Research advances in mast cell biology and their translation into novel therapies for anaphylaxis

Abstract

Anaphylaxis is an acute, potentially life-threatening systemic allergic reaction for which there are no known reliable preventative therapies. Its primary cell mediator, the mast cell, has several pathophysiologic roles and functions in IgE-mediated reactions that continue to be poorly understood. Recent advances in the understanding of allergic mechanisms have identified novel targets for inhibiting mast cell function and activation. The prevention of anaphylaxis is within reach with new drugs that could modulate immune tolerance, mast cell proliferation and differentiation, and IgE regulation and production. Several FDA-approved drugs for chronic urticaria, mastocytosis, and cancers are also being repurposed for the prevention of anaphylaxis. New therapeutics have not only shown promise in potential efficacy for preventing IgE-mediated reactions, but in some cases, they are able to further inform us of mast cell mechanisms in vivo. This review summarizes the most recent advances in the treatment of anaphylaxis that have arisen from new pharmacologic tools and our current understanding of mast cell biology.

INTRODUCTION:

Definition, impact, and overview of current treatments

Anaphylaxis is an acute systemic allergic reaction which is rapid in onset and potentially life-threatening. Clinical manifestations of anaphylaxis occur within minutes or up to a few hours after exposure to a provoking agent and typically include cutaneous symptoms (urticaria, angioedema, erythema, and pruritus), respiratory distress, hypotension, and/or gastrointestinal symptoms (1–3). Activation of mast cells and basophils and consequent release of vasoactive mediators are generally believed responsible for these presentations, although alternative mechanisms involving other cell types have also been described (4–7). The specific signs, symptoms, and severity of the reactions vary widely between individuals, making universal diagnostic criteria challenging (3). The prevalence of anaphylaxis in the general population has been estimated at 2% to 5% in developed countries, although this is likely an underestimation due to under-recognition of anaphylaxis combined with the low sensitivity of confirmatory testing. Nonetheless, the socioeconomic impact of these reactions is significant given the number of overall cases per year, anaphylaxis-related hospitalizations (>20 per million), and the risk of death, all of which underscore the need for effective immediate and long-term management strategies (1, 3, 8).

The first-line medication for immediate treatment of anaphylaxis is epinephrine, which is usually injected intramuscularly at the onset of symptoms, regardless of the cause of the reaction or diagnostic criteria (1, 3). Epinephrine acts through several subtypes of adrenergic receptors to induce vasoconstriction, increase heart rate and contractility, and cause bronchodilation, thus counteracting the edema and hypotension of anaphylaxis (9). Additionally, epinephrine has been reported to prevent further release of mediators from mast cells and basophils that promote anaphylaxis (10). Although they have not been shown to have a reliable impact on mortality, antihistamines and/or glucocorticoids are also commonly used in emergency departments as secondary treatments and to prevent potential biphasic anaphylaxis (3). Unfortunately, even with swift and comprehensive medical treatment after an episode, anaphylaxis can still be fatal.

Preventive and long-term management of anaphylaxis includes patient education and avoidance of triggers (3, 8). Additional research efforts over several decades have provided additional insights into mechanisms and pathways leading to anaphylaxis, tolerance breakdown in allergic individuals, and the role of risk factors and cofactors, engendering novel initiatives for the prevention of anaphylaxis. Furthermore, continuing advances in the understanding of the regulation of mast cell proliferation and activation through specific receptors and downstream signaling pathways has allowed the identification of novel target molecules for study. Targeting of such molecules is geared towards reducing numbers of effector cells in anaphylaxis, their activation, or the actions of the released mediators (11, 12).

Mechanisms leading to anaphylaxis

The most common mechanism causing anaphylaxis involves the presence of IgE antibodies in sensitized individuals that specifically recognize an antigen or allergen. Among these, food allergens are the predominant triggers of anaphylaxis, particularly in children, accounting for >50% of reported cases, but certain antibiotics and other medications, latex, and stinging insect venoms are also common (3, 7). Allergen-specific IgE binds to the high-affinity receptor FcεRI predominantly present on the surface of basophils and mast cells. Recognition of allergen by its specific IgE after an exposure causes the aggregation of FcεRI which initiates a signaling cascade driven by tyrosine kinases, lipid and protein kinases, and adaptor proteins (Figure 1). Such complex signaling events culminate in the immediate release of preformed mediators such as histamine and proteases by mast cells and basophils, and the synthesis and release of additional inflammatory mediators, including prostaglandins, leukotrienes and cytokines. Together, these molecules cause vasodilation, edema, bronchospasm, hypotension, and tissue inflammation (2, 4, 13).

Figure 1: Emerging anaphylaxis therapies and their targets in mast cells.

Emerging treatments for the prevention of anaphylaxis target several mast cell pathways, including the IgE/FcεRI pathway, KIT signaling, and inhibitory receptors. A) Engagement of the inhibitory receptors CD32b or Siglec-8 with allergen-specific IgG mAbs or lirentelimab, respectively, recruits inhibitory phosphotyrosine phosphatases (SHP-1; phosphotyrosine phosphatase-1) and lipid phosphatases (SHIP; inositol polyphosphate-5-phosphatase) to suppress multiple pathways of cellular activation. B) The IgE-FcεRI pathway is a target for several biologics and tyrosine kinase inhibitors (TKIs). FcεRI engagement leads to LYN-mediated spleen-tyrosine kinase (SYK) activation, which promotes the assembly of multiprotein complexes by phosphorylating the adaptor protein linker of activating T cells (LAT). Phosphorylated LAT acts as a localization site for the adaptor protein GRB2 and the guanine nucleotide exchange protein SOS, the phospholipase Cγ₁ (PLCγ₁), and SH2-domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), among others. FYN phosphorylates GRB2 to recruit and activate the phosphatidylinositol 3-OH kinase/protein kinase B (PI3K), which in turn recruits Bruton’s tyrosine kinase (BTK) through its pleckstin homology (PH) domain. BTK contributes to the activation of PLCγ, resulting in activation of protein kinase C (PKC) and release of calcium from intracellular stores. GRB2/SOS initiate the RAS-RAF-mitogen-activated protein kinase (MAPK) pathway. PKC, calcium, the PI3K and the MAPK pathway orchestrate degranulation, cytokine production and eicosanoid synthesis. Additional interactions are depicted. The anti-IgE mAbs omalizumab and ligelizumab reduce free IgE and surface FcεRI expression, whereas small molecule TKIs of BTK or SYK globally inhibit FcεRI signaling in response to allergen. C) Targeting stem cell factor (SCF) or KIT inhibits mast cell growth and survival. After binding to SCF, KIT dimerizes and transphosphorylates tyrosine residues in KIT. Phosphotyrosine residues become docking sites for the recruitment of PLCγ₁, PI3K, LYN, FYN, GRB2, SHC, and SHP-1. PI3K-regulated pathways serve to promote mast cell growth, differentiation, survival, migration, adhesion, and cytokine production. Similarly, activation of the MAPK pathway following SOS- and VAV-regulated GDP–GTP exchange of RAS, and activation of STATs via Janus Kinase (JAK) also contribute to these processes. Blockade of KIT intrinsic tyrosine kinase activity with TKIs such as avapritinib, imatinib, midostaurin, or the mAb CDX-0159 prevents mast cell proliferation and survival as well as any potentiation of FcεRI activation. Black arrows denote activation. Red dotted arrows with blunted end denote inhibition. Image created with BioRender.

Apart from IgE, anaphylaxis may be caused by IgE-independent etiologies (4). Evidence from mouse models or cases of anaphylaxis in humans have demonstrated or suggested, respectively, that presence of antigen-specific IgG and IgG-receptor (FcγRI) bearing cells (i.e., neutrophils, macrophages, basophils and mast cells) may be involved in certain cases of anaphylaxis (4, 6, 7). In addition, formation of IgG immunocomplexes, such as after administration of blood products or after infusion of clinical dextran, can activate the complement cascade resulting in the generation of the anaphylatoxins C3a, C4a and C5a (5). These anaphylatoxins are capable of inducing mast cell, basophil, and neutrophil activation, wheal-and flare reactions in healthy volunteers, and enhance IgE-mediated cutaneous anaphylaxis in mice (4).

Additionally, anaphylaxis has been reported to be caused by non-immunological or immunocomplex-independent reactions such as those described for radiocontrast agents, narcotics, sulfiting agents, non-steroidal inflammatory drugs (NSAIDS), and low molecular weight molecules. The mechanisms involving some of these triggers are not completely understood or are under debate (4–7). Recent evidence has implicated the Mas-related G-protein coupled receptor X2 (MRGPRX2) expressed on mast cells in anaphylactic reactions to agents such as peptidergic drugs, neuromuscular blocking drugs used in surgical anesthesia (e.g., atracurium, mivacurium, tubocurarine), opioids, and antibiotics of the fluoroquinolone family (14–16). It has also been reported that three types of antimicrobials (antifungal agents, aminoglycosides, and sulfonamides) can activate human and mouse mast cells via MRGPRX2 and the mouse ortholog Mrgprb2, respectively, and cause Mrgprb2-dependent anaphylaxis in mice (17). However, there is much to be clarified about the involvement of this receptor in human disease and the factors determining differences in the reactions between individuals, given that all connective tissue mast cells presumably express the receptor (6, 18). Antagonists and inverse agonists for these receptors are under development (19, 20).

Regardless of the etiology of anaphylaxis, mast cells are usually at center stage, and thus therapies to intercept their activation or regulate their presence in tissues have been broadly pursued. In this review, we summarize the components of the IgE/FcεRI pathway in mast cells as the major and best understood pathway responsible for anaphylaxis, and the importance of the tyrosine kinase KIT in the regulation of mast cell activity and homeostasis. Knowledge on the complex pathways regulating mast cell activation provides identification of molecular targets for drug development to directly prevent mast cell activation, proliferation, and survival, and thus may result in the mitigation, prevention, and treatment of anaphylaxis. We will discuss the pros and cons of drugs currently in clinical trials that target mast cell activation or reduce mast cell numbers as potential therapeutic strategies for long term management of anaphylaxis.

TARGETING THE IGE/FCεRI PATHWAY:

By far, IgE-mediated responses are the most completely understood mechanisms of anaphylaxis. The high-affinity IgE receptor FcεRI is composed of an α, β, and two disulfide-linked γ chains (21). Upon recognition of a specific allergen, IgE bound to the α subunit induces receptor crosslinking and phosphorylation of the FcεRI β and γ subunits by the SRC kinase LYN, which phosphorylates spleen tyrosine kinase (SYK) (22, 23). Activation of SYK, LYN, and FYN promote the assembly of multiprotein complexes by phosphorylating adaptor proteins that in turn recruit and activate protein kinases, lipid kinases, and phospholipases as initiators of signaling cascades that lead to mast cell activation (Figure 1). Several agents in development target components of the IgE pathway, including IgE, its receptors, and key downstream kinases to prevent anaphylaxis.

Anti-IgE therapies

Given its importance in the pathogenesis of allergy, IgE is a logical target for therapeutics. Anti-IgE biologics effectively reduce availability of allergen-specific-IgEs. Because surface expression of FcεRI on mast cells and basophils is stabilized by its binding to IgE, anti-IgE therapeutics have the added benefit of downregulating FcεRI, rendering cells less sensitive to activation in the presence of allergen (24–27). Talizumab was the first anti-IgE biologic to demonstrate that reducing peanut-specific IgE levels would raise peanut threshold doses during oral food challenge (OFC) (28). Though its development was terminated, its efficacy in early trials encouraged the development of other IgE-targeting biologics including omalizumab and ligelizumab.

Omalizumab is a humanized monoclonal antibody (mAb) which binds to the IgE Cε3 domain, thus blocking binding of circulating IgE to FcεRI and to the low-affinity IgE receptor FcεRII (CD23) (29, 30). In this way, it effectively reduces free IgE and surface FcεRI expression on circulating basophils (26, 27, 31, 32). Currently FDA-approved for asthma and chronic spontaneous urticaria (CSU), it is also under investigation for food allergy. As an adjunct therapy for food oral immunotherapy (OIT), omalizumab treatment provides significant protection against adverse events, especially serious adverse events, during OIT build-up (33–36). As a monotherapy, results from early clinical trials show promise in omalizumab’s ability to reduce clinical reactivity to food allergens in food-allergic patients after several doses (several months) of therapy (37, 38). A blinded, placebo-controlled trial demonstrated that omalizumab monotherapy increased peanut-allergic adults’ mean threshold dose 81-fold after 24 weeks of treatment, compared to a 4.1-fold increase in the placebo group (39). Unfortunately, trial data indicates that patients require several administrations (at least 8 to 12 weeks) of omalizumab to achieve an increase in food tolerance, which is in line with the kinetics of its effect on circulating IgE and cell surface FcεRI expression. Additionally, sustained hyporesponsiveness achieved by OIT with adjunct omalizumab therapy may be no more durable than without once both are discontinued (40). Finally, the primary shortcoming with omalizumab monotherapy is the variability in clinical response; while most patients demonstrate substantial increases in their food threshold with omalizumab therapy, some patients do not experience any shift in tolerance. Larger studies will need to determine predictive factors for successful use of omalizumab in food allergy. The phase 3 Omalizumab as Monotherapy and as Adjunct Therapy to Multi-Allergen OIT in Food Allergic Participants trial (OUtMATCH) is currently ongoing at the writing of this review (NCT03881696).

Though several other anti-IgE biologics in recent development have failed to meet primary endpoints in clinical trials or were discontinued for strategic business reasons (41–46), ligelizumab has emerged as a promising next-generation humanized anti-IgE mAb. As a high-affinity derivative of talizumab, ligelizumab may offer a clinical advantage over omalizumab for the treatment of IgE-mediated disorders due to several unique properties. First, ligelizumab has 88-fold higher affinity for IgE as compared to omalizumab. Additionally, it binds to a different epitope in the Cε3 domain of IgE. Binding sites for both omalizumab and ligelizumab overlap those of FcεRI and FcεRII (CD23), though with functionally important differences. Whereas omalizumab’s binding site overlaps more with the CD23 binding site, ligelizumab’s overlaps more with the FcεRI binding site (47). Therefore, it prevents binding of free IgE to FcεRI more effectively than omalizumab, and can also bind to CD23-bound IgE on B cells (47). Through mechanisms that have not been fully delineated, ligelizumab thus appears to be able to prevent new IgE production (47). In phase 1 trials for asthma, ligelizumab has demonstrated greater and more durable reduction of free IgE levels, basophil surface FcεRI expression, and allergen skin prick test (SPT) responses as compared to omalizumab, as well as enhanced protective effects against passive systemic anaphylaxis (PSA) in transgenic human FcεRI mice (47, 48). These properties may translate into superior clinical efficacy for treating allergic disorders, especially in patients with very high IgE levels. Ligelizumab has received FDA breakthrough designation for CSU and is currently in phase 3 trials for CSU and asthma. It is also under investigation as a monotherapy for the treatment of food allergy in a phase 3 randomized, controlled trial in patients age 6–55 with a history of peanut allergy (NCT04984876).

Anti-IgE therapies have consistently shown favorable safety and tolerability data in trials. Of all adverse events, mild-to-moderate injection site reactions are the most common, but anaphylactic reactions were also observed in approximately 0.1–0.5% of patients in clinical trials (49–53). A recent retrospective review of reported cases found that risk factors for omalizumab-induced anaphylaxis are female sex and age 18–44, and that receiving omalizumab for asthma rather than CSU was the biggest risk factor for a life-threatening reaction (54). The mechanism of omalizumab-induced anaphylaxis is unclear, because small studies have shown that omalizumab-specific IgE has not been detected in patients experiencing these symptoms (55). Immune complex formation is the current hypothesized mechanism; though omalizumab cannot directly crosslink receptor-bound IgE, there is evidence that, in rare circumstances, it can crosslink IgE-Fc_3–4:FcεRIα complexes to induce mast cell and basophil activation (30).

However, the consequences of lowering or neutralizing total IgE levels chronically have not yet been fully studied. IgE plays a role in protection against helminths (56), and though the risk of anti-IgE therapies causing susceptibility to parasitic infections is considered negligible in developed countries, one small study showed a potential increased intestinal geohelminth infection rate with omalizumab, though without increase in morbidity or mortality (57). An additional concern is the potential protective function of IgE against cancers (58). Though data from early clinical trials suggested an increased risk of cancer with omalizumab use, a more recent pooled analysis did not confirm this finding, and instead concluded that a causal relationship between omalizumab and malignancy is unlikely (59).

Future therapies targeting IgE aim to disrupt bound IgE as well as the circulating pool. The IgE-FcεRI interaction is one of the highest known affinities in biology at a Kd of 10¹⁰–10¹¹ M−1 (21); therefore, dislocating bound IgE from FcεRI has been difficult to achieve therapeutically in the past. Novel anti-IgE Designed Ankyrin Repeat Proteins (DARPins) are in the preclinical stage and demonstrate remarkable abilities to neutralize free IgE, disrupt bound IgE, and thus prevent IgE-mediated anaphylaxis in mice (60–62). Finally, there is some evidence that anti-IgE therapies could be modestly effective in treating mast cell disorders where a role for IgE is not well-established, including idiopathic anaphylaxis and inducible urticaria (63–66). Further studies are needed to determine the utility of anti-IgE therapies in these contexts.

Tyrosine kinase inhibitors

Several tyrosine kinases in the FcεRI pathway are thought to be indispensable for cellular activation by allergens, including SYK, Bruton’s tyrosine kinase (BTK), LYN, and FYN. Early generation small molecule tyrosine kinase inhibitors (TKIs) were relatively non-selective, and off-target effects produced undesirable toxicities so that their use was largely reserved for malignancies. The relatively recent development of highly selective TKIs has not only confirmed which kinases are essential for FcεRI signaling, but it has also opened the possibility of using these drugs in humans with minimal toxicity. Targeting essential kinases in the FcεRI pathway allows for global blockade of mediator release in an allergen-independent manner. Other advantages of TKIs would include oral dosing, a rapid onset of action, and a relatively short duration of action after cessation of therapy. With short-term, episodic use, TKIs could be useful for on-demand prevention of IgE-mediated anaphylaxis, such as drug desensitizations or adjuvants for food OIT. With chronic use, they may have other disease-modifying actions as described below.

Of the essential kinases in the IgE pathway, inhibition of BTK is the best studied for preventing anaphylaxis in humans. Because BTK is a key component of the B cell receptor pathway, it has been targeted pharmacologically for the treatment of B cell lymphomas and other malignancies and there are now four FDA-approved BTK inhibitors, all of which are oral small molecule compounds. The generally favorable side effect profile, which has improved with newer, more selective inhibitors, has allowed these drugs to be repurposed for the prevention of IgE-mediated anaphylaxis. Ibrutinib and other early-generation, covalent BTK inhibitors have shown rapid and complete prevention of IgE-mediated activation of basophils in vitro (67, 68). Studies using next generation inhibitors including acalabrutinib confirmed that the effect is also seen in human mast cells at roughly the same EC50 concentrations, and that these drugs can also prevent de novo cytokine production after allergen challenge (69). Additionally, just two human equivalant oral doses of acalabrutinib given hours prior to allergen challenge entirely prevented moderate severity of human-IgE-mediated PSA and reduced the rate of mortality during severe PSA in humanized mice that have mature human leukocytes including mast cells (69). Published data using BTK inhibitors to prevent anaphylaxis in humans are promising, though still preliminary. A prospective pilot study in patients who were prescribed ibrutinib to treat their cancer found that aeroallergen SPTs were eliminated while patients were taking ibrutinib (70). A follow-up study showed that ibrutinib therapy eliminated ex vivo IgE-mediated basophil activation and dramatically reduced (or eliminated) food SPT sizes in healthy food-allergic adults without cancer in as little as two doses (70–72). The same investigators have recently completed an investigator-initiated, open-label clinical trial to test acalabrutinib’s efficacy in preventing clinical reactivity in adults with peanut allergy (NCT05038904), with publication of results anticipated in 2023.

Finally, a recent report described the use of ibrutinib to facilitate desensitization to the chemotherapy drug brentuximab-vedotin in a patient with refractory Hodgkin lymphoma who had experienced severe anaphylaxis to his 2^nd cycle of treatment (73). Standard desensitization was unsuccessful, but when the patient was given two 420 mg doses of ibrutinib immediately prior to the next desensitization attempt, the protocol was completed with only a mild urticarial reaction observed after the final step. Baseline BAT to the drug was positive and subsequently abrogated with ibrutinib therapy, supporting an IgE-dependent mechanism for the patient’s hypersensitivity.

The above trials demonstrated no adverse effects after short courses (just a few days) of BTK inhibitors. Chronic use of ibrutinib and acalabrutinib can cause rare but severe side effects including bleeding, cytopenias, infection, and arrhythmias in patients with malignancies. The development of fenebrutinib, another highly selective BTK inhibitor, for the treatment of CSU was halted due to reversible elevations in liver function enzymes during chronic dosing in trials, though this is not a class effect of BTK inhibitors, and fenebrutinib remains under development for multiple sclerosis (74). Nonetheless, chronic use of these drugs for allergic disorders may be possible with next-generation compounds that have higher selectivity for BTK. Encouragingly, the next-generation inhibitor remibrutinib was well-tolerated with few adverse events in chronic dosing during CSU trials (75). Overall, further trials using BTK inhibitors are needed to elucidate appropriate dosage and the onset and duration of clinical protection for use of these drugs to prevent anaphylaxis. To that end, remibrutinib is currently being studied in a dose-finding phase 2 study for preventing clinical reactivity to peanut during OFC in peanut allergic adults (NCT05432388).

As with BTK, SYK inhibition results in complete suppression of IgE-mediated activation of human mast cells and basophils in vitro and PSA in mice (76–82). In a mouse model of peanut allergy, SYK blockade during initial peanut exposure prevented allergic sensitization (79). When administered to mice that were already sensitized, it prevented any observable response to peanut ingestion (79), indicating that inhibitors could be used as adjunct therapies for food OIT. No SYK inhibitors have yet been tested for the prevention of anaphylaxis in humans, but they would likely have similar efficacy and rapid onset of action as BTK inhibitors. Though early SYK inhibitors were too toxic for use in humans, next-generation, more selective inhibitors have demonstrated better safety profiles. Fostamitinib is the only selective FDA-approved SYK inhibitor, approved for immune thrombocytopenia and being tested in other immune-mediated disorders. Side effects include nausea, diarrhea, infections, elevated liver enzymes, hypertension, chest pain, and neutropenia. Other oral SYK inhibitors are in clinical trials for cancers and autoimmune diseases, including tamatinib, lanraplenib, and entospletinib, in addition to the topical SYK inhibitor GSK2646264, which is in trials for CSU and cold urticaria (83). Finally, an intranasal formulation of the SYK inhibitor R112 reduced seasonal allergic rhinitis symptoms in a phase 2 trial without serious adverse effects, though development has been halted as it did not reduce intranasal mast cell mediator release in response to intranasal allergen challenge (84, 85).

Other kinases have been explored preclinically for inhibition of IgE-mediated activation of mast cells and basophils, including FYN, LYN, and the lipid kinase phosphatidylinositol 3-OH kinase (PI3K). Although several oral PI3K inhibitors are in trials for allergic rhinitis and asthma (NCT01066611, NCT00836914, NCT01653756), none are in development for the prevention of anaphylaxis.

Therapies to engage inhibitory receptors

Multiple receptors containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or ITIM-like motifs interact with the FcεRI pathway to dampen IgE-dependent responses, including sialic acid-binding immunoglobulin-like lectins (Siglecs), CD32b, CD200Ra, CD300a, allergin-1, and others (86). Engagement of these receptors recruits phosphotyrosine phosphatases (SHP-1 and SHP-2) and inositol phosphatases (SHIP) which then dephosphorylate a variety of kinases to provide direct negative feedback on the IgE/FcεRI pathway (Figure 1). Though numerous therapies targeting inhibitory receptors are in development, only those targeting Siglecs and CD32b have reached clinical trials.

Siglecs have a relatively restricted expression profile in vivo, including leukocytes, myelinated cells, and placental trophoblasts (87). Multiple Siglecs are expressed by human mast cells, including Siglec-3 (CD33), −6, −7, and −8 (88–90). All of these receptors have been shown to suppress IgE-mediated mast cell activation in vitro (91–95). Among these, Siglec-8 is by far the best characterized in terms of structure and function. Given the finding that engagement of Siglec-8 on eosinophils induces cell death, the humanized, non-fucosylated IgG1 anti-Siglec-8 mAb lirentelimab (formerly known as AK002) was tested in eosinophilic disorders (96). Though it failed to reach primary endpoints in phase 3 trials in eosinophilic gastritis and duodenitis, development shifted towards targeting other allergic disorders mediated by eosinophils and/or mast cells, including atopic dermatitis, allergic conjunctivitis, and CSU (97, 98). While Siglec-8 engagement alone does not induce cell death in mast cells, it does suppress mast cell activation, dramatically reducing histamine release and prostaglandin D2 (PGD2) production in response to IgE stimuli (91). In humanized mice, lirentelimab prevents clinical responses during PSA (99). Based on these properties, lirentelimab was tested in a phase 2 trial in indolent systemic mastocytosis, where it significantly reduced global symptoms over placebo after only 21 weeks of treatment (100). Side effects were largely limited to mild-to-moderate infusion-related reactions, consistent with other lirentelimab trials (96–98). However, no reduction in mast cell burden was observed in patients, despite evidence that lirentelimab can induce killing via antibody-dependent cellular cytotoxicity (ADCC) in vitro (99).

Engagement of Siglec-6 may be of interest in anaphylaxis since its expression is largely limited to mast cells and placental trophoblast cells (92, 101), though some recent reports suggest it may be expressed on basophils and monocytes as well (102). Co-crosslinking of Siglec-6 and FcεRI or engagement with anti-Siglec-6 antibodies were effective in preventing PSA in mice (92, 93, 101), suppressing mast cell activation in response to IgE/antigen as well as C5a, IL-33, MRGPRX2 ligands, and toll-like receptor ligands (92, 103–105), and reducing mast cell numbers in humanized mice through antibody-dependent cellular phagocytosis (ADCP) (101). However, no clinical trials have been conducted to examine the ability of anti- Siglec-6 and −8 mAbs in preventing anaphylaxis in humans.

Recent advances in the understanding of mechanisms of immune tolerance have thrown the spotlight on the low affinity IgG receptor FcγRIIb (CD32b) as a pivotal player in tolerance and thus another promising potential target for preventing anaphylaxis. Circulating allergen-specific IgG has been proposed as a protective mechanism against allergy for both natural tolerance as well as tolerance achieved through OIT and is thought to act primarily through its interaction with CD32b (106–108). Crosslinking CD32b with FcεRI by allergen binding to specific IgG and IgE, respectively, inhibits allergen-mediated activation of both mast cells and basophils (109–112). This IgG-mediated inhibition also halts the feed-forward loop which perpetuates Th2 responses and IgE production (113). Furthermore, at higher concentrations, circulating allergen-specific IgG also acts as a competitive binder of allergen to prevent its interaction with FcεRI-bound IgE (114). Taking advantage of these properties, reengineered versions of allergen-specific IgG antibodies with higher affinity for allergen and/or FcγRII receptors are in development for the treatment of allergic disorders. Clinical trials have thus far focused on aeroallergen-specific antibodies for allergic rhinitis and asthma (REGN1908/09, anti-fel d 1; and REGN5713/14/15, anti-bet v 1), showing efficacy in reducing reactivity during nasal allergen provocation in phase II trials (115–117). Furthermore, a single dose of REGN1908/09 significantly prevented reductions in FEV1 during environmental cat exposure in allergic asthmatics on day 8 after single infusion and demonstrated sustained efficacy for 85 days (118). Though several allergen-specific IgG antibodies are in development for the prevention of anaphylaxis to foods and drugs, none have yet reached clinical trials.

An alternative and allergen-independent approach to receptor aggregation has been explored using Fcε-Fcγ fusion proteins or bispecific antibodies to IgG and IgE, which are largely still in the preclinical stage (119–123). Finally, combining CD32b and IgE targeting is a potentially powerful approach to reducing total IgE levels. The humanized anti-IgE antibody AIMab7195 (also known as XmAb7195) was engineered to have very high affinity to both CD32b and IgE and was shown to reduce serum IgE levels essentially to zero in a mouse model, presumably due to efficient clearance of IgE complexes by CD32b on hepatocytes. However, as of the writing of this manuscript we are not aware that results of completed phase 1 trials in rhinitis and atopic dermatitis have been published, and its future development is uncertain. Overall, further research is needed to evaluate the efficacy of targeting CD32b for the prevention of anaphylaxis, especially given its variable expression on mast cells according to tissue location (124, 125).

MAST CELL ERADICATION APPROACHES:

Overview of SCF/KIT pathway and role in mast cell homeostasis and function

The severity of hypersensitivity reactions generally depends on the extent and duration of mast cell responses and is influenced by the number of mast cells in tissues (126), which tend to be elevated in allergic disorders such as asthma, allergic rhinitis, food allergy and atopic dermatitis (126–128). Thus, in addition to inhibition of mast cell activation, another strategy that has been considered for prevention of allergy and anaphylaxis relates to decreasing mast cell numbers. Mast cell numbers in tissues are regulated via the recruitment and differentiation of mast cell precursors, and by the engagement of growth factor and cytokine receptors that impact mast cell proliferation, maturation and survival (127–131). Multiple studies have emphasized the preeminent role of stem cell factor (SCF) and its receptor on mast cells, the tyrosine kinase receptor KIT, in the processes regulating mast cell numbers (132). In addition, activation of KIT potentiates mast cell activation by FcεRI (133). The pleiotropic effects of KIT on mast cells thus make this receptor and its ligand desirable targets to reduce mast cell numbers.

As currently understood, activation of KIT normally occurs by binding to SCF, which induces receptor dimerization and activation of the intrinsic KIT tyrosine kinase (Figure 1). KIT tyrosine kinase transphosphorylates tyrosine residues that become docking sites for the recruitment of signaling proteins, thus initiating signaling pathways for proliferation and survival (134). These include the activation of the mitogen-activated protein kinase (MAPK) and PI3K signaling pathways, and the janus kinase (JAK)/ signal transducer and activator of transcription (STAT) pathway (135–137). Other players involved in KIT-mediated proliferation and survival of mast cells include the activation of sphingosine kinases (138) and the glioma-associated oncogene homolog (GLI) family of transcription factors (139, 140).

KIT and tyrosine kinase inhibitors

The approach of reducing mast cell number by focusing on KIT to prevent severe episodes of anaphylaxis is especially timely, given the recent success of KIT-targeting TKIs in treating clonal mast cell disorders and reducing mast cell numbers and activation (141). These small molecular weight inhibitors of KIT include drugs such as imatinib, midostaurin, and avapritinib. Developed by rational drug design to target BCR/ABL, imatinib is a relatively selective drug but interacts with multiple kinase targets, including wild type (wt) KIT, but not with KIT that carries mutations in its tyrosine kinase domain (e.g. D816V). Midostaurin acts against wt KIT, oncogenic variants of KIT found in patients with systemic mastocytosis (KIT-D816V), and several additional oncogenic kinases, while more selective inhibitors such as avapritinib act upon KIT-D816V, to a lesser extent, wt KIT, and a more limited number of additional kinase targets.

Available KIT-targeting TKIs vary in their ability to limit SCF-dependent and IgE-mediated mast cell activation as well as in their toxicity profiles. In some instances, KIT-targeting drugs are degraded in vivo into metabolites of interest. Midostaurin, for example, is degraded into two major metabolites, one of which blocks SYK and thus IgE-mediated histamine release, and one of which impacts KIT and KIT-downstream signaling to inhibit the growth of mast cells (142). Therefore, newer and highly specific KIT-targeting drugs may lack activity against additional oncogenic kinases and FcεRI-downstream signaling molecules and thus are less able to suppress mast cell activation.

Importantly, only TKIs targeting KIT-D816V effectively suppress the growth and survival of mast cells and mast cell lines exhibiting the D816V mutation (143). For example, administration of avapritinib, which inhibits KIT-D816V, leads to a rapid decrease in neoplastic mast cell burden in patients with systemic mastocytosis (141, 144). Midostaurin, which also reduces mast cell burden, is reported to induce a rapid improvement of mediator-related symptoms (145), possibly due to its impact on SYK activity in mast cells, and thus on IgE-dependent and -independent mast cell activation and mediator secretion (145–147). In a phase 2 trial, midostaurin improved refractory symptoms caused by mediator release and thus the quality of life in more than 70% of all patients with indolent systemic mastocytosis (148). At high concentrations, avapritinib similarly exerts some inhibitory effects on mast cell activation and mediator release.

However, when considering mast cell reduction to treat anaphylaxis, there is limited clinical information about the ability of KIT-targeting drugs to reduce normal tissue mast cell numbers. In one report, imatinib was administered over several years to patients with chronic myeloid leukemia (CML). It was found that numbers of mast cells decreased after 12 months, and after 24 months, mast cells were almost completely absent in bone marrow sections (149). In concert, serum tryptase levels decreased to low or undetectable values. These observations strongly suggest that a potent wt KIT inhibitor, like imatinib, can eradicate normal mast cells when administered over time (149).

To date, there is also limited information about the efficacy of KIT-specific or multi-targeted TKIs in the management of patients with IgE-dependent allergic inflammation. In one study, masitinib was administered to patients with severe corticosteroid-dependent asthma. After 16 weeks, oral corticosteroid therapy could be reduced to a greater degree in patients in the treatment arm compared to the placebo group (150). Lung function parameters, however, did not change compared to placebo. Reported side effects included nausea, skin rash, peripheral edema, diarrhea, vomiting, fatigue, and pruritus. In another study, patients with poorly controlled or uncontrolled severe asthma with airway hyperresponsiveness despite maximal medical therapy were treated with imatinib for 24 weeks (151). This was found to reduce airway hyperresponsiveness more effectively in patients compared to placebo. Mast cell numbers measured in the airway fluids declined in both groups without a significant difference. Side effects included muscle cramps and hypophosphatemia, both of which were more frequent in those receiving imatinib. There was also a slight decrease in serum tryptase levels and tryptase levels in bronchial alveolar fluid.

Antibodies against SCF or KIT

In addition to TKIs, antibody-based drugs against KIT or SCF are under study (152, 153). In one example, the anti-KIT antibody CDX-0159 produced a profound suppression of plasma tryptase in healthy donors in a single ascending dose, double-blinded placebo-controlled phase 1a human volunteer study (152). The most common adverse events included a self-limited infusion reaction consisting of areas of local itching associated with erythema and hives, mild decreases in hemoglobin levels (generally within the normal range), and variable asymptomatic decreases in circulating neutrophils relative to placebo.

There are, of course, additional considerations when blocking SCF/KIT pathways. Among these are whether long-term treatment with multi-targeted agents, such as midostaurin or avapritinib, may cause unknown side effects due to off-target actions. In addition, the consequences of mast cell eradication are not known. Mast cells are thought to have multiple important functions including contributing to resistance against certain parasite and bacterial infections and in mediating acute resistance to some venoms (154–156). Finally, there are a number of cell types in addition to mast cells, including germ cells, hematopoietic stem cells, melanoblasts, and interstitial cells of Cajal in the gastrointestinal tract which display KIT (157–160). Therefore, administration of KIT TKIs over long periods could have unknown adverse effects on normal biologic functions. Mice with deficient expression of KIT have several abnormalities in addition to absence of mast cells and melanocytes, such as sterility, abnormal electrical pacemaker activity in the small intestine, and alterations in hematopoietic cells (160). It is clear that more research and clinical studies are required to determine the exact value of mast cell-depletion as a new pharmacologic concept and to translate such concepts into clinical practice in patients with mast cell-related diseases.

CONCLUSIONS AND FURTHER DIRECTIONS:

Progress in understanding the immunopathogenesis of anaphylaxis has been instrumental in guiding efforts to develop prophylactic pharmacological approaches, a goal that seems within reach. This is especially true for classic IgE-mediated anaphylaxis, for which several promising treatment options are emerging. Even more exciting is the possibility that the availability of multiple effective preventative treatments would allow for optimal tailoring of therapy to specific situations; for example, BTK and SYK inhibitors would be ideal for rapid-onset, episodic use to prevent reactivity during food or drug desensitizations, while anti-IgE and anti-KIT therapies may be best suited for long term management of allergic disorders. However, the mechanisms involved in anaphylactic presentations are complex and may involve a combination of IgE- and non-IgE-mediated pathways along with comorbidities and co-factors that may modify the severity of the reactions. Interestingly, several of the above therapies may also be effective in preventing IgE-independent reactions, including anti-IgE and anti-Siglec mAbs as discussed above. Overall, further investigation on the complexities of mast cell biology are needed to achieve more effective patient-tailored treatments.

New avenues of research include studies on MRGPRX2 receptor and demonstration of its involvement in human anaphylaxis to various drugs, the factors determining these systemic responses in some individuals but not in others, and the development of MRGPRX2 antagonists, which is underway and tested in laboratory settings (19, 20), but not yet in clinical trials as of the writing of this report.

ABBREVIATIONS USED:

ADCC

antibody-dependent cellular cytotoxicity

ADCP

antibody-dependent cellular phagocytosis

BTK

Bruton’s Tyrosine kinase

CD23

low-affinity receptor FcεRII

CSU

chronic spontaneous urticaria

OIT

food oral immunotherapy

FcεRI

high affinity receptor for immunoglobulin E

DARPin

designed ankyrin repeat protein

FYN

Fyn proto-oncogen Src family tyrosine kinase

GRB2

growth factor receptor bound protein-2

GLI

glioma-associated oncogene homolog

SHIP

inositol polyphosphate-5-phosphatase

JAK

janus kinase

LAT

linker for activation of T cells

LYN

Lyn proto-oncogen Src family tyrosine kinase

MAPK

mitogen-activated protein kinase

MRGPRX2

Mas-related G protein-coupled receptor X2

PSA

passive systemic anaphylaxis

PI3K

phosphatidylinositol 3-OH kinase/protein kinase B

PLCγ₁

phospholipase Cγ₁

PKC

protein kinase C

SCF

stem cell factor

SHC

Src homology domain containing

SHP-1

protein tyrosine phosphatase 1

SPT

skin prick test; SYK, spleen associated tyrosine kinase

STAT

signal transducer and activator of transcription

SOS

son of sevenless homolog 1

TKI

tyrosine kinase inhibitor

OFC

oral food challenge