Use of focal adhesion kinase pathway modifiers in the treatment of allergic reactions and mast cell-mediated disorders

Abstract

This disclosure provides methods, compositions, and formulations for treating, preventing, or reducing allergic reactions — including anaphylaxis — and mast cell-mediated disorders. Methods include administering to a subject a therapeutically effective amount of a focal adhesion kinase (FAK) pathway modifier, including small molecule FAK inhibitors, FAK-targeting proteolysis-targeting chimeras (PROTACs), and nucleic acid-based FAK expression modulators.

Description

USE OF FOCAL ADHESION KINASE PATHWAY MODIFIERS IN THE TREATMENT OF ALLERGIC REACTIONS AND MAST CELL-MEDIATED DISORDERSFIELD

[0001] This disclosure provides methods and compositions for treating, preventing, or reducing allergic reactions (including acute allergic reactions such as anaphylaxis) and mast cell-mediated disorders. The methods and compositions include, but are not limited to, methods and compositions for delivering an effective amount of a focal adhesion kinase (FAK) pathway modifier — including small molecule FAK inhibitors, FAK-targeting proteolysis-targeting chimeras (PROTACs), nucleic acid-based FAK expression modulators, and antibody or polypeptide FAK modifiers — to a subject in need thereof.BACKGROUND

[0002] Allergy emergencies can range from mild reactions to life-threatening conditions, with anaphylaxis being a severe and dangerous form. Anaphylaxis is a rapid-onset, systemic allergic reaction that affects multiple organ systems and can lead to fatal consequences if not treated immediately. Anaphylaxis is most commonly triggered by food allergens (such as peanuts, shellfish, and tree nuts), insect stings, certain medications, or latex. Once exposed to the allergen, a subject's immune system releases a flood of elements causing a sudden drop in blood pressure (anaphylactic shock), airway constriction, skin reactions, and gastrointestinal distress. Acute symptoms occur from within minutes to two hours after contact with the allergen, but in rare instances onset may be delayed by as much as four hours. Contact with anaphylaxis-inducing agents, and the severity of the resulting anaphylactic reaction, can be extremely unpredictable. Without prompt intervention, anaphylaxis can quickly progress to respiratory failure, cardiovascular collapse, or even death.

[0003] Recognizing the signs and symptoms of anaphylaxis is critical for timely treatment. Symptoms often include difficulty breathing due to airway swelling, wheezing, a rapid or weak pulse, dizziness, and a sudden drop in blood pressure. Skin reactions such as hives, itching, and swelling of the lips, face, or throat are common, as are nausea, vomiting, and abdominal pain. In severe cases, individuals may experience confusion, loss of consciousness, or cardiac arrest. To date, immediate administration of epinephrine has been recommended for the treatment of anaphylaxis. However, even after administration,emergency medical care is necessary in many cases, as symptoms can return in a second wave known as biphasic anaphylaxis.

[0004] While epinephrine has been the first-line treatment for anaphylaxis, there have been challenges associated with its use. Availability and delayed administration have been issues, and the cost of epinephrine injections and auto-injectors have been prohibitively expensive for many. Furthermore, while epinephrine can halt the immediate symptoms of anaphylaxis, it does not address the underlying allergic reaction, necessitating further medical management.

[0005] Beyond anaphylaxis, mast cell degranulation plays a central role in a range of allergic and inflammatory conditions including chronic urticaria, allergic asthma, mast cell activation syndrome, allergic rhinitis, and gastrointestinal allergic disorders. Current treatments for these conditions are limited in their ability to directly modulate the mast cell degranulation process itself, instead targeting downstream mediators or broadly suppressing immune function.

[0006] Allergen immunotherapy, including oral immunotherapy (OIT), has emerged as a strategy for desensitizing subjects to specific allergens. However, allergic side effects during immunotherapy — including anaphylaxis — remain a significant clinical concern and limit the safety, tolerability, and adoption of these approaches.SUMMARY

[0007] The present disclosure provides, in some embodiments, a method of preventing or reducing allergic reactions (e.g., acute allergic reaction such as anaphylaxis) to one or more allergens (e.g., an allergen associated with a food, beverage, or other orally introduced substance) in a subject in need thereof comprising administering to the subject a composition that modifies (e.g., inhibits) focal adhesion kinase (FAK) activity, function and / or expression. In some embodiments, FAK is inhibited by inhibiting FAK activity. In some embodiments, FAK is inhibited by administering a composition comprising a small molecule FAK inhibitor (FAKi), or a pharmaceutically acceptable salt thereof, disclosed herein. In some embodiments, the FAKi is an ATP-competitive inhibitor. In some embodiments, the FAKi comprises a heterocyclic ring structure (e.g., a pyrimidine or quinazoline based core) that mimics the adenine portion of ATP. In some embodiments, the FAKi forms hydrogen bonds with the backbone "hinge" region of FAK. In some embodiments, the FAKi comprises hydrophobic groups (e.g., that enhance binding affinity and / or selectivity). Examples of hydrophobic groups include, but are not limited to, aromatic rings and aliphatic chains. Insome embodiments, the hydrophobic groups extend into adjacent hydrophobic pockets near the ATP site. In some embodiments, the FAKi comprises one or more functional groups (e.g., that increase solubility and / or provide a desired pharmacokinetic profile). The disclosure is not limited by the type of functional group. For example, in some embodiments, the FAKi comprises a polar substituent (e.g., amide, sulfonamide, or carboxylic acid group) appended to the FAKi (e.g., that enhance solubility, adjust pharmacokinetic properties, and / or improve or enhance interactions with the FAK protein). Non-limiting examples of FAK inhibitors that find use in the compositions and methods of the present disclosure include, but are not limited to, Defactinib (VS-6063), GSK2256098, PF-431396, Narmafotinib, TAE-226, and PF-573228, PF4618433, PND1186, or pharmaceutically acceptable salts thereof. In other embodiments, FAK activity and / or expression is inhibited using an inhibitor of FAK production and / or activity. Exemplary agents include, but are not limited to, small molecules, isolated nucleic acids, vectors, isolated peptides, antibodies, peptide mimetics, and the like. In some embodiments, the composition comprising an inhibitor of FAK (e.g., a small molecule FAK inhibitor (FAKi) or a pharmaceutically acceptable salt thereof) is administered to the subject prior to exposure to the one or more allergens. In some embodiments, the composition comprising an inhibitor of FAK (e.g., a small molecule FAK inhibitor (FAKi) or a pharmaceutically acceptable salt thereof) is administered to the subject subsequent to exposure to the one or more allergens. In some embodiments, the composition comprising an inhibitor of FAK (e.g., a small molecule FAK inhibitor (FAKi) or a pharmaceutically acceptable salt thereof) further comprises epinephrine. In some embodiments, the composition comprising an inhibitor of FAK (e.g., a small molecule FAK inhibitor (FAKi) or a pharmaceutically acceptable salt thereof) and epinephrine is administered after exposure to the one or more allergens.

[0008] In some embodiments, the composition that inhibits FAK activity and / or expression comprises a proteolysis-targeting chimera (PROTAC) that targets FAK protein for proteasomal degradation. In some embodiments, the PROTAC is GSK215. In some embodiments, the PROTAC is FC11. In some embodiments, more than one PROTAC is used. In some embodiments, FAK-targeting PROTACs achieve dose-dependent degradation of FAK protein and / or reduce mast cell degranulation and anaphylaxis severity in vivo.

[0009] In some embodiments, the present disclosure provides a method of arresting or reducing the severity of an ongoing anaphylactic reaction in a subject, comprising administering to the subject a composition comprising a FAK inhibitor after the subject has been exposed to an allergen and after onset of one or more symptoms of anaphylaxis. In someembodiments, the composition is administered within 10 minutes, within 5 minutes, or within 1 minute after allergen exposure. While an understanding of a mechanism is necessary to practice methods of the disclosure, and the disclosure is not limited to any specific mechanism of action, in some embodiments, FAK inhibition arrests mast cell degranulation that is already in progress thereby significantly reducing anaphylaxis severity even when administered after allergen challenge.

[0010] In some embodiments, the present disclosure provides a method of preventing or reducing one or more symptoms associated with an allergic reaction in a subject in need thereof comprising administering a composition that inhibits FAK activity. In some embodiments, the composition that inhibits FAK activity comprises one or more small molecule FAK inhibitors or pharmaceutically acceptable salt thereof. The disclosure is not limited by the one or more symptoms associated with an allergic reaction prevented or reduced. Indeed, a variety of symptoms associated with an allergic reaction treated (e.g., prevented or reduced) include, but are not limited to, anaphylaxis, diarrhea, colonic inflammation, nausea, and / or abdominal pain.

[0011] In some embodiments, the present disclosure provides a method of reducing IgE-mediated mast cell degranulation in a subject in need thereof, comprising administering to the subject a composition comprising a FAK pathway modifier that inhibits FAK activity, expression, or signaling. In some embodiments, reducing IgE-mediated mast cell degranulation treats or prevents one or more conditions selected from anaphylaxis, urticaria, allergic asthma, bronchoconstriction, mast cell activation syndrome, diarrhea, and abdominal pain. In some embodiments, the FAK pathway modifier is a small molecule FAK inhibitor, a FAK-targeting PROTAC, or a nucleic acid that reduces FAK expression.

[0012] In some embodiments, the disclosure provides a composition comprising (a) one or more inhibitors of FAK (e.g., small molecule FAK inhibitor (FAKi)) and (b) one or more allergens.

[0013] In some embodiments, the present disclosure provides a method of treating an allergy (e.g., to a food, beverage, or other orally introduced substance) in a subject comprising administering a dose of a composition comprising an allergen and one or more inhibitors of FAK (e.g., small molecule FAK inhibitor (FAKi)). In some embodiments, the one or more inhibitors of FAK comprise one or more small molecule FAK inhibitors described herein or a pharmaceutically acceptable salt thereof.

[0014] In some embodiments, the present disclosure provides a method of reducing an allergic reaction to an allergen during allergen immunotherapy in a subject undergoingallergen immunotherapy, comprising co-administering to the subject a therapeutically effective amount of a FAK inhibitor with one or more doses of the allergen. In some embodiments, the allergen immunotherapy is oral immunotherapy (OIT). In some embodiments, the FAK inhibitor is administered prior to each dose of allergen. In some embodiments, co-administration of the FAK inhibitor reduces the incidence or severity of allergic side effects during the allergen immunotherapy.

[0015] In some embodiments, a composition of the disclosure (e.g., comprising one or more FAK inhibitors) is used for the treatment of an allergic medical condition for which epinephrine is useful. The disclosure is not limited by the type of medical condition. Indeed, a variety of medical conditions may be treated including, but not limited to, anaphylaxis.

[0016] In some embodiments, the disclosure provides a method of improving at least one symptom of an epinephrine-requiring medical condition in an individual in need thereof, comprising administering to the individual a formulation comprising one or more inhibitors of FAK (e.g., small molecule FAK inhibitor (FAKi)) and a pharmaceutically acceptable carrier. In a specific embodiment, the medical condition is anaphylaxis.

[0017] In another embodiment, the disclosure provides a method of treating anaphylaxis in a subject comprising administering to the subject a formulation, the formulation comprising one or more FAK inhibitors (e.g., small molecule FAK inhibitor (FAKi)) and a pharmaceutically acceptable carrier. The disclosure is not limited by the type or route of administration. Indeed, any type or route of administration may be used including, but not limited to, those currently in use for the administration of FAK inhibitors in the treatment of cancer.

[0018] In a further embodiment, the disclosure provides a method of treating a pediatric subject in need of treatment for anaphylaxis comprising administering to the pediatric subject a formulation comprising one or more FAK inhibitors (e.g., small molecule FAK inhibitor (FAKi)) and a pharmaceutically acceptable carrier.

[0019] The compositions and methods of the disclosure may be applied to any subject, but in specific embodiments the disclosure is useful for a mammal, including a human, dog, cat, horse, cow, goat, sheep, and others.

[0020] The disclosure is not limited by the type of formulation comprising a composition comprising one or more FAK inhibitors (e.g., small molecule FAK inhibitor (FAKi)) described herein. In some embodiments, the formulation is an injectable pharmaceutical composition. In some embodiments, the injectable composition comprises one or more FAK inhibitors (e.g., small molecule FAK inhibitor (FAKi)) and EDTA. In some embodiments, theinjectable composition comprises one or more FAK inhibitors (e.g., small molecule FAK inhibitor (FAKi)) and DMSO. In another embodiment, the composition further comprises at least one antioxidant (e.g., cysteine, citric acid, thioglycerol, acetylcysteine, or a combination thereof). In another embodiment, the composition further comprises at least one stabilityenhancing agent described herein. In some embodiments, the composition comprising a FAK inhibitor is formulated for delivery via an auto-injector device. In some embodiments, the auto-injector device is configured for intramuscular or subcutaneous injection. In some embodiments, the auto-injector device is configured for self-administration by the subject or administration by a non-medical professional. In some embodiments, the auto-injector device contains a composition comprising a FAK inhibitor and epinephrine.

[0021] In some embodiments, the composition comprising a FAK inhibitor is coadministered with one or more agents selected from an anti-IgE antibody (e.g., omalizumab or ligelizumab), an inhibitor of IL-4 and / or IL-13 signaling (e.g., dupilumab), an inhibitor of IL-5 and / or IL-5 receptor (e.g., mepolizumab, benralizumab, or reslizumab), or an inhibitor of thymic stromal lymphopoietin (TSLP) (e.g., tezepelumab).

[0022] The foregoing has described the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood.Additional features and advantages of the disclosure to those described above will be described hereinafter and in totality provide basis for the subject of the claims. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other compositions, formulations or structures for carrying out the same purposes of the present disclosure. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the disclosure as set forth in the appended claims. The novel features which are believed to be characteristic of the disclosure, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows gene expression of samples taken immediately after anaphylaxis was identified and compared to baseline. Left: Over 200 genes were observed to be differentiallyexpressed and met inclusion criteria. Right: Upregulated pathways associated with anaphylaxis vs baseline defined using Ingenuity pathway analysis (Qiagen). Only those exceeding the significance threshold shown were included.

[0024] FIG. 2 shows the structural domains of focal adhesion kinase (FAK), also known as PTK2, or FAK1. FAK is a ubiquitously expressed non-receptor tyrosine kinase encoded by the PTK2 gene on chromosome 8 (15 in mice) and is subdivided into three major domains: C-terminal focal adhesion targeting (FAT) domain; N-terminal cell membrane docketing domain; and central kinase domain.

[0025] FIG. 3 shows the structural domains of proline-rich tyrosine kinase 2 (Pyk2), also known as PTK2B, or FAK2. Pyk2 / FAK2 is a non-receptor tyrosine kinase encoded by the PTK2B gene on chromosome 8 (14 in mice) and is primarily expressed in neuronal, hematopoietic, and endothelial cells.

[0026] FIG. 4 shows a variety of pathologies associated with FAK.

[0027] FIGS. 5A and 5B show the structures of multiple small molecule FAK inhibitors: Defactinib (VS-6303) (FIG. 5A); and GSK2256098 (FIG. 5B).

[0028] FIG. 6 shows an in vitro model used to characterize FAK involvement with cell adhesion and degranulation.

[0029] FIG. 7 provides a schematic of the beta hexosaminidase assay used to measure P-hexosaminidase release from cells used to assess in vitro mast cell granule release.

[0030] FIG. 8 shows the effect of FAK loss-of-function (FAK / Pyk2 inhibitor PF-431396, 1-5pM for 30 minutes prior to stimulation) on mast cell degranulation. PF-431396 (FIG. 8A) inhibits degranulation in LAD2 cells (FIG. 8C) and in RBL-2H3 cells (FIG. 8B).

[0031] FIG. 9 shows the effect of FAK loss-of-function (FAK / Pyk2 inhibitor defactinib (VS-6063), l-10pM for 1 hour prior to stimulation) on mast cell degranulation.Defactinib / VS-6063 (FIG. A) inhibits degranulation in LAD2 cells (FIG. 9C) and in RBL-2H3 cells (FIG. 9B).

[0032] FIG. 10 shows the effect of FAK loss-of-function (FAK inhibitor narmafotinib, 1-lOpM for 60 minutes prior to stimulation) on mast cell degranulation. Narmafotinib (FIG. 10A) inhibits degranulation in LAD2 cells (FIG. 10C) and in RBL-2H3 cells (FIG. 10B).

[0033] FIG. 11 shows the effect of FAK loss-of-function (FAK inhibitor GSK2256098, 0.1-lOpM for 30 minutes prior to stimulation) on mast cell degranulation. GSK2256098 (FIG. 11 A) shows minimal inhibitory effect on degranulation in either LAD2 cells (FIG. 11C) or RBL-2H3 cells (FIG. 1 IB).

[0034] FIG. 12 shows flow cytometry quantification of the surface expression of CD117, FCsRl, and CD107a on LAD2 cells incubated with PF-431396 for 30 minutes prior to stimulation for 10 minutes.

[0035] FIG. 13 shows PF-431396 dose-dependent reduction of the surface expression of CD63 and CD 107a for both IgE- and non-IgE dependent stimulation of LAD2 cells.

[0036] FIG. 14 shows that the FAK inhibitor PF-431396 significantly and selectively decreased adhesion in IgE-mediated / stimulated LAD2 cells, but did not alter / reduce adhesion in IgE-independent ionomycin stimulated cells.

[0037] FIG. 15 shows degranulation-induced adhesion with IgE-dependent stimulation was significantly reduced in RBL-2H3 cells in a dose dependent manner by PF-431396, whereas there was a minimal reduction in adhesion with IgE-independent stimulation.

[0038] FIG. 16 shows in vivo models of Passive Systemic Anaphylaxis (PSA) and Active Systemic Anaphylaxis (ASA) studied during development of embodiments of this disclosure.

[0039] FIG. 17 shows reduction in anaphylaxis scores for both Passive Systemic Anaphylaxis (PSA) and Active Systemic Anaphylaxis (ASA), as well as significant reversal of decreased temperatures observed after treatment with PF-431396 compared to vehicle treated controls.

[0040] FIG. 18 shows no effect on relative FAK detection or association to cytoskeleton was observed after introduction of PF-431396 (5uM) for 30 minutes prior to stimulation (30 minutes).

[0041] FIG. 19 shows a decrease in phosphorylated FAK (pFAK) associated with actin cytoskeleton observed after introducing PF-431396 (5uM) for 30 minutes prior to stimulation. Phosphorylated FAK (pFAK) appeared more centrally located following inhibitor treatment.

[0042] FIG. 20 shows expanded characterization of FAK signaling upregulation following food anaphylaxis. FIG. 20A provides a heatmap showing expression of FAK signaling pathway members (IL5RA, PTGDR2, S1PR3, PDGFRB, S1PR5, ADGRG1, TRGC1, IL18RAP, TGFBR3, CX3CR1, IL2RB, ERBB2, PTGDR, TRDC, TRGV9) in non-reactive (pre vs. post) and reactive (pre vs. post) patient comparisons. FIG. 20B shows FAK expression across key mucocutaneous tissue types (skin, tongue, small intestine) as demonstrated by the human tissue reference atlas Tabula Sapiens (Reference: Tabula Sapiens Consortium et al., Science. 2022 May 13;376(6594):eabl4896). FIGS. 20A and 20B supplement FIG. 1 by providing gene-level resolution of the FAK signaling pathway upregulation, and by confirming FAK expression in tissues relevant to allergen exposure and anaphylaxis, respectively).

[0043] FIG. 21 shows FAK pathway genes associated with FCsRl-regulated degranulation. FIG. 21 A: Top panel: Violin plot of Spearman correlation values for anti-IgE, ionomycin, and unstimulated conditions showing that cells with degranulation RNA signatures have highest correlation with FAK pathway gene upregulation in the anti-IgE condition. Bottom panels: Scatter plots showing correlation of mast cell degranulation gene sets (dark squares) with FAK pathway genes; slope = 0.88 for anti-IgE vs. unstimulated (left); slope = 0.98 for ionomycin vs. unstimulated (right). FIG. 2 IB: Heatmap of FAK signaling pathway member gene expression across Control, Anti-IgE, and Ionomycin conditions, with hierarchical clustering. Genes shown include PARVB, CDC42, PIK3R3, GRB2, VEGFB, TLN, VASP, MYL12A, BAIAP2, PRKCA, CCND3, RAC2, RAP1A, MYL12B, ACTG1, BRAF, ACTB, BAD, ITGA9, PFN1, PPP1CC, RAC1, PPP1CA, PTK2, ITGB1, PIK3CA, THBS1, ACTN4, GSK3B, DIAPH1, VCL, TLN1, PTK2B, ITGA2B, LAMA2, PAK2, CTNNB1, BCAR1, ARHGAP35, VAV1, MAPK1, MAP2K1, HRAS, PRKCB, and RAPGEF1. FIG. 21C: Dot plot showing percent expressed and average expression of FCsRI signaling pathway module genes (PIK3CD, PRKCD, NRAS, HRAS, MAPK1, MAPK14, MAP2K1, VAV1, PIK3R3, PRKCB, GRB2, FCER1G) across Unstimulated, Anti-IgE, and Ionomycin conditions. FIG.21D: Spearman correlation heatmap of genes positively and significantly associated with FCsRI signaling pathway modules in each treatment group. FIG. 21E: -loglO(FDR) heatmap for enrichment analysis on genes with top 200 Spearman correlation with FCsRI modules in unstimulated, anti-IgE, and ionomycin gene sets. Enrichment terms identified include: positive regulation of GTPase activity (GO: 0043547), regulation of GTPase activity (G0:0043087), plasma membrane bounded cell projection morphogenesis (G0:0120039), regulation of small GTPase mediated signal transduction (G0:0051056), vesicle-mediated transport (G0:0016192), protein modification process (G0:0036211), regulation of cell adhesion (G0:0030155), regulation of cell activation (G0:0050865), ephrin receptor signaling pathway (G0:0048013), actin filament organization (G0:0007015), actin cytoskeleton reorganization (G0:0031532), protein phosphorylation (G0:0006468), protein autophosphorylation (G0:0046777), Fc receptor signaling pathway (G0:0038093), antigen receptor-mediated signaling pathway (G0:0050851), and regulation of leukocyte degranulation (GO: 0043300).

[0044] FIG. 22 shows FCsRI signaling pathway modules are more associated with cell adhesion related functions in IgE-stimulated than unstimulated and ionomycin-stimulated conditions. FIG. 22A: Boxplot of FCsRI module expression in unstimulated, anti-IgE, and ionomycin treatment groups. FIG. 22B: Scatter plot of FCsRI genes log(FC) fromunstimulated to anti-IgE and unstimulated to ionomycin; red dots indicate genes with adjusted p < 0.05 in unstimulated and ionomycin comparison, squared dots indicate genes with adjusted p < 0.05 in unstimulated and anti-IgE comparison. FIG. 22C: Spearman correlation density plot for genes positively and significantly associated with FCsRI signaling pathway modules in each treatment; solid grey shaded curves indicate genes in mast cell degranulation pathway; dashed curves indicate other genes.

[0045] FIG. 23 shows expanded in vitro degranulation with BMMC, surface markers, and dual inhibitor comparison. FIG. 23 A: Beta-hexosaminidase release in response to stimulation from RBL-2H3 (left), BMMC (center), and LAD2 (right) cells pretreated with dose range of PF-431396. FIG. 23B: Beta-hexosaminidase release in response to stimulation from RBL-2H3 (left), BMMC (center), and LAD2 (right) cells pretreated with dose range of defactinib. FIG. 23C: CD63 (left) and CD107a (right) surface expression in response to stimulation from LAD2 cells pretreated with dose ranges of PF-431396. FIG. 23D: CD63 (left) and CD107a (right) surface expression in response to stimulation from LAD2 cells pretreated with dose ranges of defactinib.

[0046] FIG. 24A shows viability of RBL-2H3, BMMC, and LAD2 cells following pretreatment for one hour with dose range of PF-431396, as measured by CELLTITERGLO® luminescent assay. FIG. 24B shows viability of RBL-2H3, BMMC, and LAD2 cells following pretreatment for one hour with dose range of defactinib, as measured by CELLTITERGLO® luminescent assay. FIG. 24C: Gating strategy for flow cytometric analysis of CD63, CD107a surface expression on LAD2 cells following stimulation, with target population defined by singlet live cells, and separately evaluated for FCsRl, CD117, and CD63 or CD 107a expression.

[0047] FIG. 25A shows FAK autophosphorylation (Tyr 397) in RBL-2H3 cells rapidly increases with IgE- and non-IgE-mediated activation. Top blot uses Cell Signaling antibody (3283), bottom blot uses Abeam antibody (ab81298). Time points: Vehicle, Im, 5m, 10m, 15m for DNP-HSA (lOOng / mL) and Ionomycin (5pg / mL). FIG. 25B shows FAK autophosphorylation (Tyr 397) in RBL-2H3 cells due to early IgE- and non-IgE-mediated activation (5m) is effectively inhibited by FAK kinase inhibitors defactinib (D, lOpM) and PF-431396 (P, 5pM). Conditions: Vehicle (V), Defactinib (D), PF-431396 (P) for Unstimulated, DNP-HSA (lOOng / mL), and Ionomycin (5pg / mL).

[0048] FIG. 26 shows in vitro degranulation is augmented by FAK activation. FIG. 26A: Beta-hexosaminidase release in response to stimulation from RBL-2H3 cells pretreated with dose range of adhesamine (CAS 462605-73-8) for 24 hours. Conditions: Unstimulated, DNP-HSA (lOOng / mL), and lonomycin (5pg / mL) with Vehicle, O.lpM, IpM, and lOpM adhesamine. FIG. 26B: Beta-hexosaminidase release in response to stimulation from RBL-2H3 cells pretreated with dose range of ZINC40099027 (CAS 1211825-25-0) for one hour. Conditions: Unstimulated, DNP-HSA (lOOng / mL), and lonomycin (5pg / mL) with Vehicle, O.lpM, IpM, and lOpM ZINC40099027.

[0049] FIG. 27 shows FAK inhibition interrupts activation-induced adhesion and cell spreading. FIG. 27A: Adhesion of semi-adherent LAD2 cells to a fibronectin matrix following stimulation (unstimulated, anti-IgE lOOng / mL, ionomycin Ipg / mL) and dosedependent FAK inhibition with PF-431396 (O.OOlpM, O.OlpM, O.lpM). FIG. 27B: Adhesion of adherent RBL-2H3 cells to a fibronectin matrix following stimulation (unstimulated, DNP-HSA lOOng / mL, ionomycin 5pg / mL) and FAK inhibition with PF-431396 (LOpM, 5.0pM). FIG. 27C: Representative immunofluorescence morphology of RBL-2H3 cells under three conditions: unstimulated / vehicle (top row), algE / vehicle (middle row), and algE / defactinib lOpM (bottom row). Each row shows four channels: DAPI (nuclear, blue), Phalloidin (actin, green), pFAK Y397 (Abeam, red), and Merge. Scale bar = 40pm. FIG. 27D: Representative immunofluorescence morphology of LAD2 cells under same three conditions as FIG. 27C. Each row shows DAPI, Phalloidin, pFAK, and Merge. Scale bar = 10pm.

[0050] FIG. 28 shows in vivo temperature declines and anaphylaxis scores are decreased by FAK inhibition in both passive and active anaphylaxis models. FIG. 28A: Graphic representation of passive systemic anaphylaxis (PSA) and active systemic anaphylaxis (ASA) models with FAK inhibitor dosing protocol. PSA: Anti-DNP IgE (Day 0) — DNP-HSA challenge (Day 1) with FAKi 5h prior. ASA: OVA-alum sensitization (Day 0) — Ovalbumin challenges (Days 14-28) with FAKi 5h prior to final challenge. FIG. 28B: Maximum anaphylaxis scores over 60-minute monitoring period. Top: PSA model showing scores for Untreated, DNP, DNP+PF-431396, and DNP+defactinib. Bottom: ASA model showing scores for Untreated, Ova, Ova+PF-431396, and Ova+defactinib. FIG. 28C: Maximum temperature change (AC) at 15 minutes post-challenge. Top: PSA. Bottom: ASA. Four groups per model as in FIG. 28B. FIG. 28D: Temperature (°C) over 60-minute monitoring period. Top: PSA. Bottom: ASA. Groups: Untreated, allergen-only, +PF-431396, +defactinib. FIG. 28E: Left: Diarrhea score in ASA model. Right: Percentage of mice with diarrhea in ASA model.

[0051] FIG. 29 shows in vivo anaphylaxis scoring and temperature decline are not affected by FAK inhibition in histamine model. Mice were treated with 6-8mg histamine via tail vein injection. FAK inhibitors PF-431396 and defactinib (lOmg / kg) were introduced via twointraperitoneal doses 5h prior to challenge. FIG. 29A: Maximum anaphylaxis scores over 60-minute monitoring period (Vehicle, Histamine, Histamine+PF-431396). FIG. 29B: Maximum temperature change (AC) over 60-minute monitoring period. FIG. 29C: Temperature (°C) over 60-minute monitoring period (Vehicle, Histamine, Histamine+PF-431396).

[0052] FIG. 30 shows active systemic anaphylaxis model yields significant anaphylaxis scoring and temperature declines across six challenges prior to introduction of FAK inhibition. FIG. 30A shows graphic representation of active systemic models. FIG. 30B: Maximum anaphylaxis scores across challenges 1-6 (Vehicle vs. Ova groups). FIG. 30C: Temperature change at 15 minutes following challenge across challenges 1-6. FIG. 30D: Percentage of mice with diarrhea across challenges 1-6.

[0053] FIG. 31 shows FAK degradation via PROTAC contributes to reduced degranulation in vitro and significant amelioration of anaphylaxis in vivo. FIG. 31 A. FAK protein quantification via western blot following pretreatment of LAD2 cells with dose range of GSK215 PROTAC or vehicle (0.01% DMSO / v) for 24 hours, normalized to GAPDH expression and plotted as percentage of vehicle-treated control. FIG. 3 IB. Betahexosaminidase release in response to stimulation from LAD2 cells pretreated with GSK215 PROTAC. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, p < 0.0001. Error bars indicate S.E.M., statistics based on analysis via multiple unpaired t tests. FIG. 31C. FAK protein quantification via western blot following pretreatment of LAD2 cells with dose range of FC11 PROTAC or vehicle (0.01% DMSO / v) for 24 hours, normalized to GAPDH expression and plotted as percentage of vehicle-treated control. FIG. 3 ID. Beta-hexosaminidase release in response to stimulation from LAD2 cells pretreated with FC11 PROTAC. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, p < 0.0001. Error bars indicate S.E.M., statistics based on analysis via multiple unpaired t tests. FIG. 31E-F. Temperature change over 60-minute monitoring period and temperature change at 15-minutes following challenge (E) as well as maximum anaphylaxis scores over 60-minute monitoring period (F) in passive systemic anaphylaxis model treated with vehicle or GSK215 PROTAC and compared to untreated controls.

[0054] FIG. 32 shows in vitro degranulation and in vivo anaphylaxis are arrested by FAK inhibition following stimulation in a time-dependent manner. Sensitized RBL-2H3, LAD2, and BMMC cells were stimulated then treated with vehicle or lOpM defactinib at Im, 5m, and 10m post-stimulation. FIG. 32A: Beta-hexosaminidase release from sensitized RBL-2H3 (left), LAD2 (center), and BMMC (right) cells. Cells were first stimulated, then treated with vehicle or lOpM defactinib at Im, 5m, and 10m post-stimulation. Conditions: Unstimulatedand stimulated (DNP-HSA for RBL-2H3 / BMMC; Anti-IgE for LAD2) with Vehicle and defactinib at each post-stimulation timepoint. FIG. 32B: In vivo PSA rescue model. Top left: Graphic representation of passive systemic model with post-challenge rescue dosing (defactinib at Im and 5m post-challenge). Top center: Maximum anaphylaxis scores over 60-minute monitoring period (Untreated, DNP, DNP+defactinib Im, DNP+defactinib 5m). Top right: Maximum temperature change (AC) at 15 minutes post-challenge. Bottom:Temperature (°C) over 60-minute monitoring period (Vehicle, DNP, +defactinib Im, +defactinib 5m).DETAILED DESCRIPTIONDefinitions

[0055] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0056] The term "FAK pathway modifier" as used herein refers to any agent that modifies FAK activity, FAK expression, and / or FAK signaling. FAK pathway modifiers include, but are not limited to, small molecule FAK kinase inhibitors, proteolysis-targeting chimeras (PROTACs) that target FAK protein for degradation, nucleic acid molecules (e.g., siRNA, shRNA, antisense oligonucleotides) that reduce FAK mRNA expression, antibodies that bind FAK or proteins involved in FAK signaling, and polypeptide inhibitors of FAK activity or production. A FAK pathway modifier may modify FAK1 (PTK2), FAK2 (Pyk2 / PTK2B), or both.

[0057] The term "proteolysis-targeting chimera" or "PROTAC" as used herein refers to a heterobifunctional molecule comprising (a) a ligand that binds a target protein (e.g., FAK) and (b) a ligand that recruits an E3 ubiquitin ligase, thereby promoting ubiquitination and subsequent proteasomal degradation of the target protein. Unlike kinase inhibitors, which inhibit FAK catalytic activity while leaving the protein intact, PROTACs eliminate FAK protein, thereby abolishing both kinase-dependent and kinase-independent (e.g., scaffolding) functions of FAK. Non-limiting examples of FAK-targeting PROTACs include GSK215 and FC11.

[0058] The term "mast cell degranulation" as used herein refers to the process by which mast cells release the contents of their intracellular granules — including histamine, proteases, cytokines, and other mediators — into the extracellular environment. Mast cell degranulationmay be IgE-mediated (e.g., triggered by crosslinking of FcsRI by antigen-bound IgE) or IgE-independent (e.g., triggered by complement fragments, neuropeptides, or calcium ionophores such as ionomycin). Markers of mast cell degranulation include, but are not limited to, betahexosaminidase release and surface expression of CD63 and CD107a (LAMP-1).

[0059] The term "auto-injector" as used herein refers to a medical device designed to deliver a pre-measured dose of a pharmaceutical composition via injection, typically intramuscular or subcutaneous injection, and configured for self-administration or administration by a nonmedical professional.

[0060] The term "allergen immunotherapy" as used herein refers to the repeated administration of increasing doses of an allergen to a subject with the goal of inducing immunological tolerance or desensitization to the allergen. Allergen immunotherapy includes, but is not limited to, subcutaneous immunotherapy (SCIT), sublingual immunotherapy (SLIT), oral immunotherapy (OIT), and epicutaneous immunotherapy (EPIT).

[0061] The term "ongoing anaphylactic reaction" as used herein refers to an anaphylactic episode in which a subject has been exposed to an allergen and has developed one or more signs or symptoms of anaphylaxis, and in which the anaphylactic process — including mast cell degranulation and release of allergic mediators — is actively occurring at the time of therapeutic intervention.

[0062] As used herein the terms "disease" and "pathologic condition" are used interchangeably, unless indicated otherwise herein, to describe a deviation from the condition regarded as normal or average for members of a species or group (e.g., humans), and which is detrimental to an affected individual under conditions that are not inimical to the majority of individuals of that species or group. Such a deviation can manifest as a state, signs, and / or symptoms (e.g., that occur during an acute allergic reaction such as shortness of breath, diarrhea, nausea, fever, pain, blisters, boils, rash, immune suppression, inflammation, etc.) that are associated with any impairment of the normal state of a subject or of any of its organs or tissues that interrupts or modifies the performance of normal functions. A disease or pathological condition may be caused by or result from contact with an antigen / allergen (e.g., food allergen), may be responsive to environmental factors (e.g., malnutrition, industrial hazards, and / or climate), may be responsive to an inherent or latent defect in the organism (e.g., genetic anomalies) or to combinations of these and other factors.

[0063] The terms "a medical condition for which epinephrine is useful," "an epinephrine-requiring medical condition," and the like are used interchangeably herein to refer to anymedical condition wherein administration of epinephrine to an individual having the condition has previously been shown to have a pharmacologically beneficial effect, such as improving at least one symptom of the medical condition. For example, the medical condition may be acute hypersensitivity, such as an anaphylactic reaction (e.g., resulting in anaphylaxis) to one or more drugs; to animal serums, such as from a bee, wasp, or ant; to plant allergens, including peanuts; and to other allergens. The disclosure is not limited by the type of medical condition. In some embodiments, the medical condition comprises an asthmatic condition, such as bronchospasm.

[0064] The terms "host," "subject," or "patient" are used interchangeably herein to refer to an individual to be treated by (e.g., administered) the compositions and methods of the present disclosure. Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans. In the context of the disclosure, the term "subject" generally refers to an individual who will be administered or who has been administered one or more compositions of the present disclosure.

[0065] The term "anaphylaxis" as used herein refers to an acute hypersensitivity reaction as a result of exposure to an allergen / antigen, such as a previously encountered allergen / antigen or to a drug, for example. In specific embodiments, the symptoms may include rapidly progressing urticaria, respiratory distress, vascular collapse, and / or systemic shock.

[0066] The term "solution" refers to an aqueous or non-aqueous mixture.

[0067] The term "antioxidant" as used herein refers to a material that prevents oxidation.

[0068] A "disorder" is any condition or disease that would benefit from treatment with a composition or method of the disclosure. This includes chronic and acute disorders including those pathological conditions which predispose a subject to the disorder in question. Nonlimiting examples of disorders to be treated herein include conditions such as anaphylaxis.

[0069] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0070] The term "therapeutically effective amount," as used herein, refers to that amount of the therapeutic agent sufficient to result in amelioration of one or more symptoms of a disorder, or prevent advancement of a disorder, or cause regression of the disorder.

[0071] As used herein, the terms "purified" or "to purify" refer to the removal of contaminants or undesired compounds from a sample or composition. As used herein, the term "substantially purified" refers to the removal of from about 70 to 90%, up to 100%, of the contaminants or undesired compounds from a sample or composition.

[0072] As used herein, the terms "administration" and "administering" refer to the act of giving a composition of the present disclosure to a subject. Exemplary routes of administration to the human body include, but are not limited to, through the eyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intraperitoneally, intratumorally, etc.), topically, and the like.

[0073] As used herein, the terms "co-administration" and "co-administering" refer to the administration of at least two agent(s) (e.g., a composition comprising a FAK inhibitor and one or more other agents (e.g., epinephrine or other substance with allergy mitigation properties)) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. In some embodiments, co-administration can be via the same or different route of administration. Those of skill in the art understand that the formulations and / or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and / or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.

[0074] The terms "pharmaceutically acceptable" or "pharmacologically acceptable," as used herein, refer to compositions that do not substantially produce adverse reactions (e.g., toxic, allergic or other immunologic reactions) when administered to a subject.

[0075] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, and various types of wetting agents (e.g., sodium lauryl sulfate), any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), polyethylene glycol, and the like. The compositions also can include stabilizers and preservatives. Examples of carriers, stabilizers and adjuvants have been described and are known in the art (see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., MackPubl. Co., Easton, Pa. (1975), incorporated herein by reference).

[0076] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of immunotherapeutic agents, such delivery systems include systems that allow for the storage, transport, or delivery of immunogenic agents and / or supporting materials (e.g., written instructions for using the materials, etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant immunotherapeutic agents. As used herein, the term "fragmented kit" refers to delivery systems comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain a composition comprising an immunotherapeutic composition for a particular use, while a second container contains a second agent. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term "fragmented kit." In contrast, a "combined kit" refers to a delivery system containing all of the components of an immunogenic agent needed for a particular use in a single container (e.g., in a single box housing each of the desired components). The term "kit" includes both fragmented and combined kits.

[0077] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0078] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified.

[0079] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly oneof." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0080] The term "about" as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within an acceptable standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to ± 20%, preferably up to ± 10%, more preferably up to ± 5%, and more preferably still up to ± 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term "about" is implicit and in this context means within an acceptable error range for the particular value.

[0081] Methods and compositions for treating, preventing, or reducing allergic reactions, including acute allergic reactions such as anaphylaxis, and mast cell-mediated disorders.

[0082] Food allergy affects nearly 10% of the United States population, contributing to 1% of all emergency room visits and approximately $30 billion in healthcare costs annually. Despite this significant morbidity and resource utilization treatment of food allergy remains limited, and largely relies on food avoidance with only two FDA approved therapies.

[0083] Crosslinking of the high-affinity immunoglobulin E (IgE) receptor and subsequent release of allergic mediators have been well characterized in mast cell activation.Experiments conducted during development of embodiments of this disclosure have observed that, while the allergic mediators require up to 15 minutes to induce effect, dermal epithelial changes as well as differential expression of signaling pathways can be measured immediately upon allergen exposure (See Example 1). Among these pathways, focal adhesion kinase (FAK) signaling was the most highly upregulated immediately following human food reaction (Example 1). FAK is a ubiquitously expressed non-receptor tyrosine kinase important for cytoskeletal organization as well as transcriptional regulation across a broad range of biological processes, and has been a drug target of interest in oncology clinical trials employing FAK inhibitors (e.g., defactinib). As shown in the Examples, experiments were conducted in order to determine what role FAK and FAK signaling might play in acute allergic reactions such as anaphylaxis and whether the propagation of early anaphylaxis signaling relies on adjunctive processes surrounding adhesion and FAK signaling. As disclosed in detail herein, the disclosure identifies FAK and FAK signaling as targets for treating allergy to a food, beverage, or other orally introduced substance in a subject (e.g., fortreating anaphylaxis) comprising administering a composition comprising one or more FAK inhibitors described herein or a pharmaceutically acceptable salt thereof.

[0084] Thus, the disclosure is based in part on the discovery that focal adhesion kinase (FAK) signaling is rapidly and significantly upregulated during allergic reactions, including food anaphylaxis, and that FAK plays a functional role in IgE-mediated mast cell degranulation, activation-induced mast cell adhesion, and associated allergic symptomology. FAK and FAK signaling were identified as targets for reducing allergic symptomology, including but not limited to anaphylaxis, and for reducing mast cell degranulation in mast cell-mediated disorders. Accordingly, in some embodiments the disclosure is directed towards reducing allergic symptomology and / or mast cell degranulation by modifying FAK (e.g., inhibiting FAK activity, reducing FAK expression via targeted protein degradation or nucleic acid-mediated knockdown, and / or modulating FAK signaling). In some embodiments, modification of FAK comprises modifying FAK1 (e.g., inhibiting FAK1 activity, expression, and / or signaling). In other embodiments, modification of FAK comprises modifying FAK2 (e.g., inhibiting FAK2 activity, expression, and / or signaling). In some embodiments, modification of FAK comprises modifying FAK1 and FAK2 (e.g., inhibiting FAK1 and FAK2 activity, expression, and / or signaling). In some embodiments, modification of FAK comprises targeted degradation of FAK protein (e.g., via a PROTAC).

[0085] In certain embodiments, the disclosure is directed to FAK modification.

[0086] In some embodiments, FAK modification is accomplished by inhibition of FAK production and / or activity. In some embodiments, FAK is inhibited by inhibiting FAK activity. In some embodiments, FAK is inhibited by administering a composition comprising a small molecule FAK inhibitor (FAKi) disclosed herein (e.g., in FIGS. 5, 8-11, 23, and throughout the disclosure) or a pharmaceutically acceptable salt thereof. In other embodiments, FAK activity and / or expression is inhibited using an inhibitor of FAK production and / or activity. Exemplary agents include, but are not limited to, small molecules, isolated nucleic acids, vectors, isolated peptides, antibodies, peptide mimetics, proteolysistargeting chimeras (PROTACs), and the like.

[0087] FAK-Targeting Proteolysis-Targeting Chimeras (PROTACs). In some embodiments, FAK pathway modification is achieved by targeted degradation of FAK protein using a proteolysis-targeting chimera (PROTAC). PROTACs are heterobifunctional molecules that simultaneously bind a target protein and an E3 ubiquitin ligase, thereby promoting ubiquitination and proteasomal degradation of the target protein. In some embodiments, the PROTAC targets FAKI (PTK2). In some embodiments, the PROTAC targets FAK2(Pyk2 / PTK2B). In some embodiments, the PROTAC targets both FAKI and FAK2. As demonstrated in Example 10, the FAK-targeting PROTACs GSK215 and FC11 achieved dose-dependent degradation of FAK protein in LAD2 human mast cells, reduced IgE-mediated mast cell degranulation in vitro, and significantly ameliorated anaphylaxis in a passive systemic anaphylaxis model in vivo (See FIG. 31A-E). Non-limiting examples of FAK-targeting PROTACs that find use in the compositions and methods of the present disclosure include, but are not limited to, GSK215 and FC11.

[0088] Small molecule FAK inhibitors. In some embodiments, the FAK inhibitor is a small molecule FAK inhibitor (FAKi). In some embodiments, the FAKi is an ATP-competitive inhibitor. In some embodiments, the FAKi comprises a heterocyclic ring structure (e.g., a pyrimidine or quinazoline based core) that mimics the adenine portion of ATP. In some embodiments, the FAKi forms hydrogen bonds with the backbone "hinge" region of FAK. In some embodiments, the FAKi comprises hydrophobic groups (e.g., that enhance binding affinity and / or selectivity). Examples of hydrophobic groups include, but are not limited to, aromatic rings and aliphatic chains. In some embodiments, the hydrophobic groups extend into adjacent hydrophobic pockets near the ATP site. In some embodiments, the FAKi comprises one or more functional groups (e.g., that increase solubility and / or provide a desired pharmacokinetic profile). The disclosure is not limited by the type of functional group. For example, in some embodiments, the FAKi comprises a polar substituent (e.g., amide, sulfonamide, or carboxylic acid group) appended to the FAKi (e.g., that enhance solubility, adjust pharmacokinetic properties, and / or improve or enhance interactions with FAK protein). Non-limiting examples of small molecule FAK inhibitors that find use in the compositions and methods of the present disclosure include, but are not limited to, Defactinib (VS-6063) (FIG. 5A, FIG. 9A), GSK2256098 (FIG. 5B, FIG. 11 A), PF-431396 (FIG. 8A), Narmafotinib (FIG. 10A), TAE-226 and PF-573228.

[0089] When the FAK modifier is a small molecule FAK inhibitor, the small molecule may be obtained using standard methods known to an ordinarily skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis, and in vitro translation systems, using methods well known in the art. In one embodiment, a small molecule FAK modifier of the disclosure comprises an organic molecule, inorganic molecule, biomolecule, synthetic molecule, and the like.

[0090] Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are methods ofmaking the libraries. The method may use a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery as well as biased structures for lead development.

[0091] In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core building block ensembles. The shape and rigidity of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure ("focused libraries") or synthesized with less structural bias using flexible cores.

[0092] The small molecule and small molecule compounds described herein may be present as salts even if salts are not depicted and it is understood that the disclosure embraces all salts and solvates of the inhibitors depicted or described, as well as the non-salt and non-solvate form of the inhibitors, as is well understood by the skilled artisan. In some embodiments, the salts of the inhibitors of the disclosure are pharmaceutically acceptable salts.

[0093] Where tautomeric forms may be present for any of the inhibitors described herein, each and every tautomeric form is intended to be included in the present disclosure, even though only one or some of the tautomeric forms may be explicitly depicted.

[0094] The disclosure also includes any or all of the stereochemical forms, including any enantiomeric or diastereomeric forms of the FAK modifier described. The recitation of the structure or name herein is intended to embrace all possible stereoisomers of a FAK modifier depicted or described herein. All forms of the FAK modifier are also embraced by the disclosure, such as crystalline or non-crystalline forms of the FAK modifier. Compositions comprising a FAK modifier of the disclosure are also intended, such as a composition of substantially pure FAK modifier (e.g., a small molecule FAK inhibitor), including a specific stereochemical form thereof, or a composition comprising mixtures of FAK modifier of the disclosure in any ratio, including two or more stereochemical forms, such as in a racemic or non-racemic mixture.

[0095] Nucleic acid FAK modifiers. In other embodiments, the FAK modifier is an isolated nucleic acid. In some embodiments, the FAK modifier is an siRNA, shRNA, antisense molecule, nucleic acid molecule, plasmid, or expression vector, which modulates the expression level and / or activity of a FAK protein or protein involved in FAK production or activity. The disclosure also encompasses expression vectors and methods for theintroduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0096] In some embodiments, a protein involved in FAK production or activity can be modulated by way of activating, inactivating, releasing, and / or sequestering FAK or proteins involved in FAK production or activity. As such, FAK modifying activity can be accomplished by using a transdominant positive or negative mutant.

[0097] In one embodiment, siRNA or shRNA is used to modulate the level of FAK and / or proteins involved in FAK production or activity. RNA interference (RNAi) is a process in which the introduction of double- stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by ribonuclease Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, Fire et al., 1998, Nature 391 (19):306-311;Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432: 173-178) describe a chemical modification to siRNAs that aids in intravenous systemic delivery. Optimizing siRNAs involves consideration of overall G / C content, C / T content at the termini, Tm and the nucleotide content of the 3’ overhang. See, for example, Schwartz et al., 2003, Cell, 115: 199-208 and Khvorova et al., 2003, Cell 115:209-216. The present disclosure includes methods of modulating levels of FAK and / or proteins involved in the production or activity of FAK using RNAi technology.

[0098] In another embodiment, the disclosure includes a vector comprising an siRNA or antisense polynucleotide that modulates the expression of FAK and / or protein involved in the production or activity of FAK.

[0099] The siRNA, shRNA, or antisense polynucleotide can be cloned into a number of types of vectors. For expression of the siRNA or antisense polynucleotide, at least one module in each promoter functions to position the start site for RNA synthesis.1

[0100] In order to assess the expression of the siRNA, shRNA, or antisense polynucleotide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected using a viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like. In some embodiments, the disclosure relates to a vector, comprising a nucleotide sequence of the disclosure or the construct of the disclosure. The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In a particular embodiment, the vector of the disclosure is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In specific embodiments, the expression vector is selected from a viral vector, a bacterial vector, and a mammalian cell vector. Prokaryote- and / or eukaryote-vector based systems can be employed for use with the present disclosure to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.

[0101] The expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012), and in Ausubel et al. (1997). Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.

[0102] The vector may be obtained by conventional methods known by persons skilled in the art (See, e.g., Sambrook et al., 2012). In some embodiments, the vector is a vector useful for transforming animal cells. The recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin which confer resistance to certain drugs, P-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin, for example, IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

[0103] Following generation of siRNA polynucleotide, a skilled artisan will understand that the siRNA polynucleotide will have certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, the siRNA polynucleotide may be further designed to resist degradation by modifying it to include phosphorothioate, or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like.

[0104] Any polynucleotide may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and / or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and / or the inclusion of nontraditional bases such as inosine, queosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0105] Antisense molecules and their use for modulating gene expression are well known in the art. Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule. In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby modulating the translation of genes.

[0106] The use of antisense methods to modulate the translation of genes is known in the art. Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule.

[0107] Polypeptide FAK modifiers. The disclosure also provides, in some embodiments, an isolated peptide that inhibits FAK production and / or activity. For example, in one embodiment, the peptide modifier of the disclosure inhibits the expression and / or activity of FAK and / or the expression and / or activity of proteins involved in FAK production or activity directly by binding to the proteins involved, thereby altering the normal functional activity of the proteins, and reducing FAK production and / or activity. In another embodiment, the peptide FAK modifier of the disclosure decreases FAK activity and / or production by competing with endogenous proteins involved in FAK activity or production. In yet another embodiment, the peptide modifier of the disclosure inhibits proteins involved in the production or activity of FAK by acting as a transdominant positive or negative mutant. The variants of the polypeptides according to the present disclosure may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g.,residues that are modified by the attachment of substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the present disclosure, (iv) fragments of the polypeptides and / or (v) one in which the polypeptide is fused with another polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. In view of the present disclosure, such variants are deemed to be within the scope of those skilled in the art.

[0108] Antibody FAK modifiers. The disclosure also contemplates a FAK modifier comprising an antibody, or antibody fragment, specific for FAK or proteins involved in the production or activity of FAK. In some embodiments, the antibody decreases FAK levels or activity to provide a beneficial effect.

[0109] The disclosure is not limited by the type of antibody. Antibodies may be intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain Fv molecule, or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies including monoclonal and polyclonal antibodies, fragments, and chimeras, may be prepared using methods known to those skilled in the art.

[0110] Antibodies can be prepared using intact polypeptides or fragments containing an immunizing antigen of interest. The polypeptide or oligopeptide used to immunize an animal may be obtained from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Suitable carriers that may be chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. The coupled polypeptide may then be used to immunize the animal (e.g., a pig, a mouse, a rat, or a rabbit).[OHl] Methods of Treatment.

[0112] In some embodiments, the disclosure provides methods of treating, preventing and / or reducing allergic reactions (e.g., to orally introduced allergens). For example, in some embodiments, the disclosure provides methods of treating a severe allergic reaction that occurs rapidly after exposure to an allergen (e.g., anaphylaxis). In some embodiments, the method reduces a subject's allergic reaction (e.g., anaphylaxis and / or IgE antibody responses)to an allergen (e.g., a food such as peanuts, shellfish, tree nuts, or dairy, an insect sting such as a wasp or bee sting, medication such as penicillin or Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), or to latex). Furthermore, the disclosure provides methods for treating allergic emergencies, such as anaphylaxis. In some embodiments, a method of the disclosure provides emergency relief from at least one symptom of anaphylaxis (e.g., thereby providing a time sufficient for a subject to seek professional medical assistance). Compositions of the disclosure may be included in first aid kits in professional childcare settings and homes, for example, especially where one or more persons at risk for anaphylaxis are known to dwell. They may also be conveniently carried by those who are at risk for anaphylaxis or those who are charged with caring for those who are at risk for anaphylaxis. They are also well-suited for inclusion in "crash carts" in medical emergency rooms. The methods of the disclosure are suitable for treating subjects who are at risk for allergic emergencies, such as anaphylaxis, in any of the exemplary settings mentioned herein. The disclosure is not limited by the type or route of administration. Indeed, as detailed herein, any route of administration may be used. A typical administration for anaphylaxis may be via injection under the skin, or into a muscle, although any route of administration may be suitable. Injections can be given by a health care professional in a clinic or hospital setting, or at the site of a subject experiencing severe allergic reaction (e.g., by a first responder). Alternatively, an auto-injector form, for example, provides a convenient applicator for the health-care professional or for selfadministration by subjects who suffer a severe allergic response to certain stimuli.

[0113] Arresting or Rescuing an Ongoing Anaphylactic Reaction. In some embodiments, the disclosure provides a method of arresting or reducing the severity of an ongoing anaphylactic reaction. In such embodiments, a subject has been exposed to an allergen and has developed one or more symptoms of anaphylaxis (e.g., urticaria, respiratory distress, hypotension, hypothermia, vascular collapse, or systemic shock), and a composition comprising a FAK inhibitor is administered to the subject after the onset of such symptoms. Without being bound by theory, it is believed that FAK inhibition can arrest mast cell degranulation that is already in progress, thereby halting the release of additional allergic mediators and reducing the severity of the ongoing reaction. As demonstrated in Example 11, FAK inhibition with defactinib significantly reduced degranulation even when administered 1 minute, 5 minutes, or 10 minutes after IgE-mediated stimulation in vitro, and significantly reduced anaphylaxis severity when administered 1 minute, and detectably after 5 minutes, after allergen challenge in vivo (See FIG. 32A-B). In some embodiments, a composition of the disclosure is administered within 30 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2minutes, or 1 minute after onset of symptoms of anaphylaxis. In some embodiments, the method further comprises co-administering epinephrine to the subject.

[0114] Compositions and methods of the disclosure are useful for treatment of a medical condition, such as an allergic emergency that includes treatment of anaphylaxis, for which the disclosure is especially well-suited. In addition, treatment of allergic emergency includes treatment of other allergic conditions that may be treated. For example, the symptoms of anaphylactoid reactions to drugs closely mimic those of anaphylaxis and can be treated in a similar manner. Compositions and methods disclosed herein find use in settings where it is not clear whether the reaction is a systemic immunological response (anaphylaxis) or a systemic toxic response (anaphylactoid reaction). In this sense, treatment of an allergic emergency encompasses treatment of anaphylaxis, an anaphylactoid response or both.

[0115] In one embodiment, the methods prevent and / or reduce allergic reactions to foods, beverages, or other substances a subject is exposed to. In some embodiments, the disclosure provides methods for preventing and / or reducing one or more symptoms of an allergic reaction. Examples of symptoms to allergic reactions include, but are not limited to, anaphylaxis, diarrhea, nausea, abdominal pain, and colonic inflammation. Examples of allergens include, but are not limited to, egg allergen, milk and dairy allergen, peanut allergen, tree nuts allergen, fish allergen, shellfish allergen, wheat allergen, soy allergen, sesame allergen, apple allergen, apricot allergen, carrot allergen, celery allergen, cherry allergen, peach allergen, pear allergen, plum allergen, potato allergen, anise allergen, carraway seed allergen, coriander allergen, fennel allergen, parsley allergen, banana allergen, cucumber allergen, melons allergen, zucchini allergen, kiwi allergen, citrus allergen, tomato allergen, peppers allergen, broccoli allergen, cabbage allergen, cauliflower allergen, garlic allergen, onion allergen, black pepper allergen, mustard allergen, color additives allergen, corn allergen, meat allergen, gelatin allergen, sunflower seed allergen, poppy seed allergen, avocado allergen, mango allergen, acacia gum allergen, allspice allergen, amaranth allergen, annatto allergen, Aspergillus niger allergen, Balsam of Peru allergen, barley allergen, beans allergen, beer allergen, buckwheat allergen, cardamom allergen, cassia allergen, celeriac allergen, chamomile allergen, chocolate allergen, cocoa allergen, cinnamon allergen, clove allergen, coconut allergen, coffee allergen, cottonseed allergen, cumin allergen, curry allergen, dill allergen, ethanol allergen, flax seed allergen, ginger allergen, grape allergen, guava allergen, honey allergen, royal jelly allergen, hop allergen, karaya gum allergen, lentil allergen, lupine allergen, mace allergen, maple syrup allergen, millet seed allergen, mushrooms allergen, mycoprotein allergen, nutmeg allergen, oat allergen, papaya allergen,paprika allergen, pea allergen, cayenne pepper allergen, white pepper allergen, pineapple allergen, pomegranate allergen, psyllium allergen, quinine, rape seed allergen, rice allergen, rye allergen, spinach allergen, squash allergen, strawberry allergen, beet allergen, tragacanth gum allergen, turnip allergen, vanilla allergen, vitamin A, vitamin E, wine allergen, yeast allergen, pharmaceuticals, and / or fragments thereof.

[0116] In some embodiments, the method comprises administering to the subject an effective amount of a composition comprising a FAK modifier (e.g., a small molecule FAK inhibitor (FAKi)). In one embodiment, the method comprises administering to the subject an effective amount of a composition comprising one or more FAKi from Defactinib (VS-6063), GSK2256098, PF-431396, Narmafotinib, TAE-226, PF-573228, a combination thereof, or a pharmaceutically acceptable salt thereof. In one embodiment, the method comprises administering to the subject an effective amount of one or more inhibitors of FAK. In one embodiment, the method comprises administering to the subject an effective amount of PF-431396 or a pharmaceutically acceptable salt thereof. In one embodiment, the method comprises administering to the subject an effective amount of one or more antagonist of FAK2.

[0117] In one embodiment, the method comprises administering a composition comprising a FAK modifier (e.g., a small molecule FAK inhibitor (FAKi)) to a subject before, during, or after exposure to one or more allergens. Examples of administering the composition to the subject before exposure to the one or more allergen include, but are not limited to, administering the composition 24 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, or 30 seconds before exposure to one or more allergens. In one embodiment, the method comprises administering the composition to the subject more than one time before exposure to the one or more allergens. Examples of administering the composition to the subject after exposure to the one or more allergens include, but are not limited to, administering the composition 24 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, or 30 seconds after exposure to one or more allergens. In one embodiment, the method comprises administering the composition to the subject more than one time after exposure to the one or more allergens. In some embodiments, the composition is administered after exposure to one or more allergens and after onset of one or more symptoms of an allergic reaction (e.g., anaphylaxis). In such embodiments, the compositionmay be administered within 30 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute after onset of symptoms.

[0118] In one embodiment, the method comprises administering a composition comprising a FAK modifier (e.g., a small molecule FAK inhibitor (FAKi)) to a subject one or more times a day. Examples of administering the composition one or more times a day include, but are not limited to, administering the composition 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times a day.

[0119] In one embodiment, the method comprises administering a composition comprising a FAK modifier (e.g., a small molecule FAK inhibitor (FAKi)) to a subject prior to, with, or after a meal. Examples of administering the composition prior to a meal include, but are not limited to, administering the composition 24 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, and 30 seconds before a meal. Examples of administering the composition after a meal include, but are not limited to, administering the composition 24 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, and 30 seconds after a meal.

[0120] The compositions of the disclosure may be administered to a subject in need in a wide variety of ways. Modes of administration include orally, intraoperatively, intravenous, intravascular, intramuscular, subcutaneous, intracerebral, intraperitoneal, soft tissue injection, surgical placement, arthroscopic placement, and percutaneous insertion, e.g., direct injection, cannulation, or catheterization. Any administration may be a single application of a composition of disclosure or multiple applications. Administrations may be to a single site or to more than one site in the subject treated. Multiple administrations may occur essentially at the same time or separated in time. In some embodiments, administration is via an autoinjector device. In some embodiments, the auto-injector device is configured for intramuscular or subcutaneous injection. In some embodiments, the auto-injector device is configured for self-administration by the subject or administration by a non-medical professional. In some embodiments, the auto-injector device contains a composition comprising a FAK inhibitor and epinephrine.

[0121] In some embodiments, the method comprises administering to a subject a therapeutically effective amount of a composition comprising a FAK modifier (e.g., a small molecule FAK inhibitor (FAKi)) to a subject. The disclosure contemplates the administration of the FAK modifier (e.g., a small molecule FAK inhibitor (FAKi)) to a subject in apharmaceutical composition and / or formulation. The disclosure encompasses the use of pharmaceutical compositions to practice the methods disclosed herein. Such a pharmaceutical composition may comprise at least one composition of the disclosure or a salt thereof in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one composition of the disclosure or a salt thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The composition comprising an inhibitor of FAK (e.g., a small molecule FAK inhibitor (FAKi) may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0122] A pharmaceutical composition comprising an inhibitor of FAK (e.g., a small molecule FAK inhibitor (FAKi) or a pharmaceutically acceptable salt thereof) may be co-administered with one or more other agents (e.g., epinephrine or other substance with allergy mitigation properties). The disclosure is not limited by the agent(s) co-administered with an inhibitor of FAK of the disclosure. In some embodiments, epinephrine is co-administered with an inhibitor of FAK. In other embodiments, the agent co-administered with an inhibitor of FAK disclosed herein is an agent with allergy mitigation properties (e.g., an anti-IgE antibody such as Omalizumab (Xolair) or ligelizumab; an inhibitor of IL-4 and / or IL-13 signaling such as Dupilumab (Dupixent); an inhibitor of IL-5 and / or IL-5 receptor such as Mepolizumab (Nucala), Benralizumab (Fasenra) or Reslizumab (Cinqair); or an inhibitor of thymic stromal lymphopoietin (TSLP) such as Tezepelumab (Tezspire)). The disclosure is not limited to these particular agents or substances. Indeed, any agent or substance that alleviates a sign or symptom of allergy may be co-administered with an inhibitor of FAK disclosed herein. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. In some embodiments, co-administration can be via the same or different route of administration. Those of skill in the art understand that the formulations and / or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s),and / or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.

[0123] Pharmaceutical compositions that are useful in the methods of the disclosure may be suitably developed for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. A composition useful within the methods of the disclosure may be directly administered to the skin, lungs, nasal cavity and / or nasal mucosa, vagina, or any other tissue of a mammal. Other contemplated formulations include liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically based formulations. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the allergy symptom treated, the type and / or age of the subject being treated, and the like.

[0124] Although the disclosure is principally directed to the administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist may design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and other primates, and mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0125] In one embodiment, the compositions utilized in the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions comprise a therapeutically effective amount of a FAK modifier (e.g., a small molecule FAK inhibitor (FAKi)) of the disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that are useful include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0126] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In one embodiment isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of an injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0127] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, vaginal, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined with other active agents, e.g., analgesic agents.

[0128] Pharmaceutical preparations of the disclosure may include other ingredients including one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; pharmaceutically acceptable polymeric or hydrophobic materials; or other ingredient known in the art and described, for example in Genaro, ed. (1985, Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0129] A composition comprising a FAK modifier (e.g., a small molecule FAK inhibitor (FAKi)) of the disclosure may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the disclosure include but are not limited to those selected from benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof.

[0130] In one embodiment, the composition includes an antioxidant and a chelating agent that inhibits the degradation. Exemplary antioxidants are BHT, BHA, alpha-tocopherol, andascorbic acid in the range of about 0.01% to 0.3%. In one embodiment, the BHT is in the range of 0.03% to 0.1% by weight by total weight of the composition. In one embodiment, the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g., disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%. In one embodiment, chelating agents may be in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelflife of the formulation. While BHT and disodium edetate are the exemplary antioxidant and chelating agent respectively, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0131] Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyl eneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0132] Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition for use in the disclosure may comprise each of the components described regarding liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0133] Powdered and granular formulations of a pharmaceutical preparation of the composition utilized in the disclosure may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

[0134] A pharmaceutical composition for use in the disclosure may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

[0135] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after exposure to an allergen, or, prior to or after signs and symptoms of allergic reaction (e.g., anaphylaxis). Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further,the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0136] Administration of the compositions of the present disclosure to a subject, such a mammal, including a human, may be carried out using known procedures, at dosages and for periods of time effective to prevent or treat symptoms of allergy (e.g., anaphylaxis). An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a composition comprising a FAK modifier (e.g., a small molecule FAK inhibitor (FAKi)) for use in the disclosure is from about 1 and 5,000 mg / kg of body weight / per day. In other non-limiting examples, a dose may comprise from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0137] Dosage levels of the active ingredients in the pharmaceutical compositions of the disclosure may be varied so as to obtain an amount of the active ingredient that is effective toachieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

[0138] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of a composition comprising a FAK modifier (e.g., a small molecule FAK inhibitor (FAKi)) of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0139] In some embodiments, it is advantageous to formulate a composition comprising a FAK modifier (e.g., a small molecule FAK inhibitor (FAKi)) in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of an allergy symptom in a subject.

[0140] FAK Pathway Modification for Reducing Mast Cell Degranulation. In some embodiments, the disclosure provides methods of reducing IgE -mediated mast cell degranulation in a subject in need thereof. Without being bound by theory, the present disclosure demonstrates that FAK pathway modification — whether by kinase inhibition, protein degradation, or expression knockdown — reduces mast cell degranulation as measured by beta-hexosaminidase release, surface expression of degranulation markers CD63 and CD107a, and activation-induced mast cell adhesion (See Examples 2, 5-8, 10-11). In some embodiments, reducing IgE-mediated mast cell degranulation is useful for treating or preventing one or more conditions including, but not limited to, anaphylaxis, urticaria, allergic asthma, bronchoconstriction, mast cell activation syndrome, allergic rhinitis, diarrhea, colonic inflammation, nausea, and abdominal pain. In some embodiments, the subject has a mast cell-mediated disorder. In some embodiments, the FAK pathway modifier reduces mast cell surface expression of CD63 and / or CD107a. In some embodiments, the FAK pathway modifier reduces activation-induced mast cell adhesion. In some embodiments, the FAK pathway modifier inhibits FAK autophosphorylation at tyrosine 397 (Y397). Insome embodiments, the FAK pathway modifier reduces beta-hexosaminidase release from mast cells.

[0141] FAK Inhibition as an Adjunct to Allergen Immunotherapy. In some embodiments, the disclosure provides methods of reducing allergic reactions during allergen immunotherapy. Allergen immunotherapy, including oral immunotherapy (OIT), sublingual immunotherapy (SLIT), and subcutaneous immunotherapy (SCIT), involves the repeated administration of increasing doses of an allergen to induce tolerance. A significant limitation of allergen immunotherapy is the risk of allergic side effects, including anaphylaxis, during the dose escalation and maintenance phases. In some embodiments, a FAK inhibitor is coadministered with one or more doses of an allergen during immunotherapy to reduce the incidence or severity of allergic side effects. In some embodiments, the FAK inhibitor is administered prior to each dose of allergen. In some embodiments, the FAK inhibitor is a small molecule FAK inhibitor selected from defactinib, PF-431396, narmafotinib, TAE-226, and PF-573228, or a pharmaceutically acceptable salt thereof. Without being bound by theory, it is believed that FAK inhibition reduces the mast cell degranulation response to allergen exposure during immunotherapy, thereby reducing allergic side effects while permitting the immunomodulatory effects of the allergen to proceed. Accordingly, in some embodiments, the disclosure provides methods of reducing an allergic reaction to an allergen during allergen immunotherapy in a subject undergoing allergen immunotherapy, comprising co-administering to the subject a therapeutically effective amount of a FAK inhibitor with one or more doses of the allergen. The disclosure is not limited by the type or route of immunotherapy. For example, in some embodiments, the allergen immunotherapy is oral immunotherapy (OIT). Similarly, the disclosure is not limited by the type of allergen. Indeed, the compositions and methods of the disclosure may be used in allergen immunotherapy setting. In some embodiments, the allergen is a food allergen. In some embodiments, the FAK inhibitor is administered prior to each dose of the allergen. In the context of allergen immunotherapy, any FAK inhibitor described herein may be used. In some embodiments, the FAK inhibitor is a small molecule FAK inhibitor such as defactinib, PF-431396, narmafotinib, TAE-226, and PF-573228, or a pharmaceutically acceptable salt thereof. While an understanding of a mechanism is not needed, in some embodiments, it is contemplated coadministration of the FAK inhibitor reduces the incidence or severity of allergic side effects during the allergen immunotherapy.

[0142] Significance of Dual FAK1 / FAK2 Inhibition. As demonstrated herein, the FAK-selective inhibitor GSK2256098 — which displays approximately 1000-fold selectivity forFAK1 over FAK2 (Pyk2 / PTK2B) — showed minimal inhibitory effect on mast cell degranulation (See, e.g., FIG. 11), in contrast to the significant effects observed with dual FAK1 / FAK2 inhibitors PF-431396, defactinib, and narmafotinib. Without being bound by theory, the disclosure provides, in some embodiments, that inhibition of FAK2 (Pyk2 / PTK2B), in addition to or instead of FAK1 alone, may contribute to anti -degranulation effects observed with dual FAK1 / FAK2 inhibitors. In some embodiments, compositions and methods of the disclosure employ a FAK pathway modifier that inhibits both FAK1 (PTK2) and FAK2 (Pyk2 / PTK2B). In some embodiments, a FAK pathway modifier that inhibits both FAK1 and FAK2 is a dual FAK / Pyk2 inhibitor such as PF-431396 or defactinib. In some embodiments, a FAK pathway modifier that reduces IgE-mediated mast cell degranulation is selected. In some embodiments, the FAK pathway modifier has an IC50 for FAK2 (Pyk2) inhibition of less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 15 nM, or less than about 1 nM.

[0143] In certain embodiments, the composition of the present disclosure provides for a controlled release of a therapeutic agent. In certain instances, controlled- or sustained-release formulations of a pharmaceutical composition of the disclosure may be made using conventional technology, using for example proteins equipped with pH sensitive domains or protease-cleavable fragments. In some cases, the dosage forms to be used can be provided as slow or controlled release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, micro-particles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the disclosure. Thus, single unit dosage forms suitable for oral administration, such as tablets, capsules, gel-caps, lozenges, and caplets, which are adapted for controlled-release are encompassed by the present disclosure. In some embodiments, the composition is formulated for immediate release.

[0144] The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration. As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration.

[0145] One of ordinary skill in the art, based on the present disclosure, can utilize the compositions and methods described to their fullest extent. The specific embodiments are therefore to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions.EXAMPLES

[0146] Examples of specific embodiments for carrying out the present disclosure are provided. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way.

[0147] Example 1 - Identification of focal adhesion kinas (FAK) subfamily involvement in allergy and anaphylaxis.

[0148] Research and experiments were conducted during development of embodiments of the present disclosure in an effort to identify new targets for treatment of allergy, and in particular, anaphylaxis. Initially, efforts were made to collect human subject samples pre-and post-food challenge, with subsequent isolation and sequencing of RNA from the subject samples (RNA-seq). As shown in FIG. 1, it was found that among 200 differentially expressed genes, FAK signaling pathways were significantly differentially upregulated using Ingenuity Pathway analysis following reaction.

[0149] FAK, named after its typical localization in focal adhesions, is ubiquitously expressed non -receptor tyrosine kinase encoded by the PTK2 gene on chromosome 8 (15 in mice) and plays a critical role in transducing signals from sites of cell contacts with extracellular matrix. Four genetic variants encoding two different FAK isoforms have been identified (See Ossovskaya et al., FEBS Lett. 2008 Jun 10;582(16):2402-2406). When activated, FAK undergoes a conformational change that exposes the autophosphorylation site Y397, which becomes the docking site for Src kinase that then phosphorylates tyrosine residues in the activation loop.

[0150] Focal adhesion kinase (FAK), also known as PTK2, or FAK1, is a ubiquitously expressed non -receptor tyrosine kinase encoded by the PTK2 gene on chromosome 8 (15 in mice). As shown in FIG. 2, FAK is subdivided into three major domains: a C-terminal focal adhesion targeting (FAT) domain that tethers to actin cytoskeleton; an N-terminal band 4.1,ezrin, radixin, moesin (FERM) homology domain that docks to cell membrane; and a central kinase domain that forms a closed conformation with FERM. FAK activation induces structural change exposing Y397 autophosphorylation site. A nuclear localization / export signal sequences directs translocation between cytoplasm and nucleus.

[0151] The only other FAK subfamily member identified to date is proline-rich tyrosine kinase 2 (Pyk2), also known as PTK2B, or FAK2. Pyk2 / FAK2 is a non-receptor tyrosine kinase encoded by the PTK2B gene on chromosome 8 (14 in mice) and is primarily expressed in neuronal, hematopoietic, and endothelial cells. As shown in FIG. 3, Pyk2, shares general structure and major phosphorylation sites with FAKE The Pyk2 / FAK2 subfamily lacks significant sequence similarity to other kinase subfamilies.

[0152] The FAK family plays an important role in regulating cell migration, adhesion, reorganization of the actin cytoskeleton, formation and disassembly of focal adhesions and cell protrusions, cell cycle progression, proliferation, and apoptosis. FAK functions in integrin signal transduction, but also in signaling downstream of numerous growth factor receptors, G-protein coupled receptors (GPCR), EPHA2, netrin receptors and LDL receptors. FAK forms multi-subunit signaling complexes with SRC and SRC family members upon activation; this leads to the phosphorylation of additional tyrosine residues, creating binding sites for scaffold proteins, effectors and substrates. Due to its many interactions and functions, FAK has been studied in the context of a range of pathologies including cancer, fibrosis, neurological disorders, cardiovascular disease and bone disease (See FIG. 4).

[0153] FAK is commonly upregulated in malignancies and has been linked to metastatic potential. Due to this subfamily’s increased expression among malignant cells / tissues and link to metastatic potential, it has been investigated in the oncology arena as a potential therapeutic target - both in isolation and combination with other antineoplastic agents.

[0154] As detailed herein, there are a variety of different inhibitors of FAK including small molecule FAK inhibitors (FAKi) that have been studied in the context of cancer treatments. FAKi have been shown to be safe and generally tolerated across 13 completed trials. For example, Defactinib (VS-6303) has completed phase I and II trials in non-hematologic malignancies with reported reversible fatigue, gastrointestinal upset, and headache in -25% of participants (dose limiting in <1%). The chemical structure of Defactinib (VS-6303) is shown in FIG. 5.

[0155] GSK2256098 has completed phase I trials in multiple malignancies with reported similar reversible AEs in -25% of participants at or below MTD (1000 mg BID) withminimal blood-brain barrier penetrance in controls. Some significant adverse events included proteinuria, LFT abnormalities, and hypercholesterolemia.

[0156] Example 2 - Characterization of FAK’s role in allergy and anaphylaxis.

[0157] Due to the observation that FAK and FAK signaling pathways were significantly differentially upregulated in subject’s samples post induction of allergy and anaphylaxis (FIG. 1), additional experiments were conducted in an effort to understand and characterize FAK’s role in this process. In particular, experiments were constructed and carried out in an effort to determine the effect of FAK loss-of-function and gain-of-function on mast cell adhesion and degranulation, to define the intracellular localization and interacting partners of FAK during mast cell adhesion and degranulation, and to delineate the role of FAK in transcriptional programs associated with mast cell activation.

[0158] In vitro experiments related to FAK loss-of-function and gain-of-function on mast cell adhesion and degranulation

[0159] Methods. In vitro models: RBL-2H3 rat basophilic leukemia line expressing the rat FCsRl receptor was obtained from ATCC and cultured in MEM media with 15% FBS, 1% P / S; cells were used between passages 5 and 20. LAD2 human MC leukemia line expressing CD117 and human FCsRl was obtained from the NIH and cultured in StemPro-34 complete media with 200mM L-glutamine, 1% P / S, and 100 ng / mL hSCF; cells were used between passages 4 and 8. Cells were plated at variable densities as indicated, allowed to adhere (RBL-2H3) or starved of growth factor (LAD2) for 4-6 hours, sensitized by incubation with species-specific monoclonal antibody overnight, then stimulated using DNP-HSA (RBL-2H3) or anti-IgE (LAD2). lonomycin stimulation served as IgE-independent positive control. Minimal to no change in viability was observed over included dose ranges. Healthy cell viability was visually confirmed throughout the assay and prior to lysis. FIG. 6 provides a pictograph of the in vitro model. Statistics. Carried out in GraphPad Prism software vlO.2.2 using one-way or two-way ANOVA with multiple-comparison test.Statistical significance is defined as *P <0.05, **P<0.01, ***P< 0.001, ****p <0.0001. All data are mean± s.e.m., unless stated otherwise.

[0160] Mast cell granule release: The beta hexosaminidase assay (measuring P-hexosaminidase release from cells) shown in FIG. 7 is a gold standard for assessing in vitro mast cell granule release and was utilized to assess the effect of FAK inhibitors on degranulation across a dose range. The assay measures P-hexosaminidase released from MCs after their stimulation, which hydrolyzes p-nitrophenyl-acetyl-D-glucosamine (pNAG) into p-nitrophenol and N-acetyl-D-glucosamine. The amount of P-hexosaminidase in the sample isproportional to the amount of p-nitrophenol that will be formed. In a high pH environment of a “stop solution“ (glycine solution), p-nitrophenol exists as fully deprotonated p-nitrophenolat and can be detected by its light absorbance at 405 nm. P-hexosaminidase release from sensitized RBL-2H3 (FIG. 7, center left) and LAD2 (FIG. 7, center right) cells plated at 5,000 cells / well and incubated with Tyrode’s buffer containing vehicle, PF-431396 (30 minutes), or defactinib (60 minutes) prior to 60-minute stimulation. P-hexosaminidase release was calculated by [%absorbance of supernatant / (supernatant + lysate)].

[0161] Analysis of the ability of different FAK inhibitors to alter cell degranulation.

[0162] A plurality of FAK inhibitors were tested for the ability to alter cellular degranulation in vitro.

[0163] PF-431396. The effect of FAK loss-of-function (FAK / Pyk2 inhibitor PF-431396, l-5pM for 30 minutes prior to stimulation) on mast cell degranulation is shown in FIG. 8. PF-431396 (FIG. 8A) has previously been described to inhibit activating phosphorylation of FAK subfamily members with ~5-fold selectivity for FAK (FAK IC502nM, Pyk2 IC50 1 InM). Selective inhibition was observed for IgE-mediated degranulation in LAD2 cells (FIG. 8C), with more significant inhibition of IgE-mediated degranulation at lower doses in RBL-2H3 cells (FIG. 8B).

[0164] Defactinib (VS-6063). The effect of FAK loss-of-function (FAK / Pyk2 inhibitor defactinib (VS-6063), l-10pM for 1 hour prior to stimulation) on mast cell degranulation is shown in FIG. 9. Defactinib (VS-6063) (FIG. 9A), has been described to have near identical inhibitory effect on activating phosphorylation of FAK subfamily members (FAK IC50 0.6nM, Pyk2 IC500.6nM). Selective inhibition was observed for IgE-mediated degranulation in LAD2 cells (FIG. 9C), and dose-dependent reduction was observed in the significantly higher baseline degranulation that occurred in sensitized RBL-2H3 cells (FIG. 9B).

[0165] Narmafotinib. The effect of FAK loss-of-function (FAK inhibitor narmafotinib, 1-lOpM for 60 minutes prior to stimulation) on mast cell degranulation is shown in FIG. 10. Narmafotinib (FIG. 10A), currently in clinical trials for treatment of cancer, has been described to have elevated selective inhibitory effect on activating phosphorylation of FAK over even its subfamily member Pyk2 (FAK IC502-7nM, Pyk2 IC50 550nM). Selective inhibition was observed for IgE-mediated degranulation in LAD2 cells (FIG. 10C), and dosedependent reduction was seen in the significantly higher baseline degranulation observed in sensitized RBL-2H3 cells (FIG. 10B).

[0166] GSK2256098. The effect of FAK loss-of-function (FAK inhibitor GSK2256098, 0. l-10pM for 30 minutes prior to stimulation) on mast cell degranulation is shown in FIG.11. GSK2256098 (FIG. 11A), currently in clinical trials for treatment of cancer has been estimated to display about a 1000-fold specificity for FAK over Pyk2. Minimal inhibitory effect on degranulation was observed in either LAD2 cells (FIG. 11C) or RBL-2H3 cells (FIG. 1 IB).

[0167] Quantifying surface markers of degranulation by flow cytometry.

[0168] Next, surface marker expression on stimulated cells was assessed in order to confirm the beta-hexosaminidase findings described above and in an effort to elucidate / identify components of the degranulation process impacted by FAK inhibitors. Cells were seeded in multiwell plates, then 4-6h later cells in each experimental well were sensitized with IgE. Next, cells were treated with agonist / antagonist or vehicle, and stimulated with anti-IgE or ionomycin. Finally, cells were incubated with an antibody cocktail and then surface marker expression was analyzed on a flow cytometer.

[0169] Specifically, LAD2 cells were plated at 100,000 cells / well, incubated with Tyrode’s buffer containing vehicle or PF -431396 for 30 minutes prior to stimulation for 10 minutes. Staining was performed and analysis by flow cytometry carried out for singlet cells, viability, and analysis of the surface expression of CD117, FCsRl, CD 107a, and CD63.

[0170] As shown in FIG. 12, nearly 100% of LAD2 cells express CD117 (c-kit) and FCeRl, with a proportion of cells expressing CD63 and CD107a on their surface in response to activation. Expression of these surface markers on the cell surface is reduced in a dosedependent manner after treatment with FAK inhibitor PF-431396.

[0171] Additional experiments were performed in order to characterize IgE- and non-IgE dependent stimulation and cell surface expression of these markers. PF-431396, at various amounts from 0.001-0. IpM for 30 minutes prior to stimulation, resulted in a dose-dependent reduction in the surface expression of CD63 and CD 107a for both IgE- and non-IgE dependent stimulation (See FIG. 13).

[0172] Characterizing activation-induced cellular adhesion using fluorometry.

[0173] Experiments were conducted in order to determine the effect of FAK inhibitors on cell adhesion in response to cellular activation in vitro. Cellular adhesion was measured using a cellular adhesion assay that measured the presence of hydrolyzed calcein AM (a cellpermeant compound) that fluoresces (at 475 / 500-550) when hydrolyzed by intracellular esterases in live cells.

[0174] Sensitized RBL-2H3 cells (FIG. 15) and LAD2 cells (FIG. 14) were labeled with calcein-AM dye, incubated with Tyrode’s buffer containing the concentrations of PF-431396 shown for 60 minutes, then transferred to fibronectin-coated multi-well plates prior tostimulation. Percent adhesion was determined by detecting the ratio of fluorescent signal remaining after gentle washing of the growth surface to remove non-adherent cells.

[0175] As shown in FIG. 14, exposure to PF-431396 resulted in a significant and selective decrease in IgE-mediated / stimulated adhesion in LAD2 cells, but did not alter / reduce adhesion in IgE-independent ionomycin stimulated cells.

[0176] Degranulation-induced adhesion with IgE-dependent stimulation was significantly reduced in RBL-2H3 cells in a dose dependent manner by PF-431396, whereas there was a minimal reduction in adhesion with IgE-independent stimulation. (See FIG. 15), The RBL-2H3 cell plates were inverted and gently centrifuged following inhibitor (or vehicle) treatment and stimulation.

[0177] Example 3 -In vivo studies to characterize FAK’s role in allergy and anaphylaxis.

[0178] Experiments were conducted to analyze FAK role in passive / active systemic anaphylaxis in vivo. Materials and Methods. BALB / c mice were obtained from Jackson Laboratories. Both passive and active systemic anaphylaxis models employed a mixture of male and female mice between 3 and 5 weeks of age, which were starved for 4 hours prior to oral challenge. All animal care and experimentation were approved by the Institutional Animal Care and Use Committee of University of Medicine. Mice were sensitized with intravenous DNP-IgE (passive) or OVA-alum (active). In the passive model, mice were challenged with DNP-HSA via oral gavage the day following sensitization. In the active model, beginning two weeks following sensitization, mice were challenged with oral ovalbumin three times weekly for two weeks. FAK inhibitor was introduced via two intraperitoneal doses of 30mg / kg within 24 hours prior to challenge in both models, and in the case of active model is also introduced after mice have already exhibited significant and increasing reactions to successive ova challenges (See FIG. 16).

[0179] A substantial reduction in anaphylaxis scores for both Passive Systemic Anaphylaxis (PSA) and Active Systemic Anaphylaxis (ASA), as well as significant reversal of decreased temperatures, were observed after treatment with PF-431396 compared to vehicle treated controls, even in the effector-phase dosing (See FIG. 17).

[0180] Example 4 - Evaluation of morphology and protein interactions.

[0181] In order to understand and evaluate how FAK inhibition alters in vitro morphology and protein interactions, immunofluorescence was employed to assess differential subcellular localization and proximity to structures, proteins, and degranulation machinery with FAK inhibitor introduction.

[0182] In vitro assays were performed on sterile, fibronectin-coated glass coverslips in multi-well plate format prior to mounting and confocal imaging on Nikon A1HS instrumentation maintained by the University of Michigan institutional Microscopy core. For preparation, LAD2 cells (25,000 cells / well) were fixed with 4% PF A, permeabilized with 0.1% Triton-X, blocked with 3% BSA in PBS, and stained prior to mounting and confocal imaging. Tissue samples were paraffin embedded and slide mounted by the University of Michigan Tissue & Molecular Pathology Core, with subsequent processing as above. Image processing was performed with Image J software.

[0183] After incubating with PF -431396 (5uM) for 30 minutes prior to stimulation (30 minutes), no effect on relative FAK detection or association to cytoskeleton was observed (See FIG. 18). A decrease in phosphorylated FAK (pFAK) associated with actin cytoskeleton was observed after introducing PF-431396 (5uM) for 30 minutes prior to stimulation (See FIG. 19). Phosphorylated FAK (pFAK) appeared more centrally located following inhibitor treatment indicating that FAK inhibition drove nuclear localization.

[0184] Example 5 — Single-Cell RNA Sequencing Confirms Association of FAK Pathway Genes with FCsRl -Regulated Degranulation

[0185] In order to further characterize the transcriptomic association between FAK pathway genes and mast cell degranulation, and to extend the observations described in Example 1 and shown in FIG. 1, single-cell RNA sequencing (scRNAseq) was performed on LAD2 human mast cell leukemia cells. Samples underwent standard degranulation stimulation as described in Example 2. After 30 minutes post-stimulus (or control), samples were disassociated in Accutase and immediately submitted for scRNAseq at 100,000 cells per replicate with viability >90-95% in all cases.

[0186] Additional analysis of the human RNA-seq data described in Example 1 revealed prominent upregulation of several FAK signaling pathway members involved in cell migration, adhesion, and cytoskeletal organization when comparing reactive patients to non-reactive patients (FIG. 20 A). These upregulated FAK pathway members included IL5RA, PTGDR2, S1PR3, PDGFRB, S1PR5, ADGRG1, TRGC1, IL18RAP, TGFBR3, CX3CR1, IL2RB, ERBB2, PTGDR, TRDC, and TRGV9. FAK expression was also confirmed across key mucocutaneous tissue types relevant to allergen exposure and anaphylaxis — including skin, tongue, and small intestine — as demonstrated by the human tissue reference atlas Tabula Sapiens (FIG. 20B).

[0187] As shown in FIG. 21 A, in each condition (unstimulated, anti-IgE, and ionomycin), cells that showed a degranulation RNA signature showed high correlation with upregulationof FAK pathway genes. Mast cell degranulation gene sets were positively correlated with FAK pathway gene expression, with Spearman correlation values being highest in the anti-IgE-stimulated condition (slope = 0.88 for anti-IgE vs. unstimulated; slope = 0.98 for ionomycin vs. unstimulated).

[0188] However, the three conditions (unstimulated, anti-IgE, and ionomycin) showed differences in specific FAK pathway gene expression (FIG. 2 IB). Heatmap analysis revealed differential expression patterns among key FAK signaling pathway members including PARVB, CDC42, PIK3R3, GRB2, VEGFB, TLN, VASP, MYL12A, BAIAP2, PRKCA, CCND3, RAC2, RAP1A, MYL12B, ACTG1, BRAF, ACTB, BAD, ITGA9, PFN1, PPP1CC, RAC1, PPP1CA, PTK2, ITGB1, PIK3CA, THBS1, ACTN4, GSK3B, DIAPH1, VCL, TLN1, PTK2B, ITGA2B, LAMA2, PAK2, CTNNB1, BCAR1, ARHGAP35, VAV1, MAPK1, MAP2K1, HRAS, PRKCB, and RAPGEF1, among others.

[0189] Boxplot analysis of FCsRI module expression demonstrated differential module expression across unstimulated, anti-IgE, and ionomycin treatment groups. A dot plot showed percent expressed and average expression of FCsRI signaling pathway module genes (PIK3CD, PRKCD, NRAS, HRAS, MAPK1, MAPK14, MAP2K1, VAV1, PIK3R3, PRKCB, GRB2, FCER1G) across the three conditions (FIG. 21C). Spearman correlation heatmap analysis of genes positively and significantly associated with FCsRI signaling pathway modules in each treatment group revealed that genes in the unstimulated, anti-IgE, and ionomycin FCsRI signaling pathway module gene sets were significantly positively associated with CXCR4 signaling pathway modules (FIG. 2 ID).

[0190] Enrichment analysis on genes with the top 200 Spearman correlation with FCsRI modules identified distinct enrichment terms across the three conditions (FIG. 2 IE). The anti-IgE condition was enriched for actin filament organization (G0:0007015), actin cytoskeleton reorganization (GG:0031532), protein phosphorylation (G0:0006468), vesicle-mediated transport (G0:0016192), protein autophosphorylation (G0:0046777), Fc receptor signaling pathway (G0:0038093), and regulation of leukocyte degranulation (G0:0043300). The unstimulated condition was enriched for positive regulation of GTPase activity(GO: 0043547), regulation of GTPase activity (GO: 0043087), and plasma membrane bounded cell projection morphogenesis (G0:0120039). These findings provide that FCsRI signaling pathway modules are more associated with cell adhesion-related functions in IgE-stimulated than unstimulated and ionomycin-stimulated conditions (see also FIG. 22A-C).

[0191] Example 6 — Expanded In Vitro Characterization of FAK Inhibition on Mast Cell Degranulation Across Multiple Cell Lines

[0192] The in vitro degranulation experiments described in Example 2 (see FIGs. 8-11) were expanded to include bone marrow-derived mast cells (BMMCs), additional dose ranges, and parallel assessment of surface markers of degranulation using both PF-431396 and defactinib.

[0193] BMMCs were differentiated from mouse bone marrow precursors and cultured under standard conditions. BMMCs, along with RBL-2H3 and LAD2 cells prepared as described in Example 2, were plated, sensitized, and stimulated as previously described. Viability was confirmed across all dose ranges by CellTiterGlo® luminescent assay (FIG. 24A-B).

[0194] PF-431396. Beta-hexosaminidase release from RBL-2H3 (left), BMMC (center), and LAD2 (right) cells pretreated with a dose range of PF-431396 is shown in FIG. 23 A.Consistent with the results shown in FIG. 8, PF-431396 inhibited IgE-mediated degranulation in a dose-dependent manner across all three cell types. In BMMCs, PF-431396 significantly reduced DNP-HSA-stimulated degranulation (***p < 0.001 at 2.5pM; ****p < 0.0001 at 5.0pM) without significant effect on ionomycin-stimulated degranulation.

[0195] Defactinib (VS-6063). Beta-hexosaminidase release from RBL-2H3 (left), BMMC (center), and LAD2 (right) cells pretreated with a dose range of defactinib is shown in FIG.23B. Consistent with and expanding upon the results shown in FIG. 9, defactinib demonstrated dose-dependent inhibition of IgE-mediated degranulation across all three cell types. In BMMCs, defactinib at l.OpM, 5.0pM, and lO.OpM significantly reduced DNP-HSA-stimulated degranulation (****p < 0.0001) with minimal effect on ionomycin-stimulated degranulation at lower doses (*P < 0.05 at lOpM only).

[0196] Surface markers of degranulation — PF-431396. CD63 (left) and CD107a (right) surface expression in response to stimulation from LAD2 cells pretreated with dose ranges of PF-431396 is shown in FIG. 23C. Consistent with the flow cytometry results shown in FIGS.12-13, PF-431396 at lO.OpM significantly reduced both CD63+ surface expression (****p < 0.0001) and CD107a+ surface expression (****p < 0.0001) following anti-IgE stimulation, while effects on ionomycin-stimulated expression were less pronounced (*P < 0.05 for CD107a). Target population was defined by singlet live cells, and separately evaluated for FCsRl, CD117, and CD63 or CD 107a expression (FIG. 24C).

[0197] Surface markers of degranulation — Defactinib. CD63 (left) and CD107a (right) surface expression in LAD2 cells pretreated with defactinib is shown in FIG. 23D. Defactinib at lO.OpM significantly reduced CD63+ surface expression (****p < 0.0001) and CD107a+ surface expression (****p < 0.0001) following anti-IgE stimulation. Effects on ionomycin-stimulated expression were not significant (ns) at comparable doses.

[0198] FAK autophosphorylation. FAK autophosphorylation (Y397) was confirmed to rapidly increase with both IgE- and non-IgE-mediated activation in RBL-2H3 cells, as assessed by western blot using both Cell Signaling antibody (3283) and Abeam antibody (ab81298) (FIG. 25 A). FAK autophosphorylation due to early IgE- and non-IgE-mediated activation (5 min) was effectively inhibited by FAK kinase inhibitors defactinib (D, lOpM) and PF-431396 (P, 5pM) (FIG. 25B).

[0199] Example 7 — In Vitro Characterization of FAK Activation on Mast Cell Degranulation

[0200] To complement the loss-of-function experiments described in Examples 2 and 6, experiments were conducted to evaluate whether FAK gain-of-function augmented mast cell degranulation in vitro.

[0201] Beta-hexosaminidase release was measured from sensitized RBL-2H3 cells incubated with vehicle or FAK-activating small molecules adhesamine (CAS 462605-73-8; 24h) and ZINC40099027 (CAS 1211825-25-0; Ih) prior to stimulation.

[0202] As shown in FIG. 26A, RBL-2H3 cells pretreated with adhesamine at 0.1 pM, IpM, and I OpM for 24 hours showed trends toward increased degranulation upon DNP-HSA stimulation (ns, p=0.1) and ionomycin stimulation (ns). Similarly, ZINC40099027 pretreatment showed a trend toward increased IgE-mediated degranulation (ns, p=0.19) (FIG.26B).

[0203] Example 8 — Expanded Analysis of FAK Inhibition on Activation-Induced Adhesion and Cell Spreading

[0204] The adhesion and morphology experiments described in Example 2 (see FIGS. 14-15, 18-19) were expanded to include additional dose ranges and immunofluorescence characterization of morphological changes with FAK inhibition.

[0205] Adhesion of semi -adherent LAD2 cells to a fibronectin matrix was increased following stimulation with anti-IgE and ionomycin, and was dose-dependently impaired by FAK inhibition with PF-431396, with more significant impairment following IgE-mediated stimulation than ionomycin (FIG. 27A). Consistent with and expanding upon the results shown in FIG. 14, PF-431396 at O.OlpM and O.lpM significantly reduced IgE-mediated adhesion (**P < 0.01 and ****p < 0.0001, respectively), while effects on ionomycin-stimulated adhesion were not significant (ns).

[0206] Adhesion of adherent RBL-2H3 cells to a fibronectin matrix was similarly increased following stimulation and was dose-dependently impaired by FAK inhibition with PF-431396, with more significant impairment following IgE-mediated stimulation (**P < 0.01,***P < 0.001) than ionomycin (ns) (FIG. 27B). These results are consistent with and expand upon the findings shown in FIG. 15.

[0207] Immunofluorescence morphology — RBL-2H3 cells. Representative immunofluorescence morphology of RBL-2H3 cells was assessed under three conditions: without stimulation (vehicle), with IgE-mediated stimulation (vehicle), and with defactinib (lOpM) treatment 30 minutes prior to IgE-mediated stimulation (See FIG. 27C). Uninhibited conditions were treated with concentration-matched vehicle (0.1% DMSO / v). Following stimulation, cells were fixed, permeabilized, blocked, and stained for nuclear material (DAPI, blue), actin (phalloidin, green), and phospho-FAK Y397 (Abeam, red) prior to imaging on a Nikon Al HS and processing with ImageJ software. Consistent with and extending the observations in FIGS. 18-19, IgE-stimulated cells demonstrated pronounced cell spreading and increased pFAK signal at the cell periphery co-localizing with actin cytoskeletal projections. Treatment with defactinib prior to IgE stimulation reduced cell spreading and attenuated peripheral pFAK signal.

[0208] Immunofluorescence morphology — LAD2 cells. Representative immunofluorescence morphology of LAD2 cells under the same three conditions was also assessed (See FIG. 27D). LAD2 cells stimulated with anti-IgE showed increased pFAK signal and altered morphology compared to unstimulated controls, effects that were attenuated by defactinib pretreatment.

[0209] Example 9 — Expanded In Vivo Studies: FAK Inhibition with Multiple Compounds Reduces Anaphylaxis in Passive and Active Systemic Models

[0210] The in vivo experiments described in Example 3 were expanded to include treatment with both PF-431396 and defactinib in both passive and active systemic anaphylaxis models, as well as additional endpoints including diarrhea scoring. BALB / c mice were obtained from Jackson Laboratories. Both passive and active systemic anaphylaxis models employed female mice between 3 and 5 weeks of age, starved for 5 hours prior to oral challenge. All animal care and experimentation were approved by the Institutional Animal Care and Use Committee.

[0211] Mice (n=10 per condition) were sensitized with intravenous DNP-IgE (passive) or intraperitoneal OVA-alum (active). In the passive model, the day following sensitization mice were challenged with oral DNP-HSA (50mg / mouse). In the active model, two weeks following sensitization mice were challenged with oral ovalbumin (50mg / mouse) seven times over two weeks. FAK inhibitors PF-431396 and defactinib (lOmg / kg) were each dosed once via intraperitoneal injection approximately 5 hours prior to final challenge (FIG. 28 A).

[0212] Maximum anaphylaxis scores over a 60-minute monitoring period were significantly reduced by both PF-431396 and defactinib in the passive systemic anaphylaxis model< 0.0001 for both compounds vs. DNP-only) (FIG. 28B, top). Consistent with and extending the findings shown in FIG. 17, these results demonstrate efficacy with two structurally distinct FAK inhibitors. In the active systemic anaphylaxis model, maximum anaphylaxis scores were also significantly reduced by both compounds< 0.0001 for PF-431396; ***P < 0.001 for defactinib) (FIG. 28B, bottom).

[0213] Temperature change at 15 minutes following challenge demonstrated significant protection against hypothermia in FAKi-treated mice compared to allergen-challenged vehicle controls in both passive (****p < 0.0001 for both compounds) and active (****p < 0.0001 for PF-431396; ***p < 0.001 for defactinib) models (See FIG. 28C). Temperature monitoring over the full 60-minute period confirmed sustained protection against anaphylaxis-induced temperature declines in FAKi-treated groups in both models (FIG. 28D).

[0214] Diarrhea scoring in the active anaphylaxis model showed trends toward reduction with FAK inhibitor treatment (FIG. 28E).

[0215] The active systemic anaphylaxis model was validated by confirming significant anaphylaxis scoring and temperature declines across six successive ovalbumin challenges prior to introduction of FAK inhibition (FIG. 30A-D).

[0216] To assess whether FAK inhibition acts upstream of histamine release rather than blocking histamine receptor signaling, a histamine challenge model was employed. Mice were treated with 6-8 mg / mouse histamine via tail vein injection. FAK inhibitors PF-431396 and defactinib (lOmg / kg) were introduced via intraperitoneal injection 5 hours prior to challenge. In vivo anaphylaxis scoring and temperature decline were not affected by FAK inhibition in the histamine model (FIG. 29A-D). Maximum anaphylaxis scores were significantly elevated in both histamine and histamine+PF-431396 groups compared to vehicle (****p < 0.0001), with no significant difference between histamine and histamine+PF-431396 (ns) (FIG. 29B). Maximum temperature change was similarly unaffected by FAK inhibition in the histamine model (**P < 0.01 for histamine vs. vehicle; ns for histamine+PF-431396 vs. histamine) (FIG. 29C). Temperature monitoring over 60 minutes confirmed that FAK inhibition did not alter histamine-induced temperature declines (FIG. 29D). These results provide that FAK inhibition acts upstream of histamine release at the mast cell level rather than by blocking downstream histamine-mediated effects.

[0217] Example 10 — FAK Degradation Reduces Degranulation In Vitro and Anaphylaxis In Vivo

[0218] To determine whether reduced FAK protein expression, as opposed to kinase inhibition alone, could reduce mast cell degranulation and anaphylaxis, experiments were conducted using a targeted protein degradation approach. These experiments complement and extend the kinase inhibition data described in Examples 2, 6, and 9 by demonstrating that the observed anti-degranulation and anti -anaphylaxis effects are attributable to FAK pathway modulation through an independent mechanism of action.

[0219] LAD2 cells were treated with FAK -targeting PROTACs in growth media for 24h, then harvested for relative FAK protein quantification by western blot and parallel betahexosaminidase release. FIG. 31A shows FAK protein quantification via western blot following pretreatment of LAD2 cells with dose range of GSK215 PROTAC or vehicle (0.01% DMSO / v) for 24 hours, normalized to GAPDH expression and plotted as percentage of vehicle-treated control. FIG. 3 IB. Beta-hexosaminidase release in response to stimulation from LAD2 cells pretreated with GSK215 PROTAC. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, p < 0.0001. Error bars indicate S.E.M., statistics based on analysis via multiple unpaired t tests. FIG. 31C. FAK protein quantification via western blot following pretreatment of LAD2 cells with dose range of FC11 PROTAC or vehicle (0.01% DMSO / v) for 24 hours, normalized to GAPDH expression and plotted as percentage of vehicle-treated control. FIG. 3 ID. Beta-hexosaminidase release in response to stimulation from LAD2 cells pretreated with FC 11 PROTAC. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars indicate S.E.M., statistics based on analysis via multiple unpaired t tests. FIG. 31E-F. Temperature change over 60-minute monitoring period and temperature change at 15-minutes following challenge (E) as well as maximum anaphylaxis scores over 60-minute monitoring period (F) in passive systemic anaphylaxis model treated with vehicle or GSK215 PROTAC and compared to untreated controls.

[0220] Example 11 — FAK Inhibition Arrests Degranulation and Anaphylaxis Following Stimulation (Rescue Paradigm)

[0221] To determine whether FAK inhibition could arrest degranulation and anaphylaxis after stimulation had already been initiated — as would be required for therapeutic intervention during an ongoing anaphylactic event — experiments were conducted using a post-stimulation rescue paradigm.

[0222] In vitro post-stimulation rescue. Sensitized RBL-2H3, LAD2, and BMMC cells were stimulated then treated with vehicle or lOpM defactinib at 1 minute, 5 minutes, and 10 minutes post-stimulation, and final beta-hexosaminidase release was calculated as previously described (See FIG. 32A). In RBL-2H3 cells, defactinib introduced at 1 minute post-DNP-HSA stimulation significantly reduced degranulation < 0.0001), with persistent effect at 5 minutes < 0.0001) and 10 minutes (****p < 0.0001). In LAD2 cells, defactinib at 1 minute post-anti-IgE stimulation significantly reduced degranulation (****p < 0.0001), with reduced effect at 5 minutes (ns) and 10 minutes. In BMMCs, significant reduction was observed at 1 minute (***P < 0.001) post-stimulation, with reduced effect at later timepoints. Beta-hexosaminidase release was compared to collapsed mean of time-matched vehicle treatment.

[0223] In vivo post-challenge rescue. Passively sensitized mice (n=6 per condition) were challenged with oral DNP-HSA (50mg / mouse), then dosed once with defactinib (lOmg / kg) via intraperitoneal injection at 1 minute and 5 minutes post-challenge (See FIG. 32B).Maximum anaphylaxis scores over the 60-minute monitoring period were reduced in defactinib-treated mice at both 1 minute and at 5 minutes post-challenge compared to DNP-challenged controls. Temperature change at 15 minutes following challenge demonstrated substantial protection at both intervention timepoints. Temperature monitoring over 60 minutes confirmed sustained rescue effect, with defactinib-treated groups maintaining temperatures closer to untreated controls than to DNP-only challenged mice.

[0224] While the present disclosure has been shown and described with reference to preferred and various alternate embodiments, it will be readily understood by persons skilled in the art that various changes in form and details can be made without departing from the spirit and scope of the present disclosure.

[0225] For reasons of completeness, various aspects of the disclosure are set out in the following numbered clauses:

[0226] Clause 1. A method of treating or preventing anaphylaxis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a focal adhesion kinase (FAK) pathway modifier.

[0227] Clause 2. The method of clause 1, wherein the FAK pathway modifier inhibits FAK kinase activity.

[0228] Clause 3. The method of clause 1 or 2, wherein the FAK pathway modifier is a small molecule FAK inhibitor or a pharmaceutically acceptable salt thereof.

[0229] Clause 4. The method of clause 3, wherein the small molecule FAK inhibitor is an ATP-competitive inhibitor.

[0230] Clause 5. The method of clause 3 or 4, wherein the small molecule FAK inhibitor comprises a heterocyclic ring structure.

[0231] Clause 6. The method of clause 5, wherein the heterocyclic ring structure comprises a pyrimidine or quinazoline-based core.

[0232] Clause 7. The method of any one of clauses 3-6, wherein the small molecule FAK inhibitor is selected from the group consisting of defactinib, PF-431396, narmafotinib, TAE-226, and PF-573228, or a pharmaceutically acceptable salt thereof.

[0233] Clause 8. The method of clause 3, wherein the small molecule FAK inhibitor is defactinib or a pharmaceutically acceptable salt thereof.

[0234] Clause 9. The method of clause 3, wherein the small molecule FAK inhibitor is PF-431396 or a pharmaceutically acceptable salt thereof.

[0235] Clause 10. The method of clause 3, wherein the small molecule FAK inhibitor is narmafotinib or a pharmaceutically acceptable salt thereof.

[0236] Clause 11. The method of clause 3, wherein the small molecule FAK inhibitor is TAE-226 or a pharmaceutically acceptable salt thereof.

[0237] Clause 12. The method of clause 3, wherein the small molecule FAK inhibitor is PF-573228 or a pharmaceutically acceptable salt thereof.

[0238] Clause 13. The method of clause 1, wherein the FAK pathway modifier inhibits both FAK1 (PTK2) and FAK2 (Pyk2 / PTK2B).

[0239] Clause 14. The method of clause 1, wherein the FAK pathway modifier reduces FAK protein expression.

[0240] Clause 15. The method of clause 14, wherein the FAK pathway modifier is a proteolysis-targeting chimera (PROTAC) that targets FAK for proteasomal degradation.

[0241] Clause 16. The method of clause 15, wherein the PROTAC is GSK215 or FC11.

[0242] Clause 17. The method of clause 14, wherein the FAK pathway modifier is an shRNA, siRNA, or antisense molecule that reduces FAK mRNA expression.

[0243] Clause 18. The method of clause 1, wherein the FAK pathway modifier is an antibody or polypeptide that inhibits FAK production or activity.

[0244] Clause 19. The method of any one of clauses 1-18, wherein administration of the composition reduces IgE-mediated mast cell degranulation in the subject.

[0245] Clause 20. The method of clause 19, wherein the reduction in IgE-mediated mast cell degranulation comprises a reduction in beta-hexosaminidase release from mast cells.

[0246] Clause 21. The method of any one of clauses 1-18, wherein administration of the composition reduces mast cell surface expression of CD63 and / or CD 107a.

[0247] Clause 22. The method of any one of clauses 1-18, wherein the composition inhibits FAK autophosphorylation at tyrosine 397 (Y397).

[0248] Clause 23. The method of any one of clauses 1-18, wherein administration of the composition reduces activation-induced mast cell adhesion.

[0249] Clause 24. The method of any one of clauses 1-23, wherein the composition is administered prior to exposure of the subject to one or more allergens.

[0250] Clause 25. The method of clause 24, wherein the composition is administered within 24 hours prior to exposure to the one or more allergens.

[0251] Clause 26. The method of clause 24, wherein the composition is administered within 12 hours, within 6 hours, or within 2 hours prior to exposure to the one or more allergens.

[0252] Clause 27. The method of any one of clauses 1-23, wherein the composition is administered subsequent to exposure of the subject to one or more allergens.

[0253] Clause 28. The method of clause 27, wherein the composition is administered within 1 minute after exposure to the one or more allergens.

[0254] Clause 29. The method of clause 27, wherein the composition is administered within 1-10 minutes after exposure to the one or more allergens.

[0255] Clause 30. The method of any one of clauses 1-29, wherein the anaphylaxis is triggered by exposure to a food allergen.

[0256] Clause 31. The method of clause 30, wherein the food allergen is selected from peanut allergen, tree nut allergen, shellfish allergen, dairy allergen, egg allergen, wheat allergen, soy allergen, sesame allergen, fish allergen, or combinations thereof.

[0257] Clause 32. The method of any one of clauses 1-29, wherein the anaphylaxis is triggered by exposure to an insect sting allergen.

[0258] Clause 33. The method of any one of clauses 1-29, wherein the anaphylaxis is triggered by exposure to a medication allergen.

[0259] Clause 34. The method of any one of clauses 1-29, wherein the anaphylaxis is triggered by exposure to a latex allergen.

[0260] Clause 35. The method of any one of clauses 1-34, further comprising coadministering epinephrine to the subject.

[0261] Clause 36. The method of any one of clauses 1-34, further comprising coadministering to the subject one or more agents selected from an anti-IgE antibody, an inhibitor of IL-4 and / or IL- 13 signaling, an inhibitor of IL-5 and / or IL-5 receptor, an inhibitor of thymic stromal lymphopoietin (TSLP), or combinations thereof.

[0262] Clause 37. The method of clause 36, wherein the anti-IgE antibody is omalizumab.

[0263] Clause 38. The method of any one of clauses 1-37, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0264] Clause 39. The method of any one of clauses 1-38, wherein the composition is administered via injection.

[0265] Clause 40. The method of clause 39, wherein the composition is administered via an auto-inj ector.

[0266] Clause 41. The method of any one of clauses 1-38, wherein the composition is administered orally, sublingually, intranasally, or transdermally.

[0267] Clause 42. The method of any one of clauses 1-40, wherein the composition is formulated for immediate release.

[0268] Clause 43. The method of any one of clauses 1-42, wherein the subject is a human.

[0269] Clause 44. The method of clause 43, wherein the subject is a pediatric human.

[0270] Clause 45. The method of clause 43, wherein the subject has a known allergy and is at risk of anaphylaxis upon re-exposure to an allergen.

[0271] Clause 46. The method of clause 43, wherein the subject has previously experienced at least one anaphylactic episode.

[0272] Clause 47. A method of arresting or reducing the severity of an ongoing anaphylactic reaction in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising a focal adhesion kinase (FAK) inhibitor, wherein the composition is administered after the subject has been exposed to an allergen and after onset of one or more symptoms of anaphylaxis.

[0273] Clause 48. The method of clause 47, wherein the FAK inhibitor is a small molecule FAK inhibitor selected from defactinib, PF-431396, narmafotinib, TAE-226, and PF-573228, or a pharmaceutically acceptable salt thereof.

[0274] Clause 49. The method of clause 47 or 48, wherein the composition is administered within 1 minute after exposure to the allergen.

[0275] Clause 50. The method of clause 49, wherein the composition is administered within 5 minutes after exposure to the allergen.

[0276] Clause 51. The method of clause 49, wherein the composition is administered within 1-10 minutes after exposure to the allergen.

[0277] Clause 52. The method of any one of clauses 47-51, wherein the one or more symptoms of anaphylaxis comprise at least one of hypothermia, respiratory distress, urticaria, hypotension, vascular collapse, or systemic shock.

[0278] Clause 53. The method of any one of clauses 47-52, wherein administration of the composition reduces mast cell degranulation that is already in progress at the time of administration.

[0279] Clause 54. The method of any one of clauses 47-53, wherein the composition is administered via injection or auto-injector.

[0280] Clause 55. The method of any one of clauses 47-54, further comprising coadministering epinephrine to the subject.

[0281] Clause 56. The method of clause 55, wherein the FAK inhibitor and epinephrine are administered simultaneously, sequentially, or in a single co-formulated composition.

[0282] Clause 57. The method of any one of clauses 47-56, wherein the subject is a human.

[0283] Clause 58. The method of any one of clauses 47-57, wherein the allergen is a food allergen, an insect venom allergen, a medication allergen, or a latex allergen.

[0284] Clause 59. A method of reducing IgE-mediated mast cell degranulation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a FAK pathway modifier that inhibits FAK activity, FAK expression, or FAK signaling.

[0285] Clause 60. The method of clause 59, wherein the FAK pathway modifier is a small molecule FAK inhibitor, a FAK-targeting proteolysis-targeting chimera (PROTAC), or a nucleic acid that reduces FAK expression.

[0286] Clause 61. The method of clause 59 or 60, wherein reducing IgE-mediated mast cell degranulation treats or prevents anaphylaxis.

[0287] Clause 62. The method of clause 59 or 60, wherein reducing IgE-mediated mast cell degranulation treats or prevents one or more conditions selected from urticaria, allergic asthma, bronchoconstriction, mast cell activation syndrome, diarrhea, and abdominal pain.

[0288] Clause 63. The method of any one of clauses 59-62, wherein the FAK pathway modifier reduces mast cell surface expression of CD63 and / or CD 107a.

[0289] Clause 64. The method of any one of clauses 59-62, wherein the FAK pathway modifier reduces activation-induced mast cell adhesion.

[0290] Clause 65. The method of any one of clauses 59-64, wherein the subject has a mast cell-mediated disorder.

[0291] Clause 66. The method of clause 65, wherein the mast cell-mediated disorder is systemic mastocytosis, mast cell activation syndrome (MCAS), urticaria pigmentosa, or IgE-mediated food allergy.

[0292] Clause 67. The method of any one of clauses 59-66, wherein the FAK pathway modifier inhibits FAK autophosphorylation at tyrosine 397 (Y397).

[0293] Clause 68. The method of any one of clauses 59-67, wherein the reduction in IgE-mediated mast cell degranulation is measured by a reduction in beta-hexosaminidase release from mast cells.

[0294] Clause 69. The method of any one of clauses 59-68, wherein the subject is a human.

[0295] Clause 70. A method of reducing an allergic reaction to an allergen during allergen immunotherapy in a subject undergoing allergen immunotherapy, comprising coadministering to the subject a therapeutically effective amount of a FAK inhibitor with one or more doses of the allergen.

[0296] Clause 71. The method of clause 70, wherein the allergen immunotherapy is oral immunotherapy (OIT).

[0297] Clause 72. The method of clause 70, wherein the allergen immunotherapy is sublingual immunotherapy (SLIT).

[0298] Clause 73. The method of clause 70, wherein the allergen immunotherapy is subcutaneous immunotherapy (SCIT).

[0299] Clause 74. The method of any one of clauses 70-73, wherein the allergen is a food allergen.

[0300] Clause 75. The method of clause 74, wherein the food allergen is peanut allergen, tree nut allergen, shellfish allergen, dairy allergen, egg allergen, wheat allergen, soy allergen, sesame allergen, or fish allergen.

[0301] Clause 76. The method of any one of clauses 70-75, wherein the FAK inhibitor is administered prior to each dose of the allergen.

[0302] Clause 77. The method of clause 76, wherein the FAK inhibitor is administered within 30 minutes, within 1 hour, or within 2 hours prior to each dose of the allergen.

[0303] Clause 78. The method of any one of clauses 70-77, wherein the FAK inhibitor is a small molecule FAK inhibitor selected from defactinib, PF-431396, narmafotinib, TAE-226, and PF-573228, or a pharmaceutically acceptable salt thereof.

[0304] Clause 79. The method of any one of clauses 70-78, wherein co-administration of the FAK inhibitor reduces the incidence or severity of allergic side effects during the allergen immunotherapy.

[0305] Clause 80. The method of clause 79, wherein the allergic side effects comprise at least one of urticaria, bronchoconstriction, gastrointestinal symptoms, or anaphylaxis.

[0306] Clause 81. The method of any one of clauses 70-80, wherein co-administration of the FAK inhibitor permits administration of a higher dose of the allergen than could be safely administered without the FAK inhibitor.

[0307] Clause 82. The method of any one of clauses 70-81, wherein the subject is a human.

[0308] Clause 83. The method of clause 82, wherein the subject is a pediatric human.

[0309] Clause 84. A pharmaceutical composition comprising: (a) a FAK inhibitor or a pharmaceutically acceptable salt thereof; (b) epinephrine; and (c) a pharmaceutically acceptable carrier.

[0310] Clause 85. The pharmaceutical composition of clause 84, wherein the FAK inhibitor is a small molecule FAK inhibitor selected from defactinib, PF-431396, narmafotinib, TAE-226, and PF-573228, or a pharmaceutically acceptable salt thereof.

[0311] Clause 86. The pharmaceutical composition of clause 84 or 85, wherein the composition is formulated for injection.

[0312] Clause 87. The pharmaceutical composition of clause 86, wherein the composition is contained in an auto-injector device.

[0313] Clause 88. The pharmaceutical composition of clause 87, wherein the auto-injector device is a pre-filled syringe or a pen-type auto-injector.

[0314] Clause 89. The pharmaceutical composition of any one of clauses 84-88, wherein the composition is formulated for immediate release.

[0315] Clause 90. The pharmaceutical composition of any one of clauses 84-89, wherein the FAK inhibitor and epinephrine are present in a single dosage unit.

[0316] Clause 91. The pharmaceutical composition of any one of clauses 84-89, wherein the FAK inhibitor and epinephrine are present in separate dosage units configured for concurrent or sequential administration.

[0317] Clause 92. The pharmaceutical composition of any one of clauses 84-91, wherein the composition is stable at room temperature for at least 12 months.

[0318] Clause 93. The pharmaceutical composition of any one of clauses 84-92, wherein the pharmaceutically acceptable carrier is an aqueous carrier.

[0319] Clause 94. A composition comprising: (a) one or more FAK inhibitors or a pharmaceutically acceptable salt thereof; and (b) one or more allergens.

[0320] Clause 95. The composition of clause 94, wherein the one or more allergens comprise a food allergen.

[0321] Clause 96. The composition of clause 95, wherein the food allergen is selected from peanut allergen, tree nut allergen, shellfish allergen, dairy allergen, egg allergen, wheat allergen, soy allergen, sesame allergen, and fish allergen.

[0322] Clause 97. The composition of clause 94, wherein the one or more allergens comprise a non-food allergen selected from insect venom allergen, a pollen allergen, a dust mite allergen, a mold allergen, a pet dander allergen, and a latex allergen.

[0323] Clause 98. The composition of any one of clauses 94-97, wherein the FAK inhibitor is a small molecule FAK inhibitor selected from defactinib, PF-431396, narmafotinib, TAE-226, and PF-573228, or a pharmaceutically acceptable salt thereof.

[0324] Clause 99. The composition of any one of clauses 94-98, wherein the composition is formulated for oral administration.

[0325] Clause 100. The composition of any one of clauses 94-98, wherein the composition is formulated for sublingual administration.

[0326] Clause 101. The composition of any one of clauses 94-98, wherein the composition is formulated for subcutaneous injection.

[0327] Clause 102. The composition of any one of clauses 94-101, further comprising a pharmaceutically acceptable carrier.

[0328] Clause 103. The composition of any one of clauses 94-102, wherein the composition is for use in allergen immunotherapy.

[0329] Clause 104. The method of any one of clauses 1-83, wherein the FAK pathway modifier or FAK inhibitor is administered at a dose of about 1 mg / kg to about 100 mg / kg body weight.

[0330] Clause 105. The method of any one of clauses 1-83, wherein the FAK pathway modifier or FAK inhibitor is administered as a single dose or as multiple doses.

[0331] Clause 106. The method of any one of clauses 1-83, wherein the FAK pathway modifier or FAK inhibitor reduces FAK autophosphorylation at Y397 in mast cells by at least 50% as measured in a cell-based assay.

[0332] Clause 107. The method of any one of clauses 1-46, wherein treating or preventing anaphylaxis comprises reducing one or more symptoms selected from hypotension, bronchoconstriction, urticaria, angioedema, vomiting, and loss of consciousness.

[0333] Clause 108. The method of any one of clauses 1-46, wherein the subject has been previously diagnosed with a food allergy, insect venom allergy, or drug allergy placing them at risk for anaphylaxis.

[0334] Clause 109. A method of prophylactically treating a subject at risk of anaphylaxis, comprising administering to the subject a FAK pathway modifier prior to anticipated exposure to a known allergen of the subject.

[0335] Clause 110. The method of clause 109, wherein the FAK pathway modifier is a small molecule FAK inhibitor selected from defactinib, PF-431396, narmafotinib, TAE-226, and PF-573228, or a pharmaceutically acceptable salt thereof.

[0336] Clause 111. A method of stabilizing mast cells in a subject at risk of anaphylaxis, comprising administering a FAK pathway modifier to the subject in an amount sufficient to reduce IgE-FcsRI-mediated mast cell activation.

[0337] Clause 112. The method of clause 111, wherein the FAK pathway modifier inhibits FAK autophosphorylation at Y397 in mast cells.

[0338] Clause 113. A kit for treating or preventing anaphylaxis, comprising: (a) a FAK inhibitor or a pharmaceutically acceptable salt thereof; (b) optionally, epinephrine; and (c) an auto-injector device or instructions for use.

[0339] Clause 114. The kit of clause 113, wherein the FAK inhibitor is defactinib, PF-431396, narmafotinib, TAE-226, PF-573228, or a pharmaceutically acceptable salt thereof.

[0340] Clause 115. The kit of clause 113 or 114, wherein the FAK inhibitor and epinephrine are each pre-loaded in separate auto-injector devices.

[0341] Clause 116. The kit of any one of clauses 113-115, further comprising instructions for administering the FAK inhibitor prior to anticipated allergen exposure or upon onset of anaphylaxis symptoms.

[0342] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

What is claimed is:

1. A method of treating or preventing anaphylaxis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a focal adhesion kinase (FAK) pathway modifier.

2. The method of claim 1, wherein the FAK pathway modifier inhibits FAK kinase activity.

3. The method of claim 2, wherein the FAK pathway modifier is a small molecule FAK inhibitor or a pharmaceutically acceptable salt thereof.

4. The method of claim 3, wherein the small molecule FAK inhibitor is an ATP-competitive inhibitor.

5. The method of claim 4, wherein the small molecule FAK inhibitor comprises a heterocyclic ring structure.

6. The method of claim 5, wherein the heterocyclic ring structure comprises a pyrimidine or quinazoline based core.

7. The method of claim 3, wherein the small molecule FAK inhibitor is selected from the group consisting of defactinib, PF-431396, narmafotinib, TAE-226, and PF-573228, or a pharmaceutically acceptable salt thereof.

8. The method of claim 3, wherein the small molecule FAK inhibitor is defactinib or a pharmaceutically acceptable salt thereof.

9. The method of claim 3, wherein the small molecule FAK inhibitor is PF-431396 or a pharmaceutically acceptable salt thereof.

10. The method of claim 1, wherein the FAK pathway modifier inhibits both FAK1 (PTK2) and FAK2 (Pyk2 / PTK2B).

11. The method of claim 1, wherein the FAK pathway modifier reduces FAK protein expression.

12. The method of claim 11, wherein the FAK pathway modifier is a proteolysis-targeting chimera (PROTAC) that targets FAK for proteasomal degradation.

13. The method of claim 12, wherein the PROTAC is GSK215.

14. The method of claim 11, wherein the PROTAC is FC11.

15. The method of claim 1, wherein the FAK pathway modifier is an antibody or polypeptide that inhibits FAK production or activity.

16. The method of claim 1, wherein administration of the composition reduces IgE-mediated mast cell degranulation in the subject.

17. The method of claim 16, wherein the reduction in IgE-mediated mast cell degranulation comprises a reduction in beta-hexosaminidase release from mast cells.

18. The method of claim 1, wherein administration of the composition reduces mast cell surface expression of CD63 and / or CD 107a.

19. The method of claim 1, wherein the composition inhibits FAK autophosphorylation at tyrosine 397 (Y397).

20. The method of claim 1, wherein administration of the composition reduces activation-induced mast cell adhesion.

21. The method of claim 1, wherein the composition is administered prior to exposure of the subject to one or more allergens.

22. The method of claim 21, wherein the composition is administered within 24 hours prior to exposure to the one or more allergens.

23. The method of claim 1, wherein the composition is administered subsequent to exposure of the subject to one or more allergens.

24. The method of claim 23, wherein the composition is administered within 10 minutes after exposure to the one or more allergens.

25. The method of claim 1, wherein the anaphylaxis is triggered by exposure to a food allergen.

26. The method of claim 25, wherein the food allergen is selected from the group consisting of peanut allergen, tree nut allergen, shellfish allergen, dairy allergen, egg allergen, wheat allergen, soy allergen, sesame allergen, fish allergen, and combinations thereof.

27. The method of claim 1, wherein the anaphylaxis is triggered by exposure to an insect sting allergen, a medication allergen, or a latex allergen.

28. The method of claim 1, further comprising co-administering epinephrine to the subject.

29. The method of claim 1, further comprising co-administering to the subject one or more agents selected from the group consisting of an anti-IgE antibody, an inhibitor of IL-4 and / or IL- 13 signaling, an inhibitor of IL-5 and / or IL-5 receptor, and an inhibitor of thymic stromal lymphopoietin (TSLP).

30. The method of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.

31. The method of claim 1, wherein the composition is administered via injection.

32. The method of claim 31, wherein the composition is administered via an auto-injector.

33. The method of claim 1, wherein the composition is formulated for immediate release.

34. The method of claim 1, wherein the subject is a human.

35. The method of claim 34, wherein the subject is a pediatric human.

36. A method of arresting or reducing the severity of an ongoing anaphylactic reaction in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising a focal adhesion kinase (FAK) inhibitor, wherein the composition is administered after the subject has been exposed to an allergen and after onset of one or more symptoms of anaphylaxis.

37. The method of claim 36, wherein the FAK inhibitor is a small molecule FAK inhibitor selected from the group consisting of defactinib, PF-431396, narmafotinib, TAE-226, and PF-573228, or a pharmaceutically acceptable salt thereof.

38. The method of claim 36, wherein the composition is administered within 10 minutes after exposure to the allergen.

39. The method of claim 38, wherein the composition is administered within 5 minutes after exposure to the allergen.

40. The method of claim 36, wherein the one or more symptoms of anaphylaxis comprise at least one of hypothermia, respiratory distress, urticaria, hypotension, vascular collapse, or systemic shock.

41. The method of claim 36, wherein administration of the composition reduces mast cell degranulation that is already in progress at the time of administration.42 The method of claim 36, wherein the composition is administered via injection or auto-inj ector.

43. The method of claim 36, further comprising co-administering epinephrine to the subject.

44. The method of claim 36, wherein the subject is a human.

45. A method of reducing IgE-mediated mast cell degranulation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a FAK pathway modifier that inhibits FAK activity, FAK expression, or FAK signaling.

46. The method of claim 45, wherein the FAK pathway modifier is a small molecule FAK inhibitor, a FAK-targeting proteolysis-targeting chimera (PROTAC), or a nucleic acid that reduces FAK expression.

47. The method of claim 45, wherein reducing IgE-mediated mast cell degranulation treats or prevents one or more conditions selected from anaphylaxis, urticaria, allergic asthma, bronchoconstriction, mast cell activation syndrome, diarrhea, and abdominal pain.

48. The method of claim 45, wherein the FAK pathway modifier reduces mast cell surface expression of CD63 and / or CD 107a.

49. The method of claim 45, wherein the FAK pathway modifier reduces activation-induced mast cell adhesion.

50. The method of claim 45, wherein the subject has a mast cell-mediated disorder.

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