discover anti-allergen antibodies.For example, animal immunizations have been used to clone such antibodies.This technology was described by Atanasio, Orengo, and colleagues for raising anti-Fel d 1 and anti-Bet v 1 antibodies for cat and birch allergy, respectively [13,14].These researchers used their proprietary platform of transgenic mice harboring human immunoglobulin loci, thus, overcoming the need to humanize antibodies and streamlining antibody development.In an alternative approach, we and other researchers have instead focused on the isolation of anti-allergen antibodies directly from allergic patients.This approach has the added benefit of isolating antibodies against the immunodominant allergens and epitopes that are mediating the disease in patients, thus, increasing the likelihood of isolating functionally neutralizing antibodies.One of the technologies used for patient-derived antibody discovery is phage display, with random heavy and light chain pairings.This approach has led to the isolation, for example, of antibodies from patients vaccinated with hypoallergenic Bet v 1 fragments [15].Recent years have seen an increased use of single-cell technologies applied to B cells.The advantage of B-cell screening is that it maintains the cognate heavy and light chain antibody pairing, preserving those favorable properties that have been selected by the human immune systems in terms of affinity, specificity, and biophysical properties [12].Single B-cell screening technologies have been used to isolate potent neutralizing antibodies against peanut allergens [8,16,17].The concomitant advent of antibody repertoire sequencing technologies has also increased our understanding of antiallergen antibody repertoire diversity and evolution.Interestingly, different studies described that some immunodominant allergenic epitopes force selected V(D)J combinations resulting in convergent evolution of antibody germlines [17,18].

Choosing the memory B-cell source to clone human-derived anti-allergen mAbs
IgE B cells are extremely rare and the current model states that IgE-secreting cells are continuously generated from IgG B-cell precursors in the presence of the appropriate microenvironment [19].This is also supported by the overall similar epitope coverage observed between IgE and IgG from allergic patients, and the clonal relationship between the rare IgE B cells and IgG memory B cells [20,21].Part of the IgE serological memory could come from rare circulating IgE plasmablasts or bone marrow resident long-lived plasma cells [17,22].However, it is likely that the majority of the IgE response occurs in the diseaserelevant tissue by locally switching from IgG memory B cells, for example, in the gut in peanut allergic patients [23].It is still unclear whether the switch from IgG to IgE is mainly driven by extrinsic factors, such as the cytokine milieu, or an intrinsic property of a subset of memory IgG B cells.The recent description of a CD23-expressing memory B-cell subset that is prone to IgE switching supports, at least in part, the importance of intrinsic factors [24,25].The overlap between IgG and IgE memory B-cells repertoires is also corroborated by findings by us and others that antibodies derived from IgG memory B cells are able to compete with a patient's IgE binding to the major allergens [8,16].Overall, in parallel to their therapeutic potential, development of patient-derived anti-allergen therapeutics is also improving our understanding of allergy antibody responses and their clinical relevance.

Neutralizing allergens with antibody cocktails
An important finding driven by the recent developments of anti-allergen antibody therapies is that a limited number of antibodies can significantly compete with most patient-derived IgE, outside the patient pool from which these antibodies were derived, thus, demonstrating the dominance of the targeted epitopes and allergens [13, 14,16].This property allows for the development of anti-allergen antibody therapies, as an excessive number of antibodies would be challenging due to manufacturing, regulatory, and pharmacoeconomic hurdles.It is interesting in this respect that all the anti-allergen therapies being developed so far have been applied to allergies in which one major allergen has been postulated such as cat and birch allergy, with respectively Fel d 1 and Bet v 1 as potentially the unique major allergens driving the disease [13,14].While efforts move to tackle allergies where more than one major allergen has been described, it will be interesting to see how researchers in this field can develop a limited number of monoclonal antibodies while targeting as broadly as possible the disease-driving allergens and epitopes.One approach to circumvent the development and manufacturing of multiple single mAbs is to directly manufacture antibody cocktails in a single bioreactor batch by co-expression from mixture of different cell lines.This production method, which enables the creation of complex antibody mixtures, has recently been utilized to produce a recombinant polyclonal mixture of up to 25 different mAbs [26].By employing this approach, even complex allergies caused by a larger number of allergens and allergenic epitopes could be treated through passive immunotherapy.Yet, this approach remains challenging in terms of chemistry, manufacturing, and controls (CMC) aspects and regulatory requirements.Alternatively, the number of different antibody molecules can be reduced by combining several V regions into bispecific or even multispecific molecules.Over the past few decades, advances in antibody engineering technologies have greatly facilitated the construction of such molecules [27,28].While this approach may significantly reduce production costs and streamline the regulatory process, it may also likely require considerable development efforts.

Anti-allergen antibodies efficacy: beyond simple neutralization
It is not unlikely that another enabling factor in the development of anti-allergen therapies is represented by the increased use and understanding of passive immunization approaches made in infectious diseases.Use of monoclonal antibodies against Ebola and SARS-CoV-2 have showcased the potential of the passive immunotherapy approach.In addition, the use of monoclonal antibodies during the SARS-CoV-2 pandemic has highlighted that antibody efficacy in restricting infection goes beyond simple neutralization and competition with cellular virus receptors [29].Effector function, antigen presentation, and restriction of viral replication are all antibody-mediated mechanisms that work together to prevent pathogen spread [29].Similarly, anti-allergen antibody therapies are expected to exhibit a range of additional modes of action beyond neutralization (Fig. 1), albeit this is likely to remain the main mechanism of action.
These additional modes of action, together with its association with protection from allergic disease in patients, are likely the reason why IgG4 was selected as the backbone of anti-allergen antibody therapeutics.As opposed to IgG1 with effector-incompetent Fc regions often used in other antibody therapeutics [8,13,14,16,17], IgG4 still retains binding to a subset of immunoglobulin receptors.In addition, IgG4 shows lower complement activation, which provides an improved safety profile when compared to effector-competent IgG1 [30].Most importantly, IgG4 maintains binding to the Fcγ receptor IIB (FcγRIIB also known as CD32B) which plays a crucial role in modulating the allergic response.FcγRIIB is the only receptor containing the immune tyrosine inhibitory motif (ITIM) and can suppress mast cell and basophil activation [31].This suppressive activity is thought to be an important mechanism of action of IgG during allergen desensitization.However, it is difficult to dissect the relative contribution of IgG between allergen neutralization or by FcγRIIB engagement in patients.In cellular assays, blocking FcγRIIB has been shown to impair efficacy of anti-allergen antibodies [32].In mouse studies, the evidence is less consistent as it has been both shown that anti-allergen antibod-K Anti-allergen monoclonal antibodies for the treatment of allergies 291 Fig. 1 Proposed modes of action of anti-allergen antibodies in allergic disease prevention and immune modulation.Allergic effector cells, such as basophils and mast cells, bind allergen-specific IgE via the high-affinity IgE receptor Fcε receptor I (FcεRI) on their cell surface.Once allergen-induced cross-linking of IgE on the allergic effector cell has occurred, the cells degranulate and release inflammatory mediators, for instance histamines, leukotrienes and type 2 inflammatory cytokines (IL-13, IL-4), leading to the clinical manifestation of an allergy.Allergen neutralization is proposed to be the main mode action of anti-allergen antibodies with an immediate onset of protection, directly upon the therapeutic administration.Anti-allergen monoclonal antibodies target and neutralize allergenic IgE-epitopes on the surface of the allergens, thus, preventing IgE cross-linking and activation of basophils or mast cells.In addition, the presence of both the inhibitory IgG-receptor FcγRIIB on allergic effector cells and the allergen-specific IgG allows allergen-induced cross-aggregation of the inhibitory Fc(γ)RIIB with the activating Fc(ε)RI receptor, thereby, inhibiting cell activation and degranulation.A consequence of allergen neutralization by anti-allergen antibodies may also be the redirection of antigen presentation by anti-allergen IgG antibodies, which could promote tolerance induction or a state of clinically sustained unresponsiveness.On the one hand, antiallergen IgG antibodies disrupts the formation of allergen-IgE complexes by preventing the binding of these complexes to the low-affinity IgE receptor FcεRII (CD23), which is expressed on antigen-presenting cells such as B cells and dendritic cells.As a result, antigen presentation to allergen-specific T helper 2 cells (TH2 cells) will be blocked, suppressing the type 2 inflammation and IgE production.On the other hand, allergen capture by anti-allergen antibodies could direct antigen presentation towards Fc gamma receptor (FcyR)-carrying cells of the myeloid compartment, which may induce the formation of regulatory T and B cells and promote IgG formation in the absence of type 2 inflammation.Image created with biorender.commini-review ies have decreased efficacy when blocking FcγRIIB but in other instances retain efficacy without a functional Fc, suggesting that at least in certain circumstances neutralization plays a key role [16,33].
However, natural IgG4 are unique in their ability to undergo Fab arm exchange in circulation, which randomly combines two IgG4 halves making IgG4 de facto monovalent and bispecific in patients [34].This monovalency also decreases the likelihood to form immune complexes, which is one of their antiinflammatory properties To avoid having a heterogenous product with uncontrollable Fab arm exchange in patients, the hinge region of therapeutic IgG4 is generally modified to completely abolish Fab arm exchange [35].

Potential immunomodulatory role of anti-allergen antibodies
Regardless of the relative importance of FcγRIIB receptor in driving the therapeutic response to antiallergen antibodies, retaining some effector function might be beneficial for the potential of combining anti-allergen antibody therapies with AIT, i.e., passive and active immunotherapy.This is especially attractive in food immunotherapy for which side effects are significant and a major barrier to their wider uptake.Early approaches utilizing hyperimmune serum tested this hypothesis clinically in both ragweed-allergic patients and honeybee venom-allergic patients.Administration of hyperimmune sera allowed to reach 10-fold higher allergen doses during rush desensitization protocols, while at the same time maintaining induction of allergen specific IgGs [36,37].In addition, Burton and colleagues found that FcγRIIB plays a significant role in inducing desensitization and a tolerogenic T-cell response in a mouse model of combination therapy of anti-allergen antibodies with AIT [38].This effect was correlated with decreased degranulation and suppression of IL-4 secretion likely from FcγRIIB expressing cells, and was concomitant with an increase in anti-inflammatory T regulatory cells.It is possible that another contributing factor to increased tolerance induction in the presence of antiallergen antibodies will be driven by an effect on antigen presentation.Anti-allergen antibodies will block facilitated allergen presentation by B cells through IgE:FcεRII [39,40].On the other hand, anti-allergen IgGs might shift allergen presentation to the myeloid compartment through Fc-mediated receptors which, in the context of a different cytokine milieu, could promote induction of tolerance by engagement of different T-cell subsets.A similar immunomodulatory role of anti-allergen IgGs has been shown for maternal IgG [41].In general, anti-allergen IgG could promote tolerance induction or a state of sustained unresponsiveness, while protecting against side-effects and simplifying AIT clinical protocols, as observed in the earlier clinical studies with hyperimmune serum [36,37].

Conclusion
Overall, fueled by novel antibody discovery technologies, anti-allergen antibody therapies are now being developed that are likely to change the therapy landscape of allergies as they progress through clinical testing.Anti-allergen antibody therapy, or passive immunization, has the potential to quickly and safely protect against allergic reactions, while reaching a bigger population of allergic patients currently excluded from AIT.In addition, the contribution of anti-allergen antibodies, when combined with the allergen or with AIT, in inducing tolerance offers a unique opportunity to immediately protect patients while working on permanent disease modification.