Significance IgE antibody interaction with allergen proteins is the main driver for life-threating allergic responses. Using a nanoparticle-based method, we identified the most crucial binding sites for IgEs on peanut proteins by testing a small population of patient serum with a clinical history of severe allergies to peanuts. We then synthesized inhibitors, we call covalent heterobivalent inhibitors, which specifically and permanently prevent IgEs from binding to peanut proteins and triggering allergic responses. This is significant as it seems that only a few binding sites on peanut allergen proteins drive allergic responses. Also, this is a functional example of selective IgE inhibition to a relevant food allergen and establishes cHBIs as a class of allergy therapeutics.

Abstract Allergies are a result of allergen proteins cross-linking allergen-specific IgE (sIgE) on the surface of mast cells and basophils. The diversity and complexity of allergen epitopes, and high-affinity of the sIgE–allergen interaction have impaired the development of allergen-specific inhibitors of allergic responses. This study presents a design of food allergen-specific sIgE inhibitors named covalent heterobivalent inhibitors (cHBIs) that selectively form covalent bonds to only sIgEs, thereby permanently inhibiting them. Using screening reagents termed nanoallergens, we identified two immunodominant epitopes in peanuts that were common in a population of 16 allergic patients. Two cHBIs designed to inhibit only these two epitopes completely abrogated the allergic response in 14 of the 16 patients in an in vitro assay and inhibited basophil activation in an allergic patient ex vivo analysis. The efficacy of the cHBI design has valuable clinical implications for many allergen-specific responses and more broadly for any antibody-based disease.

Allergy symptoms can vary from a rash to a potentially fatal anaphylactic reaction. Food allergies, especially peanut allergy, fall into the latter. These types of allergic reactions (IgE-dependent type-I hypersensitivity reactions) can lead to life-threatening systemic inflammatory responses such as anaphylactic shock (1, 2). The critical step in a peanut-allergy reaction is the binding of peanut allergens to allergen-specific IgE antibodies (sIgEs) attached to the high-affinity Fc epsilon receptors (FcεRIs) on mast cell surfaces that results in their cross-linking. This cross-linking triggers intracellular signaling that culminates in cellular degranulation that releases preformed mediators stored in cytoplasmic granules, including vasoactive amines, neutral proteases, proteoglycans, cytokines, and chemokines (3).

Currently, in the clinic, there are no medications that can prevent the degranulation response from mast cells, aside from a nonspecific IgE depletion therapy, wherein omalizumab, a monoclonal antibody, binds and inhibits all IgE from binding the surface receptor. Although it has shown effectiveness in clinical treatment of asthma, omalizumab has not been approved for the treatment of peanut allergies (4, 5). Furthermore, the inhibition of all IgE can lead to nonspecific immune suppression, leading to increases in parasitic infection and cancer (6, 7). A targeted and specific sIgE inhibitor would alleviate these concerns. One strategy for inhibition of peanut-allergy reactions (and any other food allergy) is to prevent the binding of allergen epitopes to sIgEs, but there are currently no functional examples of specific sIgE inhibitory compounds in the clinic or scientific literature.

The major challenge in developing targeted sIgE inhibitors is to identify the critical immunogenic epitopes from the large number of potential epitopes on allergen proteins (8, 9). Literature reports of several sIgE binding epitopes of the two most immunogenic peanut proteins, Ara h 2 and Ara h 6, exist (10, 11). These studies, however, did not assess the relative importance of those epitopes for IgE-dependent activation, instead only determining that they bind sIgEs. Here, we developed a physiologically relevant, nanoparticle-based assay system we termed nanoallergens, to identify the most critical immunogenic epitopes of Ara h 2 and Ara h 6. Next, we used this information to rationally design a class of sIgEs inhibitors, named covalent heterobivalent inhibitors (cHBIs), to irreversibly bind to and inhibit peanut allergen/sIgE interactions, thereby selectively and exhaustively inhibiting peanut-allergy reactions (SI Appendix, Fig. S1). We demonstrated, by using peanut allergic patient sera, that cHBIs specifically and irreversibly target the most crucial sIgEs for allergic responses and yield almost complete inhibition of degranulation responses to peanuts in cellular assays with patient serum. This study presents compelling preclinical data for further development and assessment of the cHBI strategy as inhibitors of peanut allergies and other food allergies to improve patient outcomes.

Discussion Here, we described the design and evaluation of cHBIs, a selective sIgE inhibitor, to target and permanently inhibit the antigen binding site of allergen-specific IgE antibodies that recognize specific allergen epitope sequences. The most critical component of cHBI design required the identification of the immunodominant epitopes that are critical mediators during the initiation of degranulation responses. Using the nanoallergen platform, we identified these immunodominant peanut-allergen epitopes for a diverse set of serum samples in a functional cellular degranulation assay. The results showed that the immunodominant epitopes were commonly shared by all of the 16 subjects. Therefore, two cHBI molecules to mimic these two epitopes were synthesized and evaluated. One of the most significant and surprising results was that two cHBIs, targeting only two of the peanut epitopes, was sufficient to inhibit degranulation responses to crude peanut extract with over 80% efficiency in 14 of 16 patient samples, although this effect was dependent upon the concentration of allergen challenge. Given that patients generate IgE to multiple proteins and multiple epitopes within each protein, the inhibition of responses by only two epitopes highlights that we still know very little about engagement of IgE by complex allergens. In our previous work we demonstrated that low-affinity epitopes can cooperate with high-affinity epitopes in IgE engagement and degranulation responses (32). Moreover, we previously observed that certain peanut epitopes trigger degranulation at significantly lower concentrations of nanoallergens, but when used in combination with an epitope of lower potency, there is a synergistic enhancement of degranulation (12). This combinatorial aspect is important and contrasts with many studies that use “allergens” with high numbers of multimerized epitopes. This work suggests several things that are important in considering allergen reactivity. First, we suggest that there are public epitopes, reactivity that is shared across a broad portion of the peanut-allergic human population, in IgE recognition of peanut proteins. Second, we suggest that these public allergen epitopes are required for allergen responses and can be therapeutically targeted to inhibit responses. This finding potentially eliminates a major obstacle in designing allergen-specific inhibitors, suggesting that inhibitors would not need to be personalized and that common reagents could be used by a broader segment of the peanut-allergic population than initially perceived. This will be a critical concept to explore in other allergens to determine if our approach to inhibitor generation can be broadly applied to allergy treatment. The second most significant result is increase in efficacy and the lack of complications or off-target effects due to incorporation of ITC in the cHBI design. From our experience with allergen model systems, we predicted that without a permanent covalent linkage, the epitope mimics would be unlikely to sufficiently inhibit sIgEs due to the short half-life of peptide-based therapeutics and the eventual dissociation of the inhibitor from sIgEs (33). One major concern is that cHBIs will indiscriminately conjugate proteins and trigger nonspecific inflammatory responses or side effects. While we did not observe any acute toxicity in our mouse experiments and have demonstrated that these inhibitors are very selective in vitro, further studies concerning the chronic toxicology of these inhibitors on immune responses is necessary. Based on the data from this study, we expect cHBIs to be nontoxic and the irreversible action of cHBI inhibitors could potentially yield long-lasting inhibition, since IgE bound to mast cells and basophils can remain in the body for months (34). The cHBI design would be a significant improvement over current allergy therapeutics. Drugs that are currently available, such as epinephrine or antihistamines, only transiently attenuate the intensity of the degranulation response and allergic symptoms. This type of approach to immune inhibition is nonspecific and results in blocking immune components that could be important for antipathogen or antitumor immunity. Attempts at developing specific allergy inhibitors have not been successful due to the complexity of the immune response and the challenges arising from the multivalent nature of the interactions. This study describes the cHBI design, which provides a solution to these issues by using a lesser-known binding site on the F ab arm of antibodies with a multivalent design, and presents a successful example of a specific allergy inhibitor to be used on a clinically relevant protein-based allergen.

Materials and Methods Materials. NovaPEG Rink amide resin, 5(6)-carboxy-fluorescein, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU), Fmoc-Lys(IvDde)-OH, all Fmoc protected amino acids, and 10 kDa 0.5 mL centrifugal filters were purchased from EMD Millipore. N,N-dimethylformamide (DMF) (>99.8%), dichloromethane (DCM) (>99.8%), N,N-diisopropylethylamine (DIEA), methanol, hydrazine, piperidine, trifluoroacetic acid (TFA), triisopropylsilane (TIS), tryptamine, 2-naphthaleneacetic acid, ethylene diamine, biotin, di-tert-butyl carbonate (BOC 2 O), 4-(dimethylamino)pyridine (DMAP), succinic anhydride, carbon disulfide (CS 2 ), diisopropylazocarboxylate (DIAD), 2,4-dinitro-1-fluorobenzene (DNFB), acetonitrile, acetic acid, methanol, carbonate-bicarbonate buffer, Tween 20, indole-3-butyric acid (IBA), biotin, PBS, streptavidin conjugated to HRP (Step- HRP), defattened peanut meal (crude peanut extract), carboxyfluorescein (CF), and cytochalasin D were purchased from Sigma-Aldrich. IBA-Boc is a Boc protected variant of IBA, which was synthesized separately in house. Ara h 1, Ara h 2, biotin-Ara h 2, Ara h 3, and Ara h 6 proteins were purchased from Indoors Biotechnologies. DSPC, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000) (ammonium salt), membranes, and all mini extruder components were purchased from Avanti Polar Lipids (Alabaster). High-binding and non-binding 96-well plates were purchased from Corning. RPMI media, minimum essential media, penicillin-strep solution, l-glutamine, and Amplex Red ELISA kits were purchased from Life Technologies. BSA, G418 disulfide salt, and FBS were purchased from Gemini Biosciences. The 96-well tissue culture plates were purchased from Falcon. Fmoc-N-amido-dPEG 2 -acid (EG 2 ), Fmoc-N-amido-dPEG 4 -acid (EG 4 ) and Fmoc-N-amido-dPEG 8 -acid (EG 8 ) were purchased from Quanta Biodesign. Fluorescein isothiocyanate (FITC) was purchased from Toronto Research Chemistry. Anti-human cyclinA IgEs (clone BF683) were purchased from BD Biosciences. Mouse IgGPenicillin (monoclonal antibody clone P2B9) anti-human IgE (clone XTE4) was purchased from Abcam anti-DNP IgE (clone SPE-7) was purchased from Sigma-Aldrich. Anti-FITC mouse IgG-HRP conjugate was purchased from Jackson Immunoresearch. All antibodies used for BAT (visibility dye, anti-HLA-DR, anti-CD123, anti- CD63, anti-CD107a and anti-CD203c) were purchased from Fisher. ACK lysis buffer was purchased from Lonza. Human serum was obtained from two different sources. Peanut-allergic patient serum (sera 1, 2, 3, and 4) and healthy nonallergic control patient serum were purchased from Plasma Lab International. Sera 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 were taken from patients enrolled in an oral peanut immunotherapy (OIT) clinical trial administered at Massachusetts General Hospital under the direction of Wayne Shreffler (NCT01324401). Samples were obtained from patients before OIT and selected from patients having sIgEs for peanuts >50 kU/L as determined by ImmunoCAP. Blood from human subjects was obtained by venipuncture from patients with a clinical history of severe allergic reactions to peanuts or from healthy volunteers. All volunteers consented and all studies were approved by either the Massachusetts General Hospital Institutional Review Board or the Indiana University Institutional Review Board. Methods. Nanoallergen synthesis and characterization. We utilized the epitope-lipid synthesis strategy as seen in previous publications (12). We synthesized epitope-lipids of all five Ara h 2 epitopes and five Ara h 6 epitopes using standard Fmoc solid-phase peptide synthesis (SPPS) chemistry on NovaPEG Rink amide resin. Residues were activated with HBTU and DIEA in DMF for 5 min and allowed to couple to resin for 30–45 min. Coupling efficiency was monitored using a Kaiser test. The Fmoc-protected residues were deprotected with a 3-min application of piperidine in DMF three times. After synthesis of epitope as a linear peptide, an EG 2 spacer, three lysines, three additional EG 6 spacers, a tryptophan residue, an Fmoc-Lys(Fmoc)-OH and palmitic acid (C16) tails were added in that order to the resin. Molecules were cleaved from the resin using a 95/2.5/2.5 TFA/water/TIS mixture for two cycles for 45 min each. We purified the molecules using RP-HPLC on an Agilent 1200 series system with a semipreparative Zorbax C18 column or Zorbax C3 column with either acetonitrile or 2-propanol gradients in the mobile phase. We monitored the column eluent with a diode array detector allowing a spectrum from 200 to 400 nm to be analyzed. The purified product was characterized using a Bruker Autoflex III Smartbeam matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Liposomal nanoallergens were prepared using a procedure as previously described (35). Epitope lipids were then incorporated into liposomes using dry film hydration and extrusion. The liposomes were composed of 2% epitope lipid, 5% polyethylene glycol 2000(mPEG2000)–DSPE, 5% cholesterol, and 93% DSPC. The lipid mixture was dissolved in chloroform, dried with nitrogen lyophilized for 30 min, rehydrated in PBS at 60 °C, and then extruded through a 100-nm polycarbonate filter (Avanti). The liposomes were characterized by DLS analysis via a 90Plus nanoparticle size analyzer (Brookhaven Instruments Corp.), using 658 nm light observed at a fixed angle of 90° at 20 °C. All nanoallergens had particle sizing between 115 and 125 nm, depending on the epitope lipid (Fig. 2C and SI Appendix). cHBI synthesis. All inhibitors (cHBI and FITC-cHBI) were synthesized using SPPS (23), with several modifications. First, the epitope was synthesized as a linear peptide using Fmoc-amino acids activated with a 3.6-fold excess of HBTU with 20-fold DIEA for 5 min before addition. Note that for FITC-cHBI, before epitope synthesis, Fmoc-Lys-IvDdE-OH was added, then deprotected and Fmoc-EG 2 -OH was added. The Fmoc was deprotected and a HBTU-activated CF was added. Then the IvDdE group was deprotected with 2% hydrazine in DMF and epitope synthesis was performed. Following epitope synthesis for all cHBIs, a Fmoc-EG 8 -OH spacer was added, followed by conjugation of Fmoc-Lys(IvDdE)-OH. To the Fmoc-protected amine, IBA-BOC was added. Following IBA-Boc addition, the IvDdE group of lysine was deprotected using 2% hydrazine in DMF in the same fashion. Fmoc-EG 4 -OH spacer was added to the ε-amine of this lysine. After deprotection of the Fmoc group of the EG 4 spacer, ITC domain was added, just before cleavage from resin. The deprotected amines were chemically modified into ITC moieties using a modified procedure from Munch et al. (36). Briefly, resins with deprotected primary amines were washed in anhydrous DMF three times. A 10-fold excess CS 2 with a 20-fold excess of DIEA was added in DMF and allowed to react for 30 min. Resin was then drained and washed once with anhydrous DMF. One milliliter of DMF with a 20-fold excess of DIEA was added to resin and cooled to ∼0 °C in a −20 °C freezer. Then, a 2-fold excess of Boc 2 O and 0.2-fold of DMAP was added to the vessel and allowed to react for 20 min at −20 °C. The vessel was removed, allowed to warm to room temperature for 30 min, and then washed with DMF, DCM, and diethyl ether and allowed to dry in a vacuum chamber. See SI Appendix, Fig. S5 for further details. Molecules were cleaved from the resin using a 95/2.5/2.5 TFA/water/TIS mixture for two cycles for 45 min each. The resulting solution was evaporated using a Buchi rotor-evaporator to remove TFA, rehydrated in 50/50 ACN/water, and purified by RP-HPLC using an Agilent 1200 series HPLC with a Zorbax C18 semiprep column using an ACN/water gradient between 20% and 60% ACN in 10 min with a flow rate of 4 mL/min. Product was collected, evaporated, lyophilized, and redissolved in DMSO. Concentration was determined by absorbance at 280 nm or 335 nm. All molecules were characterized using high-resolution MicroTOF MS analysis. Purity was determined by analytical RP-HPLC using Zorbax Eclipse XBD-C18 with a 20–60% ACN gradient; all molecules were assessed to have >95% purity through analysis of a 220-nm signal. The following molecules used in this study were analyzed using high-resolution mass spectroscopy using a Bruker microTOF II mass spectrometer. Note that all molecular weights have +1 mass increase from the addition of a proton. cHBIArah2/ep5 (C 126 H 207 N 27 O 49 S) was determined to be 2916.45 m/z (2915.43 m/z expected); cHBIArah6/ep5 (C 99 H 170 N 18 O 39 S) was determined to be 2269.17 m/z (2268.16 m/z expected); FITC-cHBIArah2/ep5 (C 160 H 242 N 30 O 59 S) was determined to be 3561.67 m/z (3560.66 m/z expected); and FITC-FITC-cHBIArah6/ep5 (C 133 H 205 N 21 O 49 S) was determined to be 2914.40 m/z (2913.39 m/z expected). Cell culture. RBL-SX38 cells (cells expressing human FcεRI) were a generous gift from Jean-Pierre Kinet, Harvard University, Cambridge, MA. RBL-SX38 were cultured in MEM (Gibco) with 10% FBS (Gemini BioProducts), l-glutamine, penicillin-streptomycin, and 1.2 mg/mL of G418 salt (Sigma) as previously described (37). RBL-2H3 cells (ATCC) were cultured in a similar manner but without the addition of G418. Degranulation assays. Degranulation assays were performed as previously described using nanoallergens, Ara h proteins, or crude peanut extract as the allergen with some modifications (12). RBL-SX38 cells were plated (100 µL each, 50,000 cells per well) into 96-well dishes for 24 h and then incubated with 10% of patient sera (sera 1–16 or a serum taken from healthy volunteer as control) in cell culture media for an additional 24 h before the degranulation assay. Cells were then washed and incubated in RBL media for 1 h. Cells were then washed, incubated in tyrodes buffer with 0.5 mg/mL BSA and 1 µM Cytochalasin D, and challenged with allergens or lysed with Triton X (as a positive control). After allergen challenge, the supernatant was incubated with pNAG solution for 45 min, the reaction halted with a stopping solution (1.5 mg/mL glycine, pH 10.7), and the degranulation measured by reading the absorbance at 405 nm after subtracting the background signal at 630 nm. To evaluate the cHBI inhibitors, RBL-SX38 cells were similarly plated and primed with patient serum. However, before incubation with allergen, cells were incubated with cHBIs (15 min–24 h), and similarly challenged with allergens as described above. Note that concentrations of crude peanut extract used for inhibition assays shown in Fig. 3A varied, given the sensitization of the patient to crude peanut extract. A total of 1 ng/mL was used for serum 6 and sera 8–16, 100 ng/mL was used for sera 3 and 7, and the remaining sera 1, 2, and 4 were challenged with 1,000 ng/mL. For DNP-BSA degranulation assays, a similar procedure was used with the following exceptions: RBL-2H3 cells were used instead of RBL-SX38 cells, and cells were incubated with a 25%/75% mixture of anti-DNP IgE and anti-cyclin A IgE for 24 h instead of patient serum, and DNP-BSA was used as the allergen. Percent degranulation was calculated as previously decribed (32). Briefly, experimental signals were calculated as a percentage between positive control (100%) and negative control (0%). Degranulation maximum (D max ) and EC 50 values were calculated using Origin 7 software using Hill’s curve fit for sigmoidal curves using percent degranulation values. Note that if nanoallergen did not induce significant degranulation above error (determined as SD of triplicate experiments, P < 0.05), it was deemed to have “no response.” If a significant signal was present but there was insufficient data to determine an EC 50 value, the EC 50 value was estimated to be greater than an EC 50 value calculated assuming that the highest signal reached was D max . ELISA assay. Binding interactions between cHBI and antibodies were detected using a sandwich ELISA. Before the ELISA test, patient serum was incubated at various concentrations with FITC-cHBIs at 37 °C for 5 h. Then, a high-binding 96-well ELISA plate was coated with 100 μL of 2 nM of anti-IgE antibody for 2 h in bicarbonate buffer (pH = 10). Plates were washed to remove unbound antibody. Wells were blocked with a blocking buffer (5% BSA, 0.2% Tween-20 solution in PBS) for 1 h, washed, and incubated with serum-cHBI mixture mixed 1:1 with the blocking buffer for 16 h. The plate was washed again and incubated with an anti-FITC mouse IgG-HRP conjugate (diluted 1:5,000 in blocking buffer) for 1 h. The plate was washed again and an Amplex Red Kit was used to quantify the ELISA signal using a SpectraMax M5 spectrophotometer according to the manufacturer’s instructions. For the Ara h 2 competitive binding inhibition, a similar ELISA was performed with some modifications. Before the ELISA test, patient serum was incubated in various concentrations with unlabeled cHBIs at 37 °C for 5 h. Then, a high-binding 96-well ELISA plate was coated with 100 μL of 2 nM of anti-IgE antibody for 2 h in bicarbonate buffer. Plates were washed with PBS to remove unbound antibody. Wells were blocked with the blocking buffer for 1 h, washed, and incubated with serum-cHBI mixture mixed 1:1 with the blocking buffer for 16 h. The plate was washed again and incubated with biotin-tagged HRP (diluted 1:5,000 in blocking buffer) for 1 h. The plate was washed again, incubated with streptavidin-HRP conjugate, washed, and an Amplex Red Kit was used to quantify the ELISA signal using a SpectraMax M5 spectrophotometer according to the manufacturer’s instructions. Animal studies. C57BL/6 female mice (7- to 8-wk old) were obtained from Harlan Biosciences. Mice were maintained in pathogen-free conditions, and studies were approved by the Indiana University Institutional Animal Care and Use Committee. Toxicology studies were carried out in a similar manner as previously published in the University of Indiana In Vivo Therapeutics Core (32). Basophil activation test. The BAT assay protocol was used as previously described (38). Briefly, blood collected in vacutainer tubes containing heparin was processed within 24 h of collection. For basophil activation: 100 µL of whole blood was diluted 1:1 in serum-free RPMI containing cHBIs for 4 h at 37 °C. Cells were activated with 100 ng/mL of crude peanut extract or media (blank) for 30 min at 37 °C. RBs were lysed using ACK lysis buffer and remaining cells were stained and fixed for flow cytometry. Basophils were selected as viability dye negative, SSClow, HLA-DR−, and CD123+. Each sample was analyzed in triplicate with a cutoff of 1,000 basophils per sample. Basophil activation was assessed by measuring fluorescence of CD63, CD107a, and CD203c. Percentage maximal CD63 MFI was found by calculating the percentage between the blank (negative control) and the no inhibitor (positive control) MFI for CD63.

Acknowledgments We thank Dr. Jean-Pierre Kinet for his kind gift of the RBL-SX38 cells; Mr. Douglas Zych and Mr. and Mrs. Jim and Annette Lecinski for funding; the NIH for Grants R01 AI108884 (to B.B. and M.H.K.), R01 AI129241 (to M.H.K.), U19 AI095261 (to W.G.S.), and T32 DK007519 (to A.A.Q.); the University of Notre Dame Proteomics Facility for use of their mass spectroscopy equipment; and Indiana University (IU)’s In Vivo Therapeutics Core for performing our toxicology experiments. M.J.T. was supported by VA CDA2 (IK2 CX001019). Core facility usage was supported by IU Simon Cancer Center Support Grants P30 CA082709 and U54 DK106846. A pilot grant and additional support provided by the Herman B Wells Center was in part from the Riley Children’s Foundation.

Footnotes Author contributions: P.E.D., A.A.Q., M.H.K., and B.B. designed research; P.E.D., B.K., A.A.Q., J.S., G.V., K.M.K., and M.J.T. performed research; P.E.D., A.A.Q., G.V., K.M.K., M.J.T., N.S., W.G.S., and M.H.K. contributed new reagents/analytic tools; P.E.D., A.A.Q., T.K., M.H.K., and B.B. analyzed data; P.E.D., T.K., and B.B. wrote the paper; and W.G.S. provided access to patient samples.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. S.J.G. is a guest editor invited by the Editorial Board.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1820417116/-/DCSupplemental.