Drug delivery targets and strategies to address mast cell diseases

Abstract

Introduction:

Current and developing mast cell therapeutics are reliant on small molecule drugs and biologics, but few are truly selective for mast cells. Most have cellular and disease-specific limitations that require innovation to overcome longstanding challenges to selectively targeting and modulating mast cell behavior. This review is designed to serve as a frame of reference for implementing new approaches that utilize nanotechnology or combine different classes of drugs to increase mast cell selectivity and therapeutic efficacy.

Areas covered:

Mast cell diseases include allergy and related conditions as well as malignancies. Here, we discuss the targets of existing and developing therapies used to treat these disease pathologies, classifying them into cell surface, intracellular, and extracellular categories. For each target discussed, we discuss drugs that are either the current standard of care, under development, or have shown indications for potential use. Finally, we discuss how novel technologies and tools can be used to take existing therapeutics to a new level of selectivity and potency against mast cells.

Expert opinion:

There are many broadly and very few selectively targeted therapeutics for mast cells in allergy and malignant disease. Combining existing targeting strategies with technology like nanoparticles will provide novel platforms to treat mast cell disease more selectively.

1. Introduction

Mast cells are uniquely long-lived granulocytes that are omnipresent throughout the body and fundamental to both the innate and adaptive immune response. Despite their many beneficial immunological roles, dysregulated mast cell responses contribute to a wide range of pathologies that include allergy, anaphylaxis, and cancer. A major contemporary therapeutic question remains as to whether it is possible to selectively target mast cells and their functions directly in all tissues. Healthy mast cells do not usually proliferate but do exist in tissue-specific subclasses. Pathologically, this implies that subpopulations of mast cells may respond differently depending on the therapy being applied and the disease context. This highlights the critical need for targeted therapies that can penetrate tissues to reach specific mast cell populations to address disease without disrupting immune homeostasis. Here, we discuss three main categories of drug classes and their strengths and weaknesses regarding selectivity for mast cells.

Mast cells react to and defend against parasites and likely play diverse roles in host defense and repair. They are mostly known as key effectors of allergy via activation of their FcεRI surface receptors. The basic mechanism of allergy involves sensitization of the host, typically via mucosal surfaces, to a foreign protein antigen. This process involves B cell antibody class switching, during which Immunoglobulin E (IgE) is produced by plasma cells that recognizes epitopes of the target allergen. This IgE circulates throughout the body and attaches to high affinity IgE receptors (FcεRI), awaiting future exposure to that same antigen. Once exposure occurs, the antigen will crosslink FcεRI receptors on mast cells (and basophils), triggering the rapid release of preformed and newly synthesized substances including histamine, prostaglandin D2, leukotrienes, proteases, heparin, cytokines, and others. The overall result is inflammation that manifests in symptoms such as sneezing, wheezing, hives, itchiness, swelling, and in severe cases anaphylaxis, the latter manifesting in mice as a drop in body temperature. Histology and flow cytometry can identify mast cells and their activation using selective surface markers such as CD117 (KIT), Siglec-6, and FcεRI⍺. Toluidine blue is another common stain used to visualize the granules within mast cells [1]. Commonly used surface markers to assess mast cell degranulation include CD63 and CD107a, as well as measurement of histamine, beta-hexosaminidase and tryptase in cell supernatants or in vivo.

Given their contributions to disease, mast cells are logical therapeutic targets for a wide range of pathologies including cancer. For example, mast cells can become malignant in a disease known as systemic mastocytosis [2]. This is caused by aberrations in the KIT gene, resulting in gain-of-function mutations that allow for their clonal proliferation independent of stimulation with the KIT receptor ligand, stem cell factor (SCF). By far the most common version of the mutation occurs in exon 17 of the gene and is known as D816V. Mastocytosis has been classified in several forms, some of which are life-threatening, while isolated mastocytomas tend to be self-limited [3]. Additionally, a role for mast cells in non-mast cell solid tumors also seems likely, so targeting mast cells may influence other types of cancer [4,5]. These classifications, in order of relative severity, are cutaneous mastocytosis, indolent systemic mastocytosis, systemic smoldering mastocytosis, aggressive systemic mastocytosis, mast cell leukemia, and mast cell sarcoma [3,6]. Another classification, known as systemic mastocytosis with associated hematologic neoplasm, is as severe and aggressive as other advanced forms of the disease, but displays a wider range in severity due to the variable nature of the associated neoplasms [7]. Mutations in the KIT gene are the most reliable characteristics of malignant mast cells, however histological studies of cell morphology and growth combined with detection of aberrantly expressed surface markers such as CD25, and perhaps to a lesser extent CD2, are part of the criteria needed to define mastocytosis [8,9]. As a result, bone marrow biopsies are commonly needed, but the invasiveness of this analysis requires reasonable evidence that mastocytosis is present. Despite this knowledge of mast cell function, identification markers, and roles in a wide range of immunopathology, few therapies that directly target these cells have been successfully developed [10].

Mast cells have proven to be difficult targets for direct and selective drug treatment. This is due to a combination of factors: 1) they have only a few unique cell surface and intracellular targets, and fewer are readily receptive to safe and selective modulation, 2) mast cells are long-lived, senescent or slowly dividing tissue-resident cells present throughout the body, 3) many aspects of their responsiveness and mediators are interrelated with products from other cells (e.g., IgE, SCF) and have effects on other immune cells as well as non-immune cells like the vasculature and neurons [11–13]. As a result, potential anti-mast cell therapeutics have side effects due to low specificity (e.g., KIT is expressed by cells other than mast cells) or indirectly target mast cells by antagonizing their mediators, with very few, if any, true “mast cell stabilizing” or specific mast cell depleting drugs, despite the panoply of receptors on these cells [14]. Recent developments have looked to improve efficacy, target selectivity, and specificity for mast cell disease. Here, we review two major disease pathologies associated with mast cells (allergy and malignancy) and summarize past, current, and developing drug treatment strategies for each. Furthermore, we tabulate information on these drugs and discuss their selectivity for mast cells and regulatory status (if applicable) for easy reference.

Existing mast cell therapeutics are dominated by small molecule antagonists and biologics. This strategy circumvents challenges associated with the presence of mast cells throughout the body [15]. Drugs that fall into this category, such as antihistamines, work by directly antagonizing H1 histamine receptors or via inverse agonism [16], can be orally dosed to systemically inhibit responses from allergic mast cells in tissues such as the skin, gastrointestinal tract and airways. intranasal and inhaled topical corticosteroid treatments gained prominence in the 1980s but despite their marked efficacy in allergic rhinitis and asthma have little to no direct effect on mast cells [17]. Biologics like omalizumab incompletely block binding of IgE to FcεRI⍺ at doses used clinically, and such an agent would not be expected to have any impact on non-IgE-mediated activation. And while receptor antagonists for leukotrienes exist, none are approved for antagonizing prostaglandin D2 receptors. There are also differences in mast cell phenotype depending on the tissue in which they are found. For example, mucosal mast cells only produce tryptase, while connective tissue mast cells contain tryptase, chymase, and carboxypeptidases [18,19]. Recent studies have elucidated that the variability and complexity of mast cell phenotypes can vary temporally under conditions such as type 2 inflammation [20]. Modern innovations continue to advance approaches to treatment and investigation of mast cell disease, and our discussion here aims to discuss both established therapies and more recent approaches.

2. Targets for mast cells in allergic disease

2.1. Cell surface targets

Cell surface targets include a variety of receptors that are under investigation for therapeutic mast cell modulation. These range from classical mast cell-associated receptors, such as KIT, to newer targets such as Siglecs or MRGPRX2. We have included separate figures that describe two generalized pathways governing mast cell activation. Figure 1 primarily shows G protein-coupled receptors and non-FcεRI-associated mast cell activation, whereas Figure 2 focuses primarily on FcεRI signaling. Here we describe approaches for selective targeting of mast cell receptors along with their potential advantages and disadvantages. This is not an exhaustive list of all possible targets and is instead intended to highlight key examples of existing treatment strategies and their merits for future development and application. In Table 1, we summarize additional examples of these drugs with notes for clinical development stage and mast cell selectivity.

Figure 1.

Non-FcεRI-associated mast cell activating pathways (black lines) and their inhibition (red lines) by various drugs. G protein-coupled receptors are antagonized by a variety of small molecules that interfere with ligand binding or with intracellular signaling components. Phosphatases SHP and SHIP play a significant role in several inhibitory pathways involving kinases important to mast cell signaling.

Figure 2.

Examples of small molecules and antibodies used to inhibit (red lines) various activating pathways (black) associated with FcεRI signaling. Kinases tend to be the most frequently targeted components of mast cell activating pathways and are exclusively inhibited by small molecule drugs of varying selectivity. Antibody-based therapeutics can be used to inhibit (KIT and ST2/IL-1RAcP) or trigger inhibitory signals (Siglecs).

Table 1.

Therapeutic targets and associated drugs for allergic mast cell disease

Drug Target	Receptor Expression	Drug Name	Drug Type	Delivery Route	Clinical Development	Mast Cell Selectivity	Citations
KIT (CD117)	MC, Thy, SP, Ba, X	CDX-0159	Monoclonal antibody	Subcutaneous	Phase 1	High	[28]
		Imatinib	Small Molecule	Oral	Approved	Low	[29,159,160]
		THB001	Small Molecule	Oral	Phase 1	High	[31]
Siglec-3 (CD33)	MC, E, Ba, N, M, DC	Anti-CD33-Anti-IgE	Antibody-Ligand Conjugate	Subcutaneous	Preclinical	Mid	[161]
Siglec-6	MC, Tr	AK006	Monoclonal Antibody	Subcutaneous	Preclinical	High	[53]
Siglec-7	MC, EW, NK, TCD8+, DC, Ba, M, X	-	Monoclonal Antibody	Subcutaneous	Preclinical	Low	[40]
		-	Antibody-Nanoparticle Conjugate	No in vivo work	Preclinical	Low	[162]
Siglec-8	MC, E, BaW	AK002	Monoclonal Antibody	Subcutaneous	Phase 2 and 3	High	[55,56]
		-	Ligand-allergen Liposomes	Intravenous	Preclinical	High	[54]
MRGPRX2	MC, X	Imperatorin	Small Molecule	Intravenous	Preclinical	High	[64,70,71,163]
		Paeoniflorin	Small Molecule	Intradermal	Preclinical		[72–76]
		Saikosaponin A	Small Molecule	Intraperitoneal	Preclinical		[77–80]
C3a Receptor	MC, X	BR111	Small Molecule	Intraplantar	Preclinical	Low	[83]
		SB 290157	Small molecule	Intraperitoneal	Preclinical		[164]
C5a Receptor	MC, X	PMX-53	Small Molecule	Oral	Preclinical	Low	[84]
CD300f/LIMR3	MC, E, N, M, DC	Ceramide	Lipid/Nanoparticle	Intradermal	Preclinical	Mid	[85,87,165,166]
		Sphingomyelin	Lipid/Nanoparticle	No in vivo work	Preclinical	Mid	[87]
ST2/IL-1RAcP	MC, X	Astegolimab	Monoclonal Antibody	Subcutaneous	Phase 2	Low	[93,138]
GPR35	MC, Ba, E, M, N, DC, X	Cromolyn	Small Molecule	Inhaled	Approved	Low	[94,96,97]
		Nedocromil	Small Molecule	Inhaled	Approved	Low	[94,96,97]
		Cromolyn-chitosan	Nanoparticle Payload	Oral	Not Applicable	Low	[98]
Histamine Receptor 1 (H1R)	MC, many cell types	Loratadine (Claritin)	Small Molecule	Oral	Approved	Low	[99,167–169]
		Fexofenadine (Allegra)	Small Molecule	Oral	Approved	Low	[170]
		Cetirizine (Zyrtec)	Small Molecule	Oral or Intravenous	Approved	Low	[170]
		Diphenhydramine (Benadryl)	Small Molecule	Oral or Intravenous	Approved	Low	[171]
Histamine Receptor 2 (H2R)	MC, X	Dimaprit	Small Molecule	Oral	Preclinical	Low	[99,100,167,168]
Histamine Receptor 4 (H4R)	MC, X	Toreforant	Small Molecule	Oral	Phase 2	Low	[107,167,168]
		JNJ 38758979	Small Molecule	Oral	Phase 2	Low	[104,172]
CRTh2	MC, E, Ba, X	AZD1981	Small Molecule	Oral	Phase 2	Mid	[111]
IgE	BC, Extracellular	Omalizumab	Monoclonal Antibody	Subcutaneous	Approved	-	[35,115,117,173]
		Ligelizumab	Monoclonal Antibody	Subcutaneous	Phase 3	-	[118–120]
IgE-producing Cells	BC	Quilizumab	Monoclonal Antibody	Subcutaneous	Phase 2	-	[121,122]
Tryptase	MC, Ba, Extracellular	MTPS9579A	Monoclonal Antibody	Subcutaneous or Intravenous	Phase 2	-	[127]
Chymase	MC, Ba, Extracellular	Fulacimstat	Small Molecule	Oral	Phase 2	-	[174]
Carboxypeptidase	MC, Ba, Extracellular	NvCI	Protein	Intraperitoneal	Preclinical	-	[128,175]
IL-4	Extracellular	Dupilumab	Monoclonal Antibody	Subcutaneous	Approved	-	[131,135]
IL-33	Extracellular	Itepekimab	Monoclonal Antibody	Subcutaneous	Phase 2	-	[138,139]
		Etokimab	Monoclonal Antibody	Subcutaneous	Phase 2	-	[140,141]
		IL-33trap	Fusion Protein	Intraperitoneal	Preclinical	-	[176]
TSLP	Extracellular	Tezepelumab	Monoclonal Antibody	Subcutaneous	Approved	-	[143,177]
SYK	MC, X	BI 1002494	Small Molecule	Oral	Preclinical	Low	[149]
		PRT-2761	Small Molecule	Oral	Preclinical	Low	[146]
BTK	MC, BC, DC, M	Acalabrutinib	Small Molecule	Oral	Approved	Mid	[178]
		Ibrutinib	Small Molecule	Oral	Approved	Low	[155,178]
		Zanubrutinib	Small Molecule	Oral	Approved	Mid	[154]
		Fenebrutinib	Small Molecule	Oral	Phase 2	Mid	[157]
Ras	MC, X	FTI-277	Small Molecule	Oral	Preclinical	Low	[158]

MC = Mast cells, E = Eosinophils, Ba = Basophils, T = T cells, BC = B cells, DC = Dendritic cells, NK = Natural killer cells, N = Neutrophils, M = Monocytes/Macrophages, Tr = Trophoblasts, Thy = Thymocytes, SP = Stem cells and Progenitors ^W = Weak expression, X = cell types outside of leukocytes

2.1.1. KIT

KIT facilitates the regulation of mast cell growth, proliferation, function, and differentiation (Fig. 1) [21–23]. Stem cell factor is the natural ligand of KIT, and its presence is needed for mast cell survival. It can prime mast cells for enhanced degranulation via IgE-mediated FcεRI activation [24,25]. Mast cell depletion with antibodies has previously been explored as a tool for therapeutic use [26,27]. However, the development of KIT-targeted therapeutics requires caution due to the potential for clustered cross-linking of FcγR around KIT, which could trigger degranulation instead of suppression. This has proven to be a major hurdle in the development of therapeutic anti-KIT antibodies.

2.1.1.1. Anti-KIT antibody ‘CDX-0159’

Anti-KIT Antibody ‘CDX-0159’ is a humanized monoclonal antibody developed by Celldex. Recent work by Alvarado et al details the use of CDX-0159’s modified IgG1/κ to prevent FcγR engagement from occurring while binding KIT [28]. The antibody inhibits KIT phosphorylation, which suppresses downstream signals for cell proliferation, survival, and activation leading to depletion of mast cells over time. Because KIT is expressed on other hematopoietic and non-hematopoietic cells (e.g., melanocytes, hair pigment cells), its targeting would be expected to have effects on cells other than mast cells which may or may not limit its use.

2.1.1.2. Imatinib

Imatinib, or Gleevec, is a small molecule designed to bind and inhibit receptor kinase enzymes. KIT receptor tyrosine kinase is one enzyme affected by this drug. Imatinib is approved and primarily used to treat malignant disease, however a study evaluated its use for severe asthma patients and substantial reductions in airway hyperresponsiveness [29]. This same study also showed patients experienced a depletion of mast cells and reduction in serum tryptase. It is unclear if the effect of depletion or pathway inhibition is the lead cause of decreased hyperresponsiveness in the patients evaluated. In general, KIT-targeted tyrosine kinase inhibitors will inhibit KIT-dependent mast cell activation, however only two have been shown to inhibit IgE-mediated activation: midostaurin and dasatinib [30]. This is likely due to their low selectivity for KIT receptor kinases.

2.1.1.3. THB001

THB001 is an oral small molecule wild type KIT inhibitor designed to deplete mast cells. However, there is little information publicly available on this drug. THB001 is currently undergoing phase 1a trials as a treatment for chronic urticaria [31].

2.1.2. Sialic acid-binding immunoglobulin-like lectins

Sialic acid-binding immunoglobulin-like lectins (Siglecs) comprise a family of potential targets for the treatment of mast cell disorders [32–37]. Siglecs are inhibitory receptors with differential immune cell expression (Fig. 1 and 2). Most Siglecs, but not all, have a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that recruits phosphatases to inhibit downstream kinase-mediated cascades [38]. Mast cells express Siglec-3 (CD33), -6 [39], -7 [40], and -8 [41–43] with low level expression of Siglec-2 (CD22), -5 [44], -9 [45], and -10 [45,46]. Siglec-6 expression is shared only with placental trophoblasts, which are absent outside of pregnancy, so it is otherwise quite selective for mast cells. Siglec-8 expression is shared with eosinophils. NK cells, dendritic cells, and CD8+ T cells express Siglec-7. CD33 is widely expressed among different immune cell populations, but strategies that co-localize IgE or FcεRI with CD33 ligand, as described below, can specifically isolate mast cells [34,47,48].

Current strategies to target Siglecs on mast cells involve monoclonal antibodies (mAbs) [39] or sialoside mimetics. Sialoside mimetics leverage N-terminal extracellular domains on Siglecs that bind sialic acid-containing carbohydrate structures (sialosides) with α2,3, α2,6, or α2,8 sialic acid linkages, depending on the specific Siglec [43,49,50]. Typically, these strategies aim for mast cell inhibition or elimination and can be a blend of both effects. Co-engagement with FcεRI can augment Siglec-mediated inhibition [39,40,47,48].

2.1.2.1. Antibodies

Antibodies targeting Siglecs -6, -7, and -8 inhibit human mast cell degranulation in vitro by activating the Siglec’s inhibitory signaling pathways [39,40,51–53]. Anti-Siglec-7 mAbs require anti-FcεRI mAb co-crosslinking to achieve an effect [40], whereas FcεRI co-crosslinking is not required for anti-Siglec-6 or anti-Siglec-8 mAbs but does enhance the inhibitory effect [39,54]. In addition to inhibiting mast cell degranulation [51,52,55,56], anti-Siglec-8 mAbs prevent anaphylaxis in humanized mice [57] and inhibit human bronchial ring contraction induced by FcεRI crosslinking [51].

2.1.2.2. High-affinity Siglec ligands

Several high-affinity Siglec ligands— including ligands specific for CD33 (CD33L) and Siglec-8— have been discovered through a glycan array screening [58–62]. Liposomal nanoparticles conjugated with Siglec-8 ligand selectively bind to Siglec-8 (+) cells [60]. When conjugated with CD33 or Siglec-8 ligand and IgE or antigen, liposomal nanoparticles inhibited degranulation of a human mast cell line, prevented anaphylaxis in transgenic mice, and prevented bronchoconstriction of human lung slices [47,54]. Anti-IgE antibody conjugated with CD33L also inhibits systemic anaphylaxis in humanized mice [48].

2.1.3. Mas-related G protein-coupled receptor-X2

Mas-related G protein-coupled receptor-X2 (MRGPRX2) is a receptor expressed on mast cells and is associated with their role at the neuro-immune interface (Fig. 1) [63,64]. The receptor triggers FcεRI independent activation in mast cells and thus pseudo-allergic reactions [65]. Substance P (SP) and hemokinin-1 (HK-1) have been identified as natural ligands of MRGPRX2. Charged substances like compound 48/80 (C48/80) and various drugs can activate this receptor, too [66,67]. MRGPRX2, SP, and HK-1 are upregulated in atopic dermatitis and allergic asthma [67–69]. These combined factors mark MRGPRX2 as an important candidate for drug targeting. So far, investigations into MRGPRX2-mediated mast cell inhibition have been dominated by small molecule antagonists, none of which are yet approved for clinical use.

2.1.3.1. Imperatorin

Imperatorin is a small molecule that regulates MRGPRX2 activation via inhibition of substance P-mediated calcium flux in mast cells. The molecule has been shown to inhibit FcεRI-associated degranulation in addition to modifying several other non-allergy related physiological processes [70,71]. Given imperatorin’s wide range of impacted targets and hydrophobic properties, further investigation is required to improve both its water solubility and specificity for MRGPRX2.

2.1.3.2. Paeoniflorin

Paeoniflorin is a small molecule that has been found to have neuroprotective and anti-inflammatory effects [72–74]. This molecule appears to bind MRGPRX2 and inhibit the C48/80-mediated phosphorylation of ERK1/2, PLCγ, and other kinases [75]. Additional evidence indicates that the disruption of pathways extends to FcεRI-mediated degranulation as well [76].

2.1.3.3. Saikosaponin A

Saikosaponin A is another small molecule shown to have multiple effects including protection from cardiac fibrosis, LPS-induced acute lung injury, and NF-κB inhibition [77–79]. Wang et al showed saikosaponin A to inhibit C48/80 activation in mast cells both in vitro and in vivo [80]. Their work demonstrated that the molecule does not inhibit FcεRI-mediated mast cell activation, and its association with MRGPRX2 was confirmed using MRGPRX2 HEK cells.

2.1.4. Complement components

Complement Components play major roles in innate immunity. Regarding mast cells, complement activation can trigger non-IgE mediated anaphylactic reactions via the generation of C3a and C5a anaphylatoxins [81], and may be responsible for some allergic reactions to medications such as the COVID-19 vaccines [82]. One strategy to modulate their effects would be by blocking the relevant complement receptor. A major caveat of blocking complement activity is the potentially negative impact on normal immune function, as these receptors are found across many cell types.

2.1.4.1. C3a receptor (C3aR) antagonist ‘BR111’

C3a receptor (C3aR) antagonist ‘BR111’ is a novel candidate for regulating mast cell degranulation [83]. This small molecule competitively binds the C3a receptor (C3aR), blocking mast cell degranulation and subsequent inflammatory effects on macrophages and neutrophils in vivo [83]. The BR111 molecule is the product of a slight conformational switch that favorably interacts with arginine in C3aR, whereas the other molecular conformation is an agonist of C3aR. The specificity of BR111 for C3aR remains to be investigated.

2.1.4.2. C5a receptor (C5aR) antagonist ‘PMX-53’

C5a receptor (C5aR) antagonist ‘PMX-53’ is a peptide-based dual antagonist/agonist (agonist of MRGPRX2). The drug is not viable as an inhibitor of mast cell activation, given its degranulatory interactions with MRGPRX2 [84]. Its agonist behavior is low-affinity by comparison to its antagonistic binding to C5aR. C5a-dependent mast cell inhibition and activation is independent of MRGPRX1 and MRGPRX2. At most, PMX-53 demonstrates that C5aR is a viable inhibitory target, but the molecule is still in early stages of development. An important limitation of PMX-53 is that its antagonist and agonist qualities depend on the same tryptophan and arginine residues within the peptide structure [84].

2.1.5. CD300f

CD300f (also known as LIMR3) is an ITIM-containing immunoreceptor (Fig. 1) associated with regulatory functions in neutrophils, eosinophils, macrophages, monocytes, mast cells, and dendritic cells [85]. The receptor has recently been identified as a potential inhibitor of MRGPRX2 and IgE-mediated mast cell degranulation [86,87]. Due to the wide number of cells impacted by this receptor, its development as a mast cell drug will likely require strategies to minimize other on-target effects on other cells such as suppression of innate immunity [85].

2.1.5.1. Ceramide and sphingomyelin

Ceramide and sphingomyelin are both lipid molecules and natural ligands of CD300f [85,86,88]. The work of Takamori et al demonstrated inhibition of C48/80-triggered degranulation using ceramide to bind and activate CD300f. Their work compared CD300f^−/− and wild type peritoneal mast cells to highlight the necessity of the receptor to prevent degranulation in the presence of ceramide. Long chain ceramides and sphingomyelin have poor aqueous solubility; therefore they may require the use of nanocarrier vehicles or chemical modification to effectively target CD300f [89,90]. Additionally, these lipids readily exchange between membranes, rendering them difficult to control in alternate delivery formats such as liposomes [91].

2.1.6. ST2/IL-1RAcP

ST2/IL-1RAcP is a heterodimeric cytokine receptor for IL-33 (Fig. 2). It is expressed by a wide range of leukocytes, including mast cells and other allergic effector cells. Because of their role in allergy and asthma, ST2 and IL33 have been intensively studied as candidates for therapeutic intervention in a variety of allergic pathologies [92].

2.1.6.1. Astegolimab

Astegolimab is an anti-ST2 monoclonal antibody under investigation as a therapeutic for COPD and has been studied in the context of atopic dermatitis. An asthma study published in 2021 showed efficacy in a broad group of patients, including subjects with low blood eosinophils [93].

2.1.7. G Protein-coupled Receptor 35

G Protein-coupled Receptor 35 (GPR35) is an orphan receptor with a yet unknown natural ligand (Fig. 1). Despite this, it has been investigated as the target of the mast cell stabilizer cromolyn and its derivatives, though aspects of the receptor and its agonists’ inhibitory mechanism remain undetermined. Selective targeting of GPR35 may be possible, however the receptor is expressed by a wide range of cells and tissues, so depending on the effect and efficacy of a targeted drug treatment, there may be broad spectrum effects [94].

Cromolyn was considered a breakthrough asthma medication after its discovery in 1965. Nedocromil is a long-acting derivative of cromolyn that is used in the same role as an asthma prophylactic. Cromolyn also inhibits chloride channels, which were initially believed to be a potential mechanism behind its mast cell stabilizing effect [95]. However, it is possible that cromolyn’s anti-asthmatic effects are not due to mast cell stabilization at all, but instead due to its ability to inhibit pro-inflammatory cytokine release [96,97]. Recent investigations have tried to implement modern tools to improve cromolyn. One such example has been the use of chitosan to improve the oral pharmacokinetics of cromolyn and overcome its dose administration limitations [98].

2.1.8. Histamine receptors

Histamine receptors are also G protein-coupled receptors (Fig. 1) and perhaps the oldest and most common target of day-to-day allergy management. Here we only discuss those that can directly affect mast cell secretory function. H₂ receptor agonists such as dimaprit have been used to inhibit mast cell histamine release [99] and have been observed to reduce TNFα expression in response to TLR4 activation [100]. H₂ receptors are primarily indicated as targets in acid-peptic diseases such as reflux and heart burn. H₂ antagonists are primarily used to treat these conditions and have little effect on mast cell histamine release. Mast cells and others, including cells in the brain, express H4 receptors. This receptor mediates mast cell chemotaxis and calcium flux [101,102]. H₄ receptor antagonists have been investigated, and some are approved for non-mast cell conditions [103]. However, no H₄ antagonist has been approved for allergies, largely due to side effects during pre-clinical testing or lack of drug effect in clinical trials [103,104]. Recently, a clinical-phase small molecule has demonstrated inhibitory activity on eosinophils and mast cell activation [105–107].

2.1.9. Beta receptor agonists

Beta receptor agonists such as albuterol and epinephrine are ‘rescue’ drugs which are used to treat asthma and anaphylaxis [108]. They activate β2-adrenergic receptor and stabilize mast cells by increasing intracellular cAMP, which in turn inhibits secretion of mediators by mast cells [109]. These drugs are used episodically and cannot be used chronically due to their tendency to develop tolerance [110].

2.1.10. Chemoattractant receptor-homologous molecule expressed on Th2 cells

Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a receptor found on mast cells and others involved in type 2 inflammation. The receptor binds prostaglandin D₂ (PGD2) released by degranulating mast cells, eosinophils, and basophils. Because CRTH2 is found on these cells as well, this can result in a source of positive feedback in asthma and chronic urticaria pathologies [111]. CRTH2 antagonists are small molecules that bind CRTH2, blocking PGD2-mediated activity. One example is AZD1981, a drug developed by AstraZeneca. In a phase 2 trial of asthma, atopic patients displayed significantly improved symptom scores, whereas a nonsignificant increase in expiratory flow was observed vs the placebo [112]. Another study evaluated the impact of AZD1981 on eosinophil activity in chronic spontaneous urticaria and found that the drug changed circulating eosinophil numbers and reduce itch scores. Interestingly, treatment increased CRTh2 expression on circulating eosinophils [113].

2.2. Extracellular targets

2.2.1. Immunoglobulin E

Immunoglobulin E (IgE) is an antibody isotype synthesized by plasma cells. These antibodies, when specific for allergy-related antigens, are the root cause of allergic sensitivity (Fig. 2). The Fc fragment of these antibodies uniquely contain an extra Fc domain (Cε3) that allows IgE to bind with high affinity to the FcεRI⍺ chain on mast cells, allowing mast cells to engage allergens via the Fab region of IgE. It is this cross-linking effect that triggers mast cell activation. One of the largest areas of allergy therapeutics has been in the development of IgE antagonists designed to intercept the ability of mast cells to engage antigens.

Omalizumab is a clinically approved, subcutaneously injected anti-IgE antibody for the treatment of severe asthma and chronic spontaneous urticaria [114]. It is used off label for various other indications [115]. It binds to the Cε3 domain on the Fc region of IgE, neutralizing its ability to engage FcεRI [116]. The drug has also been investigated in conjunction with desensitization protocols to reduce allergic reactions [117].

Ligelizumab is another anti-IgE antibody and has shown 88-fold higher affinity for IgE compared to omalizumab [118]. Interestingly, it also shows somewhat preferential effects where it is effective in urticaria but not so in asthma [119,120]. This is likely indicative of differences in their ability to block IgE binding to FcεRI versus FcεRII.

2.2.2. IgE-producing cells

IgE-producing cells can be targeted to reduce serum IgE. Quilizumab is an antibody that targets the M1’ extracellular epitope of transmembrane IgE [121]. M1’ is absent IgE, meaning that quilizumab will not interact with secreted IgE. Treatment led to reductions in total serum IgE that lasted 6 months after halting dosing and reduced late asthmatic airway responses by up to 36% [121]. Another trial investigating its use in refractory chronic spontaneous urticaria patients saw a reduction of serum IgE by 30%, however this was determined to be clinically insignificant [122]. This antibody is no longer in development.

2.2.3. Tryptase, chymase, and carboxypeptidase

Tryptase, chymase, and carboxypeptidase are proteases stored in mast cell granules. They are some of the hallmarks of mast cell degranulation and associated inflammation. The roles of tryptase are multifaceted, contributing to the activation of neighboring mast cells and the activation and recruitment of inflammatory cells to the site of degranulation [123,124]. Chymase release is a good indicator of mast cell activation, though there are other routes by which it can be expressed [125]. Of note, chymase converts angiotensin I to angiotensin II, contributing to vasoconstriction in anaphylaxis. Carboxypeptidase A3 (CPA3) is a member of a much larger family of enzymes. It is released in complexes with, and works in concert with, tryptase or chymase.

2.2.3.1. MTPS9579A

‘MTPS9579A’ (RG6173) is an anti-tryptase monoclonal antibody undergoing clinical investigation. The antibody dissociates the tetrameric protease into its monomer subunits, inhibiting its activity and decreasing inflammation [126]. In a phase I clinical trial, dose-dependent reductions in serum tryptase levels were observed [127].

2.2.3.2. Nerita versicolor Carboxypeptidase Inhibitor

Nerita versicolor Carboxypeptidase Inhibitor (NvCI) is a protein derived from a marine snail that has potent inhibitory effects on CPA3 activity [128]. In a mouse model of asthma, treatment with NvCI showed reduced accumulation of goblet cells, however it did not affect accumulation of inflammatory cells in the airways [128].

2.2.4. IL-4

IL-4 is a key cytokine in the proliferation and priming of mast cells for degranulation. It is the first cytokine observed to be produced by mast cells [129]. Upon binding to the IL-4 receptor, a multitude of processes are initiated, including production of leukotriene synthase, adhesion, chemotaxis, and increased expression of FcεRI [130].

Dupilumab binds and block IL-4 receptor α (IL-4Rα), which in turn inhibits IL-4 and IL-13 activity in mast cells [131]. It is a monoclonal antibody that is approved to treat severe eczema, asthma, chronic rhinosinusitis, and eosinophilic esophagitis [132–134]. Inhibition via dupilumab treatment modestly reduces serum IgE levels and decreases mast cell accumulation at sites of inflammation [135].

2.2.5. IL-33

IL-33 is the cytokine ligand of ST2 receptors associated with many allergic pathologies as mentioned above. Mast cells become primed for activation by IL-33, amplifying their cytokine production [136].

Anti-IL-33 antibody has been shown to reduce symptoms of atopic dermatitis in mice. In this study, mast cell infiltration into dermal tissue and serum IgE levels were significantly reduced using IL-33 antibody [137]. Sanofi and Regeneron’s lead clinical candidate for anti-IL-33 therapy (Itepekimab) was unable to outperform Dupilumab, however it may provide alternatives for patients unresponsive to the latter [138,139]. Another anti-IL-33 antibody, etokimab, was shown to facilitate safer peanut allergen desensitization in a phase 2 study, however it failed to meet its endpoints in an atopic dermatitis trial [140,141].

2.2.6. Thymic stromal lymphopoietin

Thymic stromal lymphopoietin (TSLP) is a cytokine heavily implicated in asthma pathology. It promotes Th2 inflammation and can create a positive feedback loop with IL-33 which drives asthmatic activity of mast cells and eosinophils [142].

Tezepelumab is an anti-TSLP antibody which has been approved for use in severe asthma patients. It blocks TSLP-mediated activation and has demonstrated improved lung function and control over asthma symptoms compared to placebo controls [143]. The drug binds the cytokine and prevents it from interacting with its target receptor on mast cells.

2.3. Intracellular targets

2.3.1. Spleen Tyrosine Kinase

Spleen Tyrosine Kinase (SYK) is an upstream tyrosine kinase involved in signal transduction for receptors expressed by a wide range of cell types in addition to immune cells [144]. In mast cells, SYK is immediately downstream of the FcεRI signaling cascade, and its phosphorylation is key to IgE-dependent mast cell degranulation. As a result, it is an attractive target for regulating allergic responses [145,146]. Unfortunately, the widespread function of SYK makes it a difficult therapeutic target, given its contribution to signaling for many receptors. Several inhibitors have been developed but in addition to the problems with affecting other cells that express SYK, these inhibitors are often non-specific and block other kinases. Below, we discuss two examples with more under investigation [147,148].

BI 1002494 is a small molecule SYK inhibitor with potent inhibitory activity. It was found in one study that 23 kinases (8% of the panel) were inhibited by this molecule [149]. The authors noted that the potency of BI 1002494 was greater in mast cells than B cells, however this is not a comprehensive evaluation given the vast number of cell types that express SYK [144].

PRT-2761 is a SYK inhibitor under investigation to treat allergic conjunctivitis via topical application. In a murine model of allergic conjunctivitis, relief of symptoms was seen on a level similar to prednisolone [146].

2.3.2. Bruton’s Tyrosine Kinase

Bruton’s Tyrosine Kinase (BTK) is mostly associated with B cell receptor signaling and adaptive immunity [150]. In the past few years, the importance of BTK signaling in immune signaling has expanded to mast cells and basophils [151]. In the context of mast cells, activation of the FcεRI pathway leads to phosphorylation of BTK, which drives further phosphorylation events, subsequent activation of transcription factors, and calcium flux [151,152]. Three approved BTK inhibitors (BTKi) are currently on the market, with several more in development [153].

Acalabrutinib (AcB) is one of three currently approved BTKi (the others being ibrutinib and zanubrutinib), albeit for hematologic malignancies [154,155]. All three are covalent small molecule inhibitors that irreversibly bind BTK with varying specificity. Recent investigations have tested AcB as a therapy to treat and protect from FcεRI-mediated anaphylaxis. Dispenza et al observed that AcB protected humanized mice from severe anaphylaxis and death. The same group also observed that Ibrutinib blocked FcεRI-mediated contraction in excised human bronchi [156]. These results have prompted a phase II clinical investigation into the use of AcB to prevent allergic reactions. Also worthy of mention is fenebrutinib, a highly selective, reversible BTK inhibitor that has shown promise in a phase 2 trial of H₁ antihistamine-refractory chronic spontaneous urticaria [157].

2.3.3. Ras

Ras is a small GTPase that is responsible for switching on signaling pathways once it is activated. In mast cells, it is an upstream part of the MAPK signaling pathway and contributes to mast cell activation via the KIT and FcεRI receptors. FTI-277 is a Ras inhibitor that decreases phosphoinositide 3-kinase γ (PI3Kγ) signaling in mast cells [158]. As a result, this molecule demonstrated attenuation of IgE-activated mast cells when co-delivered with adenosine. In addition, this inhibition did not affect C5a or SCF-dependent pathways [158]. PI3Kγ can operate in two forms: a p110γ/p84 or p110γ/p101 complex. These forms are found in multiple leukocytes, however Ras-dependent PI3Kγ is only in the p110γ/p84 format. To ensure that minimal impact is felt in non-target cells, FTI-277 would need to target only this version of the PI3Kγ complex in mast cells.

3. Targeting mast cells in malignant disease

Mastocytosis and mast cell leukemia are relatively rare conditions that are notoriously difficult to treat [179]. Only recently have therapies with improved specificity for the treatment of these diseases become available, delivering improved patient outcomes [10,159,180]. It is important to mention that mastocytosis and its subvariants present a great challenge when it comes to developing novel therapies: the pool of patients available for clinical trials is relatively small [181]. This is made even more difficult by additional constraints tied to disease subtypes or requirements for no pre-existing therapies [7–9,179]. Table 2 details selectivity for mast cells and clinical development for each drug. Primary strategies by which malignant mast cells rely on either small molecule therapeutics or antibodies and antibody-drug conjugates have additionally been provided (Fig. 3).

Table 2.

Therapeutic targets and associated drugs for malignant mast cell disease

Target	Expressed by	Drug Name	Drug Type	Delivery Method	Clinical Development	Selectivity	Citations
KIT tyrosine kinases	MC, Thy, SP, E, Ba	Bezuclastinib	Small Molecule	Oral	Phase 3	High	[190]
		Avapritinib	Small Molecule	Oral	Approved	High	[30,188,189]
		Masitinib	Small Molecule	Oral	Approved	Low	[180]
		Midostaurin	Small Molecule	Oral	Approved	Low	[180,189]
		Imatinib	Small Molecule	Oral	Approved	Low	[159,160]
KIT	MC, Thy, SP, E, Ba	LOP 628	Antibody-drug Conjugate	Intravenous	Terminated	High	[202]
Siglec-3	MC, E, Ba, M, DC	Gemtuzumab	Antibody-drug Conjugate	Intravenous	Approved	Mid	[201]
Siglec-7	MC, EW, NK, TCD8+, DC, Ba, M	-	Monoclonal Antibody	Intraperitoneal	Preclinical	Mid	[197]
Siglec-8	MC, E, BaW,	Lirentelimab	Monoclonal Antibody	Subcutaneous	Phase 3	High	[198–200]

MC = Mast cells, E = Eosinophils, Ba = Basophils, T = T cells, DC = Dendritic cells, NK = Natural killer cells, M = Monocytes/Macrophages, Thy = Thymocytes, SP = Stem cells and Progenitors, ^W = Weak expression

Figure 3.

Examples of malignant mast cell therapeutic strategies. Current clinical methods largely rely on small molecule kinase inhibitors (e.g. midostaurin, avapritinib, etc) which target and inhibit (red lines) mutant KIT activity (black lines). Historically, these drugs had broad kinase antagonistic activity responsible for many of their side effects. Recent iterations have resulted in more selective and better tolerated drugs. Receptor targeting strategies such as KIT or Siglec antibodies and antibody-drug conjugates have shown promise as potential therapeutic methods as well.

3.1. Cell surface targets

3.1.1. KIT-Associated Tyrosine Kinases

KIT-Associated Tyrosine Kinases have been the traditional therapeutic targets of mastocytosis and mast cell leukemia (Fig. 3) [10,182]. Development of tyrosine kinase inhibitors (TKIs), after the successful and revolutionary introduction of Gleevec (imatinib), became a major treatment strategy for many malignant diseases [183]. Rationally designed small molecule drugs typically have several advantages; they can be chemically synthesized, fit into specific molecular pockets, and in many cases, formulated or functionalized for multiple routes of delivery [184–186].

3.1.1.1. Masitinib

Masitinib is a TKI primarily used to treat mast cell tumors in dogs. It also modulates the activity of macrophages and appears to act more effectively in solid tumor manifestations of mast cell malignancies, somewhat limiting the scope of its use in humans. Currently, it is under investigation to treat indolent systemic mastocytosis and smoldering systemic mastocytosis [180].

3.1.1.2. Imatinib

Imatinib was the first selective TKI developed using rational design methods and functions better as a general chemotherapeutic than as a mastocytosis drug. Long term use of this TKI reduced serum tryptase levels and reduced mast cell burden in asthma patients and chronic myeloid leukemia patients [160]. This drug is limited in its capacity to treat systemic mastocytosis patients, as it is approved in patients lacking the D816V mutation or those with unknown mutation status [159].

3.1.1.3. Midostaurin

Midostaurin is a multiple TKI that was approved to treat advanced systemic mastocytosis in 2017. It is a small molecule that is dosed orally and binds a wide variety of kinases, resulting in undesirable side effects such as thrombocytopenia and neutropenia [187]. As a result, its use is generally limited to advanced systemic mastocytosis and similar conditions such as acute myeloid leukemia.

3.1.1.4. Avapritinib

Avapritinib was approved in 2021, and it is a potent TKI with greatly improved selectivity for mutant KIT tyrosine kinase compared with its predecessors. Avapritinib possesses distinct resistance profiles in mastocytosis, depending on the mutation status of KIT, so while this drug is effective for patients with the D816V mutation (>90% of patients), the remainder of patients require alternative and less selective treatments with imatinib or masitinib [188,189].

3.1.1.5. Bezuclastinib

Bezuclastinib is another small molecule that has been designed to selectively bind KIT receptor tyrosine kinase with exon 17 mutations. It is currently undergoing testing in two mastocytosis clinical studies involving advanced and nonadvanced stages of disease. This drug appears to have reduced blood-brain barrier penetration and side-effects compared with other TKIs used to treat mastocytosis [190].

3.1.2. KIT Receptor

KIT Receptor is the target of several therapies due to its surface abundance and relative specificity for mast cells (Fig. 3). In the case of mastocytosis, therapeutics which target KIT must be able to do so without dependence on a normally functioning receptor. This is due to the mutations in the KIT gene and is relevant in one of the drugs discussed below.

KIT Antibody ‘CDX-0159’ was mentioned above in the context of allergy. The use of this antibody is likely limited in the context of malignant mast cell disease (it is effective on cells dependent on SCF-KIT signaling), but at least demonstrates that anti-proliferative anti-KIT antibody treatments are possible for mast cells [28,191]. If modified with a drug payload, this antibody could potentially be used for mastocytosis. Though not a malignant disease, it is being explored to treat chronic urticaria [28,191].

3.1.3. Siglecs

Siglecs have also been shown to be viable targets for the selective elimination of mast cells (Fig. 3) [192,193]. Furthermore, in addition to systemic mastocytosis and mast cell malignancies, mast cell reduction or elimination may be useful when treating other conditions such as chronic urticaria. In addition, several Siglecs have demonstrated endocytic properties, which may be exploited for selective drug payload delivery to mast cells [39,192–194]. Because of the scarcity of natural killer (NK) cells in most tissue compartments containing mast cells, it’s expected that antibody-dependent cellular phagocytosis (ADCP) via macrophages predominates over antibody-dependent cellular cytotoxicity (ADCC) via NK cells [195,196]. Alternative strategies for reduction/elimination have also relied on growth inhibition or other cytotoxic mechanisms.

3.1.3.1. Anti-Siglec antibodies

Anti-Siglec antibodies have similar potential for mast cell-specific drug delivery and as anti-proliferative receptors. Anti-Siglec-7 mAbs inhibit growth of a human mast cell leukemia cell line in vitro and in vivo [197]. In clinical studies, the humanized afucosylated IgG1 anti-Siglec-8 antibody Lirentelimab decreases gastrointestinal mast cell counts, but modestly compared to near total elimination of eosinophils [198]. Lirentelimab also improves symptoms in patients with systemic mastocytosis [199] and chronic urticaria, even though no reduction in mast cells was observed [200].

3.1.3.2. Anti-Siglec drug conjugates

Anti-Siglec drug conjugates (ADCs) have gained interest as therapeutic strategies for malignant mast cell disease. Gemtuzumab Ozogamicin (anti-Siglec-3 with toxic drug conjugate) is one example. Interestingly, it was previously approved for resurgent acute myeloid leukemia patients but was removed for several years before receiving new approval as a combination therapy and in selective scenarios as a singular therapy [201]. This demonstrates the possibility of developing other ADCs with higher selectivity for mast cells, e.g., via Siglec-6 or Siglec-8. Indeed, treatment with an anti-Siglec-8 mAb conjugated with a toxic payload causes cell death in a human mast cell leukemia cell line HMC-1.2 [193] and Siglec-6 endocytosis has been demonstrated on primary human cells and mast cell lines [39].

4. Future directions for mast cell therapies

Currently, the cutting edge of allergy and mastocytosis therapies are dominated by small molecules and biologics. Perhaps it is unsurprising, given that mast cells have proven difficult to manipulate and that some of their functions have been difficult to unravel. It has become increasingly clear that there are still gaps in the community’s approaches to treat allergy and mastocytosis. Even with the advent of new tyrosine kinase inhibitors with improved selectivity, there is a good chance that some forms of mastocytosis will be resistant to these types of drugs. Treatment of mast cell-mediated allergies and anaphylaxis has evaded the scientific community for decades. Drugs are available to manage allergic reactions and their symptoms, but therapeutic strategies to selectively, reliably, or directly ablate or modulate mast cells are lacking. Systemic mastocytosis likely requires a targeted multi-drug approach to tackle its multifarious biology across different patients. These shortcomings indicate a need for both improved methods of mast cell-selective drug delivery and a better understanding of mast cell-driven pathology. We propose that two key tools can play this role: targeted drug delivery and improved animal models.

4.1. Nanoparticles and antibody-drug conjugates

Nanoparticles and antibody-drug conjugates are still in the early stages of development for treatment of mast cell disease. While there have been basic studies investigating the interactions of mast cells and different types of nanoparticles, few groups have employed targeted nanotherapy to address allergy or mastocytosis.

Nanoparticles include particulates of a wide range of compositions and morphologies possessing one or more dimensions less than 1000 nm [203–205]. Serving as customizable carriers for drugs and biologics, these nanoscale vehicles have become staples in the field of controlled drug delivery, where bioactive molecules can be either encapsulated within their interiors or displayed on their surfaces. The advantages of nanoparticle-based therapies (Fig. 4) include: 1) their ability to protect drug payloads from enzymatic or cellular interactions before reaching their intended target, 2) ability to present multiple cell-targeting components and thus engage different cell types or co-engage multiple targets on a single cell, 3) decrease undesirable immune interactions such as inflammation, and 4) tune drug release kinetics from nanoparticles and control bioavailability via modulation of material pharmacokinetics and pharmacodynamics (PK/PD) [206]. In many ways, nanoparticles often act as prodrugs designed to home in on their cellular target before undergoing a triggerable physical or chemical change to locally release their payload. This enables increased specificity for drugs that can typically have broad spectrum effects. This often greatly decreases the dose required to effectively treat a cell population and reduce side effects as well. While some cell types can be passively and effectively targeted by controlling nanoparticle geometry and surface chemistry [205,207–209], many require the association or attachment of targeting moieties such as antibodies, Fab’ fragments, or receptor ligands. Indeed, one of the limiting factors of cell targeting with nanoparticles can be a requirement to have identified and thoroughly characterized cell-specific targets that are externally accessible. Furthermore, most targeting strategies likely benefit from optimized nanoparticle size and shape in combination with an active targeting molecule [210]. Mast cells have a few relatively specific surface receptors, including Siglec-6, MRGPRX2, KIT, and to a lesser extent, Siglec-8 [12]. Given the multitude of existing drugs which show variable levels of selectivity for mast cells, there is a strong argument to explore the use of nanoparticles as delivery vehicles to enhance specificity and efficacy for mast cells in disease.

Figure 4.

Advantages of nanoparticles as drug delivery vehicles in the treatment of mast cell disease. (A) Nanoparticles can harbor and protect internal drug payloads from physiological threats such as enzymes, differences in pH, and unintended uptake by non-target cells. (B) Multivalent conjugation and surface presentation of targeting species enhance cell selective uptake by specific cell surface receptors. (C) The material composition and geometry of nanoparticles impacts cellular interactions and pharmacokinetics to allow optimization of drug delivery and cell selectivity. Nanoparticle composition, geometry, and morphology can range from small solid core micelles to larger more complex architectures such as vesicles, nanocages and filaments, each with unique degradation, pharmacokinetic, and pharmacodynamic properties. (D) Nanoparticle surface chemistry, size and payloads can be optimized to avoid unwanted cellular interactions such as innate inflammatory reactions or non-specific payload delivery.

4.2. Animal models

Animal models present a useful tool to understanding disease progression and therapy. The recent advancement of humanized mouse models has allowed for direct association to human biology without putting individuals at risk. There are many preclinical in vitro models to study advanced systemic mastocytosis (SM). Recent advancements in mast cell lines have allowed for further characterization of biomarkers and possible drug targets, however, in vivo tools have been lacking [10]. In order to better understand disease progression and drug therapy, an in vivo model of systemic mastocytosis is important for further validation of in vitro findings. Very few mouse models of mastocytosis have been developed as many models do not show similarities to human disease. A limitation to many of the xenograft models that have previously been used is the lack of consistency in disease development and kinetics. The KIT D816V+ human mast cell line has neoplastic characteristics seen in advanced SM, and when injected into NOD-SCID IL-2R γ^−/− (NSG) is capable of generating a unique humanized in vivo model of advanced SM, thus allowing for the study of mast cell drug targets [211]. Mast cell neoplasms in dogs are also another animal model that can be exploited for novel drug therapies as it is the leading diagnosed malignancy [212].

Mast cell-driven diseases also include food allergy, which presents another humanized mouse model to study mast cell drug targets. While there are a variety of genetic backgrounds and methods of engraftment to generate humanized mice, the NSG-SGM3 is the most useful for expansion of myeloid lineages as the mouse has three transgenes encoding human IL-3, GM-CSF and SCF. Intravenous injection of CD34+ cord blood derived human stem cells allows for stable engraftment mast cells. Once engrafted, mast cells can be sensitized with antigen specific IgE and challenged with antigen leading to anaphylaxis [156]. There are no known therapies capable of preventing anaphylaxis and this model of passive systemic anaphylaxis (PSA) provides a tool to test potential mast cell therapies.

Other possible mouse models may also include transgenic expression of human proteins of interest. One such example is the ability to express human Siglec-8 in a mast cell-specific manner in a C57BL/6 mouse [213]. The signaling properties of these targets of interest can be thoroughly elucidated and PSA models can be utilized. This method can be more cost effective than the use of humanized mice but is more limited in direct translation to humans.

5. Expert opinion

In the near term, therapeutics for mast cell disease will continue to be dominated by small molecule and antibody-based therapies. Very few drug targets are truly selective for mast cells, and fewer have clear drug development strategies. The information and advances we have discussed can provide a frame of reference in developing new strategies which target these cells. While many other leukocytes have seen attention from the nanotechnology community, mast cells have seen startlingly little. Given that multiple relatively mast cell-specific targets have been identified, selective approaches to modulate these cells should be possible. Antibody-drug conjugates, though sometimes difficult to create, may provide a potent and more immediate solution for targeted drug delivery while nanotherapies continue to be developed.

Very little is understood regarding the bio/nanointerface between mast cells and nanoparticles, which highlights the need for further investigations of these interactions. However, as described above, there is a large pool of well-studied biologics and small molecule drugs that can modulate mast cell behavior, many of which have limited clinical utility due to their side effects and lack of selectivity. By addressing these limitations, nanoparticles may therefore provide new life to drugs that have been shelved for allergy applications. In addition to drug selectivity issues, the variable and difficult-to-diagnose nature of mastocytosis has also presented challenges. Even drugs with improved selectivity may not justify the side effects incurred for indolent forms of mastocytosis, or perhaps they might lack the potency necessary to sufficiently clear mast cells with multiple mutations in the KIT gene. Ongoing clinical trials of novel KIT kinase inhibitors will be pivotal in how we address indolent forms of the disease moving forward. It is apparent that regardless of the outcome, there will be patients that would benefit from more selective therapies of combination drug therapies delivered by nanoparticles.

The development of new allergy therapies presents an interesting conundrum. As a society, we have lived to cope with allergies, yet there are serious and unresolved issues stemming from managing severe allergic reactions. Not to mention the associated costs that come with acute anaphylaxis, asthma, and chronic allergic disorders. Much of this stems from how difficult it has been to selectively treat mast cells. Many early allergy medications had complications such as crossing the blood-brain barrier or inhibiting activity in a wide variety of cells. Accurate identification and characterization of receptor expression in mast cells has only been obtained within the last 10–15 years. Despite this, these targets have not been heavily explored by many groups outside of the mast cell research field. It is important to advertise these targets as opportunities to the greater biomedical research community to stimulate novel approaches to these diseases.

Opening mast cell-related therapeutics to other areas of science and engineering will diversify, strengthen, and present new approaches to tackle these diseases. The identification of several unique mast cell receptors presents opportunities to develop targeted nanotherapies. Current hurdles to developing therapies that target some of these receptors, such as MRGPRX2, revolve around finding an appropriate ligand or binding molecule that is amenable to drug or nanoparticle conjugation. Other receptors, such as Siglec-6, can already be targeted by selective antibodies and are thus more receptive to the development of drug or nanoparticle conjugates using established methods. Regardless, these challenges are inherent to bringing potential nanotherapies to mast cell disease. By combining new technology and robust animal models with established drug molecules, new classes of allergy and mastocytosis therapies will be possible.

Article highlights.

• There is a lack of selective and potent medications available to directly regulate mast cell activity in allergy or malignant disease.

• Existing approved mast cell therapies are dominated by small molecule drugs, the majority of which have varying unintended side-effects due to a lack of mast cell selectivity.

• Powerful multi-omics approaches have aided in identifying novel mast cell targets.

• Modern methods of generating biologics, drug conjugates, and nanotherapies have opened the door to more selective mast cell therapeutics.

• Some antibody and receptor-specific strategies pose significant risk of uncontrolled rupture or activation of mast cells, potentially leading to dangerous anaphylactic responses.

• There are multiple mast cell targets which have potential to be employed for both allergic and malignant mast cell diseases, highlighting a likely benefit of collaboration and crossover between these research specialties.