Deglycosylated IGE antibodies and methods for treating IGE-mediated disorders

Abstract

This disclosure relates to compositions comprising modified IgE antibodies or Fc fragments thereof and uses thereof for inducing anti-IgE immunogenic responses and / or reducing risk of or treating IgE-mediated disorders.

Description

[0001] Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0002] DEGLYCOSYLATED IGE ANTIBODIES AND METHODS FOR TREATING IGE-MEDIATED DISORDERS

[0003] CLAIM OF PRIORITY

[0004] This application claims the benefit of U.S. Provisional Application Serial No.

[0005] 63 / 762,945, filed on February 25, 2025. The entire content of the foregoing is incorporated herein by reference.

[0006] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with government support under Grant No. AI139669 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] TECHNICAL FIELD

[0009] Provided herein are compositions and methods for preventing and treating IgE-mediated disorders.

[0010] BACKGROUND

[0011] Allergic diseases represent a major and growing global health burden, affecting approximately 20%-40% of the population worldwide. These disorders include, for example, asthma, allergic rhinitis, food allergy, and urticaria and are driven by inappropriate immune responses to otherwise innocuous environmental antigen, food antigens, or drugs. Central to the pathophysiology of allergic disease is the immunoglobulin E (IgE) antibody, which orchestrates potent inflammatory cascades upon allergen encounter, the most severe of which is anaphylaxis, which can be lifethreatening.

[0012] SUMMARY

[0013] The present disclosure relates to modified IgE antibodies or Fc fragments thereof and uses thereof for inducing anti-IgE immunogenic responses and / or preventing or treating IgE-mediated disorders. The present disclosure is based, at least in part, on the discovery that removal of a specific glycan (e.g., oligomannose) atN394 and / or mutation to remove the N-linked glycosylation site at N394 on human IgE (orAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0014] the equivalent on mouse IgE), renders IgE immunogenic and capable of inducing longterm immune protection against IgE. IgE antibodies or Fc fragments thereof that lack that glycan (e.g., oligomannose) at N394 or having an amino acid modification (e.g., deletion or mutation) to remove the N-linked glycosylation site at N394 can be administered to individuals and result in long-term anti-IgE immune protection. Administration of the modified IgE antibodies or Fc fragments thereof disclosed herein can reduce the titer of IgE antibodies in individuals having IgE-mediated disorders.

[0015] In some aspects, provided herein is a method of inducing an anti-IgE immunogenic response in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising a modified IgE antibody or an Fc fragment thereof comprising a deglycosylated N394, wherein the modified IgE antibody is a human antibody or a humanized antibody.

[0016] In some aspects, provided herein is a method of treating or preventing an IgE-mediated disorder in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising a modified IgE antibody or an Fc fragment thereof comprising a deglycosylated N394, wherein the modified IgE antibody is a human antibody or a humanized antibody.

[0017] In some embodiments, the deglycosylated N394 lacks oligomannose, optionally as a result of treatment of a native human IgE antibody or Fc fragment thereof with EndoFl or PNGaseF. In some embodiments, the deglycosylated N394 is linked to N-acetylgalactosamine, galactose, and / or sialic acid; or the deglycosylated N394 lacks N-acetylgalactosamine, galactose, and / or sialic acid. In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises a deglycosylated N383 and / or a deglycosylated N371. In some embodiments, the deglycosylated N383 and / or the deglycosylated N371 lack oligomannose. In some embodiments, the deglycosylated N383 and / or the deglycosylated N371 are linked to N-acetylgalactosamine, galactose, and / or sialic acid; or the deglycosylated N383 and / or the deglycosylated N371 lack N-acetylgalactosamine, galactose, and / or sialic acid.

[0018] In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises one or more amino acid modifications atN371, N383, and / or T396. In some embodiments, the one or more amino acid modifications at N371, N383, and / or T396 comprise one or more mutations or deletions, optionally wherein: theAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0019] mutation atN383 isN383Q, N383A, orN383D; the mutation atN371 isN371Q, N371A, orN371D; and / or the mutation at T396 is T396A or T396V.

[0020] In some aspects, provided herein is a method of inducing an anti-IgE immunogenic response in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising a modified IgE antibody or an Fc fragment thereof comprising one or more amino acid modifications at N394 and / or T396, wherein the modified IgE antibody is a human antibody or a humanized antibody.

[0021] In some aspects, provided herein is a method of treating or preventing an IgE-mediated disorder in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising a modified IgE antibody or an Fc fragment thereof comprising one or more amino acid modifications at N394 and / or T396, wherein the modified IgE antibody is a human antibody or a humanized antibody.

[0022] In some embodiments, the one or more amino acid modifications at N394 and / or T396 comprise one or more mutations or deletions, optionally wherein: the mutation at N394 is N394Q, N394A, or N394D; and / or the mutation at T396 is T396A or T396V. In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises one or more amino acid modifications at N383 and / or N371. In some embodiments, the one or more amino acid modifications at N383 and / or N371 comprise one or more mutations or deletions, optionally wherein: the mutation at N383 isN383Q, N383A, orN383D; and / or the mutation at N371 is N371Q, N371A, or N371D. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a deglycosylated N394, a deglycosylated N383, and / or a deglycosylated N371. In some embodiments, the deglycosylated N394, the deglycosylated N383, and / or the deglycosylated N371 lack oligomannose. In some embodiments, the deglycosylated N394, the deglycosylated N383, and / or the deglycosylated N371 are linked to N-acetylgalactosamine, galactose, and / or sialic acid; or the deglycosylated N394, the deglycosylated N383, and / or the deglycosylated N371 lack N-acetylgalactosamine, galactose, and / or sialic acid.

[0023] In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises the Fc Cs3 domain. In some embodiments, the Fc Cs3 domain comprises a sequence at least 80% identical to SEQ ID NO: 18, optionally wherein the Fc Cs3Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0024] domain comprises the sequence as set forth in SEQ ID NO: 18, 28, or 29. In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises an Fc Cs2 domain and / or an Fc Cs4 domain, optionally wherein the Fc Cs2 domain comprises a sequence at least 80% identical to SEQ ID NO: 17, and the Fc Cs4 domain comprises a sequence at least 80% identical to SEQ ID NO: 19, optionally wherein the modified IgE antibody or an Fc fragment thereof comprises a sequence at least 80% identical to SEQ ID NO: 25, optionally wherein the modified IgE antibody or an Fc fragment thereof comprises the sequence as set forth in SEQ ID NO:25, 26, or 27. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an antigen-binding fragment. In some embodiments, the antigen-binding fragment comprises a VHH, a Fab, or an scFv. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a heavy chain. In some embodiments, the heavy chain comprises a sequence at least 90% identical to SEQ ID NO: 14, 23 or 24. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a light chain. In some embodiments, the light chain comprises a sequence at least 90% identical to SEQ ID NO: 20.

[0025] In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises an Fc region not in an open conformation and / or in a conformation that precludes FcsRI binding. In some embodiments, the modified IgE antibody or an Fc fragment thereof does not bind to FcsRI or binds to FcsRI with an affinity lower at least 100 times (e.g., at least 1000, 5000, 10000, 50000, or 100000 times) lower than a reference control (e.g., an IgE with unmodified Fc region). In some embodiments, the modified IgE antibody or an Fc fragment thereof binds to an FcsRI with a dissociation constant KD of at least 10-1M.

[0026] In some embodiments, the composition is a vaccine composition, optionally the vaccine composition further comprises an adjuvant.

[0027] In some embodiments, the subject is not in anaphylaxis.

[0028] In some embodiments, the IgE-mediated disorder is selected from the group consisting of urticaria (e.g., chronic spontaneous urticaria), systemic lupus erythematosus (SLE), nephritis caused by SLE, bullous pemphigoid, allergic rhinitis, asthma, allergic asthma, non-allergic asthma, atopic dermatitis, gastroenteropathy, and hyper-IgE syndrome (HIES).Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0029] In some embodiments, the IgE-mediated disorder is an IgE-mediated allergy. In some embodiments, the IgE-mediated allergy is selected from the group consisting of food allergy, environmental allergy (e.g., allergies to tree, grass, weed pollens, dust mites, pet saliva / fur, and mold, and / or seasonal allergies), venom allergy, and drug allergy.

[0030] In some embodiments, the method disclosed herein further comprises administration of the modified IgE antibody or an Fc fragment thereof results in longterm immune protection (e.g., induction of antibodies and / or memory immune cells that are detectable at 1 month, 3 months, 6 months, 9 months, and / or 12 months postadministration) against the IgE antibody or an Fc fragment thereof in the subject.

[0031] In some embodiments, the method disclosed herein further comprises measuring a level of long-term immune protection by measuring levels of antibodies and / or memory immune cells that bind to IgE antibody or an Fc fragment thereof at 1 month, 3 months, 6 months, 9 months, and / or 12 months post-administration. In some embodiments, the method disclosed herein comprises measuring levels of IgM, IgGl, IgG2, and / or IgG4 that specifically bind to IgE antibody or an Fc fragment thereof.

[0032] In some embodiments, the level of long-term immune protection is measured by an assay selected from the group consisting of Enzyme-Linked Immunoassay (ELISA), surface plasmon resonance (SPR) / biolayer interferometry (BLI), chemiluminescent immunoassays (CLIA), multiplex bead-based immunoassays (e.g., Luminex), flow cytometry-based analysis, immunofluorescence assay (IF A), cytokine secretion assay, ELISpot, T cell proliferation assay, cytotoxicity assays, immunoprecipitation, Western Blot, and immunocytochemistry.

[0033] In some aspects, provided herein is a composition comprising a modified IgE antibody or an Fc fragment thereof comprising a deglycosylated N394, wherein the modified IgE antibody is a human antibody or a humanized antibody.

[0034] In some embodiments, the deglycosylated N394 lacks oligomannose. In some embodiments, the deglycosylated N394 is linked to N-acetylgalactosamine, galactose, and / or sialic acid; or the deglycosylated N394 lacks N-acetylgalactosamine, galactose, and / or sialic acid. In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises a deglycosylated N383 and / or a deglycosylated N371. In some embodiments, the deglycosylated N383 and / or the deglycosylated N371 lack oligomannose. In some embodiments, the deglycosylated N383 and / or theAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0035] deglycosylated N371 are linked to N-acetylgalactosamine, galactose, and / or sialic acid; or the deglycosylated N383 and / or the deglycosylated N371 lack N-acetylgalactosamine, galactose, and / or sialic acid.

[0036] In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises one or more amino acid modifications atN371, N383, and / or T396. In some embodiments, the one or more amino acid modifications at N371, N383, and / or T396 comprise one or more mutations or deletions, optionally wherein: the mutation atN383 isN383Q, N383A, orN383D; the mutation atN371 isN371Q, N371A, orN371D; and / or the mutation at T396 is T396A or T396V.

[0037] In some aspects, provided herein is a composition comprising a modified IgE antibody or an Fc fragment thereof comprising one or more amino acid modifications at N394 and / or T396, wherein the modified IgE antibody is a human antibody or a humanized antibody.

[0038] In some embodiments, the one or more amino acid modifications at N394 and / or T396 comprise one or more mutations or deletions, optionally wherein the mutation at N394 is N394Q, N394A, or N394D; and / or the mutation at T396 is T396A or T396V.

[0039] In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises one or more amino acid modifications atN383 and / or N371.

[0040] In some embodiments, the one or more amino acid modifications at N383 and / or N371 comprise one or more mutations or deletions, optionally wherein: the mutation atN383 isN383Q, N383A, orN383D; and / or the mutation atN371 is N371Q, N371A, orN371D.

[0041] In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a deglycosylated N394, a deglycosylated N383, and / or a deglycosylated N371. In some embodiments, the deglycosylated N394, the deglycosylated N383, and / or the deglycosylated N371 lack oligomannose. In some embodiments, the deglycosylated N394, the deglycosylated N383, and / or the deglycosylated N371 are linked to N-acetylgalactosamine, galactose, and / or sialic acid; or the deglycosylated N394, the deglycosylated N383, and / or the deglycosylated N371 lack N-acetylgalactosamine, galactose, and / or sialic acid.

[0042] In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises the Fc Cs3 domain. In some embodiments, the Fc Cs3 domain comprises aAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0043] sequence at least 80% identical to SEQ ID NO: 18, optionally wherein the Fc Cs3 domain comprises the sequence as set forth in SEQ ID NO: 18, 28, or 29. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises the Fc Cs2 domain and / or the Fc Cs4 domain, optionally wherein the Fc Cs2 domain comprises a sequence at least 80% identical to SEQ ID NO: 17, and the Fc Cs4 domain comprises a sequence at least 80% identical to SEQ ID NO: 19, optionally wherein the modified IgE antibody or an Fc fragment thereof comprises a sequence at least 80% identical to SEQ ID NO: 25, optionally wherein the modified IgE antibody or an Fc fragment thereof comprises the sequence as set forth in SEQ ID NO: 25, 26, or 27.

[0044] In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises an antigen-binding fragment. In some embodiments, the antigen-binding fragment comprises a VHH, a Fab, or an scFv. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a heavy chain. In some embodiments, the heavy chain comprises a sequence at least 80% identical to SEQ ID NO: 14, 23 or 24. In some embodiments, the modified IgE antibody or Fc fragment thereof comprises a light chain. In some embodiments, the light chain comprises a sequence at least 80% identical to SEQ ID NO: 20.

[0045] In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises an Fc region not in an open conformation and / or in a conformation that precludes FcsRI binding.

[0046] In some embodiments, the modified IgE antibody or an Fc fragment thereof does not bind to FcsRI or binds to FcsRI with an affinity lower at least 100 times (e.g., at least 1000, 5000, 10000, 50000, or 100000 times) lower than a reference control (e.g., an IgE with unmodified Fc region). In some embodiments, the modified IgE antibody or an Fc fragment thereof binds to an FcsRI with a dissociation constant KD of at least 10-1M.

[0047] In some embodiments, the composition is a vaccine composition, optionally the vaccine composition further comprises an adjuvant.

[0048] In some aspects, provided herein is a composition comprising a modified IgE antibody, wherein the modified IgE antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a sequence at least 80% identical (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0049] 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to SEQ ID NO: 14, 23 or 24 and the light chain comprises a sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 20.

[0050] In some embodiments, the heavy chain comprises a sequence at least 80% identical (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to SEQ ID NO: 14 and the light chain comprises a sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 20.

[0051] In some embodiments, the heavy chain comprises a sequence as set forth in SEQ ID NO: 14 and the light chain comprises a sequence as set forth in SEQ ID NO: 20.

[0052] In some embodiments, the heavy chain comprises a sequence at least 80% identical (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to SEQ ID NO: 23 and the light chain comprises a sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 20.

[0053] In some embodiments, the heavy chain comprises a sequence as set forth in SEQ ID NO: 23 and the light chain comprises a sequence as set forth in SEQ ID NO: 20.

[0054] In some embodiments, the heavy chain comprises a sequence at least 80% identical (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to SEQ ID NO: 24 and the light chain comprises a sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 20.

[0055] In some embodiments, the heavy chain comprises a sequence as set forth in SEQ ID NO: 24 and the light chain comprises a sequence as set forth in SEQ ID NO: 20.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0056] In some aspects, provided herein is a use of the composition described herein in inducing an anti-IgE immunogenic response in a subject, or treating or preventing an IgE-mediated disorder an IgE-mediated disorder in a subject

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0058] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0059] DESCRIPTION OF DRAWINGS FIGS. 1A and IB show conservation of IgE glycoforms. FIG. 1A shows schematic of human IgE with glycosylation sites on the heavy chains. N-linked glycan types include complex, biantennary (closed circles) and oligomannose (open circle) glycans which are composed of GlcNAc (squares), fucose (triangle), mannose (circles), galactose (circles), and sialic acid (pin diamonds). X denotes unoccupied. FIG. IB shows percentage occupancy of N-linked glycan types on total human IgE from non-allergic individuals, helminth-infected individuals, hybridoma-derived, myeloma-derived, peanut-reactive Arah2+IgE allergic individuals, peanut-reactive Arah2+IgE non-allergic individuals, Expi293 cell produced recombinant, systemic lupus erythematosus (SLE) patients as determined by glycopeptide mass spectrometry; biantennary (medium gray), oligomannose (dark gray, arrow), unoccupied (light gray), not detected (white).

[0060] FIGS. 2A and 2B show that both recombinant and hybridoma mouse IgE have oligomannose at asparagine 384. FIG. 2A shows schematic representation of mouse IgE showing the glycosylation sites on the heavy chain along with the sugar components of the glycans. FIG. 2B shows percentages of the different glycans on the heavy chain of mouse IgE.

[0061] FIGS. 3A-3E show that 3-Mannose IgE and oligomannose-containing IgE loads equally on mast cells. FIG. 3A shows representation of alpha (1-2,3)-Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0062] mannosidase digestion of oligomannose on IgE (hlgE) to generate 3 -mannose glycoform (Man-3-hIgE) and subsequent digestion by Endo Fl to release the residual sugar residues as analyzed by HPLC. FIG. 3B shows Coomassie stained WT (hlgE) or Man-3 -hlgE or enzyme’s buffer and co-factor treated hlgE (BC-hlgE) after resolving the antibodies on non-reducing SDS-PAGE gel. FIGS. 3C-3D show HPLC analyzed traces and percentage of mannose structures released from Man-3-hIgE and BC-hlgE by EndoFl. FIG. 3E shows FlowJo gating percentages of IgE loaded LAD2 cells that were treated with PBS or hlgE or BC-hlgE or Man-3 -hlgE or with EndoF 1 treated hlgE (EndoFl -hlgE).

[0063] FIGS. 4A-4H show that Man-3-IgE binds its receptors and triggers anaphylaxis. FIG. 4A shows percentage antigen-specific degranulation following IgE-crosslinking of LAD2 human mast cells sensitized with PBS or hlgE or buffer-control hlgE (BC-hlgE), Man-3 -hlgE or EndoFl -hlgE (n=6). FIG. 4B shows percentage of hlgE detected on sensitized LAD2 cells following sensitization with PBS or hlgE or buffer-control hlgE (BC-hlgE), Man-3 -hlgE or EndoFl -hlgE (n=6) and representative histograms as determined by flow cytometry. FIG. 4C shows dye leakage into sensitized mouse ears following OVA-specific PC A following sensitization with PBS or mlgE or buffer-control mlgE (BC-mlgE), Man-3-mIgE orEndoFl-mlgE (n=8). FIG.

[0064] 4D shows OVA-specific ELISA absorbances of Man-3 -a-OVA-mlgE or BC-a-OVA-mlgE. FIGS. 4E-4F show dose-dependent binding and KD of BC-hlgE (FIG. 4E) or Man-3 hlgE (FIG. 4F) to hFcsRIa as determined by BLI. FIG. 4G and 4H show dosedependent binding and KD of BC-hlgE (FIG. 4G) or Man-3-hIgE (FIG. 4H) to CD23 as determined by BLI. All experiments are represented as mean ± SEM with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparison test.

[0065] FIGS. 5A-5E show equal loading of 1-Mannose IgE and oligomannosecontaining IgE on mast cells. FIG. 5A shows sites of cleavage of alpha (1-2, 3,6)-Mannosidase enzyme on oligomannose attached to IgE (hlgE) to produce IgE with 1-Mannose structure (Man-l-hlgE) and digestion of this 1-Mannose IgE by Endo Fl to release the sugars which are then analyzed by HPLC. FIG. 5B shows Coomassie stained migration of hlgE or Man- 1 -hlgE or enzyme’s buffer and co-factor treated hlgE (BC-hlgE) on non-reducing SDS-PAGE gel. FIGS. 5C-5D show HPLC peak resolution of the sugars and percentages of sugars released by EndoFl digestion of Man- 1 -hlgE orAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0066] BC-hlgE FIG. 5E shows FlowJo gating percentages of IgE loaded LAD2 cells that were treated with PBS or hlgE or BC-hlgEl or Man-l-hlgE or EndoFl -hlgE.

[0067] FIGS. 6A-6H show that Man-l-IgE binds its receptors and triggers anaphylaxis. FIG. 6A shows percentages of antigen-specific degranulation following IgE-crosslinking of LAD2 human masts cells sensitized with PBS or BC-hlgE, Man-1-hlgE or EndoFl-hlgE (n=6). FIG. 6B shows percentages of hlgE detected on sensitized LAD2 cells following sensitization with PBS, BC-hlgE, Man-l-hlgE or EndoFl-hlgE (n=6) and representative histograms as determined by flow cytometry. FIG. 6C shows dye leakage into sensitized mouse ears following OVA-specific PCA following sensitization with PBS, BC-mlgE, Man-l-mlgE or EndoFl -mlgE (n=8). FIG. 6D shows OVA-specific ELISA absorbances of Man-l-oc-OVA-mlgE or BC-a-OVA-mlgE. FIGS. 6E-6F show dose-dependent binding and KD of BC-hlgE (FIG. 6E) or Man-3 -hlgE (FIG. 6F) to hFcsRIa as determined by BLI. FIGS. 6G-6H show dosedependent binding and KD of BC-hlgE (FIG. 6G) or Man-3-hIgE (FIG. 6H) to CD23 as determined by BLI. All experiments are represented as mean ± SEM with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 obtained by one-way ANOVA with Dunnett’s multiple comparison test.

[0068] FIGS. 7A-7H show that Man-O-IgE does not bind to high affinity receptor and does not trigger anaphylaxis. FIG. 7A shows percentage antigen-specific degranulation following IgE-crosslinking of LAD2 human mast cells sensitized with PBS, Man-l-BC-hlgE or Man-O-hlgE or EndoFl-hlgE (n=6). FIG. 7B shows percentage of hlgE detected on sensitized LAD2 cells following sensitization with PBS, Man-l-BC-hlgE or Man-O-hlgE or EndoFl-hlgE (n=6) and representative histograms as determined by flow cytometry. FIG. 7C shows dye leakage into sensitized mouse ears following OVA-specific PCA following sensitization with PBS, Man-l-BC-mlgE, Man-O-mlgE or EndoFl -mlgE (n=4-8). FIG. 7D shows OVA-specific ELISA absorbances of Man-O-a-OVA-mlgE or Man-l-BC-a-OVA-mlgE. FIGS. 7E-7F show dose-dependent binding and KD of Man- 1 -BC-hlgE (FIG. 7E) or Man-O-hlgE (FIG. 7F) to hFcsRIa as determined by BLI. FIGS. 7G-7H show dose-dependent binding and KD of Man- 1 -BC-hlgE (FIG. 7G) or Man-O-hlgE (FIG. 7H) to hCD23 as determined by BLI. All experiments are represented by mean ± SEM with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 obtained by one-way ANOVA with Dunnett’s multiple comparison test.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0069] FIGS. 8A-8E show that 0-Mannose IgE loads significantly less than Man-1-IgE on mast cells. FIG. 8 A shows digestion site of P-Mannosidase on Mannose- 1-IgE, following digestion of the high mannose structure by alpha (l-2,3,6)-Mannosidase to generate 0-Mannose structure and digestion of this 0-Mannose IgE by PNGaseF to release the residual sugars which are then analyzed by HPLC. FIG. 8B shows Coomassie stained non-reducing SDS-PAGE gel showing migration of Man-l-hlgE or Man-l-hlgE treated with beta-mannosidase and its buffer (Man-O-hlgE) or with only the buffer (Man-l-BC-hlgE). FIGS. 8C-8D show HPLC-based peak resolution and percentages of the sugars released by PNGaseF digestion of OVA-specific Man-l-BC-mlgE or Man-O-mlgE. FIG. 8E shows FlowJo gating percentages of human IgE loaded onto LAD2 cells that were treated with PBS or Man-l-BC-hlgE or Man-O-hlgE or EndoFl -hlgE.

[0070] FIGS. 9A-9I show that EndoFl treated human IgE shows altered conformation. FIG. 9A shows digestion site of EndoFl on oligomannose of IgE to release the sugars. FIG. 9B shows Coomassie stained non-reducing SDS-PAGE gel showing migration of hlgE or EndoFl and its buffer treated hlgE (EndoF 1 -hlgE) or only buffer treated hlgE (BC-hlgE). FIGS. 9C-9D show HPLC traces and percentages of the sugars released from the oligomannose of hlgE due to digestion of hlgE with Endo Fl (EndoFl -hlgE) or only its buffer (BC-hlgE). FIG. 9E shows size exclusion chromatogram of buffer treated a-OVA-hlgE (BC-a-OVA-hlgE) or EndoFl and buffer treated a-OVA-hlgE (EndoF 1-a-OVA-hIgE). FIG. 9E shows 2D class averages of negative staining microscopy images of EndoF 1-a-OVA-hIgE and BC-a-OVA-hlgE. FIG. 9G shows superimposed major conformations of EndoF 1-a-OVA-hIgE and BC-a-OVA-hlgE. FIG. 9H shows cloud images of major conformation of a-OVA-hlgE with oligomannose and without oligomannose. FIG. 91 shows cloud images of minor conformation of a-OVA-hlgE with oligomannose and without oligomannose.

[0071] FIGS. 10A-10H show FCE2-FCS3 conformation in oligomannose-deficient IgE. FIGS. 10A and 10B show representative processed images of negative staining microscopy generated pictures of major conformation of BC-a-OVA-hlgE and EndoF 1-a-OVA-hIgE. FIGS. 10C and 10D show representative processed images of negative staining microscopy generated pictures of minor conformation of BC-a-OVA-hlgE and EndoF 1-a-OVA-hIgE. FIG. 10E and 10F show 90° rotated images of the major conformations of BC-a-OVA-hlgE and EndoF 1-a-OVA-hIgE. FIGS. 10G andAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0072] 10H shows 90° rotated images of the minor conformations of BC-a-OVA-hlgE and EndoFl -a-OVA-hlgE.

[0073] FIGS. 11A-11L show oligomannose-deficient-IgE retaining CD23 binding.

[0074] FIGS. 11 A-l 1C show PyMOL modelling of binding of processed major conformation images of BC-a-OVA-hlgE (FIG. 11 A), EndoFl -a-OVA-hlgE (FIG. 1 IB) and minor conformation image of EndoFl -a-OVA-hlgE (FIG. 11C) to hFcsRIa. (FIGS. 11D-1 IF) show PyMOL modelling of binding of processed minor conformation images of BC-a-OVA-hlgE (FIG. 1 ID), EndoFl -a-OVA-hlgE (FIG. 1 IF) and major conformation image of EndoFl -a-OVA-hlgE (FIG. HE) to hCD23 . FIGS. 11G-11H show dose-dependent binding and KD of BC-a-OVA-hlgE (FIG. 11G) or EndoFl -a-OVA-hlgE (FIG. 11H) to hFcsRIa as determined by BLI. FIGS. 111-11 J show dosedependent binding and KD of BC-a-OVA-hlgE (FIG. Ill) or EndoFl -a-OVA-hlgE (FIG. 11 J) to hCD23 as determined by BLI. FIG. 1 IK shows human PBMC loading of BC-a-OVA-hlgE and EndoFl -a-OVA-hlgE on basophils and B cells (n=3). FIG. 1 IL shows peritoneal mast cell and B cell loading of a-TNP-mlgE treated with buffer (BC-a-TNP-mlgE) or Endo Fl and buffer (EndoFl -a-TNP-mlgE) (n=4). Data shows mean ± SEM with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 obtained by 2-way ANOVA with Sidak’s multiple comparison test.

[0075] FIGS. 12A-12E show that EndoFl treated IgE interacts with CD23 and loads onto effector cells. FIG. 12A shows CD23-specific ELISA absorbances of EndoFl buffer treated human IgE (BC-a-OVA-hlgE) or EndoFl and its buffer treated human IgE (EndoFl -a-OVA-hlgE). FIG. 12B shows FlowJo gating strategy for BC-a-OVA-hlgE or EndoFl -a-OVA-hlgE loading onto human PBMC basophils and B cells (n=3). FIG. 12C shows percentage of mouse FcsRIa and mouse CD23 on peritoneal mast cells and B cells (n=4). FIG. 12D shows gating strategy of mouse FcsRIa and CD23 on mouse peritoneal mast cells and B cells. FIG. 12E shows gating strategy for checking loading of BC-a-TNP-mlgE and EndoFl -a-TNP-mlgE on mouse peritoneal mast cells and B cells and representative histograms of IgE loaded peritoneal mast cells and B cells in mice that were sensitized with PBS or BC-a-TNP-mlgE or EndoFl-a-TNP-mlgE. Data shows mean ± SEM with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 obtained by 2-way ANOVA with Sidak’s multiple comparison test.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0076] FIGS. 13A-13H show that oligomannose-deficient IgE is immunogenic in vivo. Mice were given PBS or 50pg of BC-a-OVA-mlgE or 50pg of EndoFl -a-OVA-mlgE and bled on day 14 (FIG. 13A). FIGS. 13B-13E show day 14 serum a-OVA-mlgE-specific IgGl, IgG2a, IgG2b and, IgG3 as determined by ELISA (n=4). FIG. 13F shows PSA in mice systemically sensitized with 20pg a-OVA-mlgE 4 days post ELISA and then systemically challenged with Img of OVA 3 days later. FIG. 13G shows temperature loss following systemic allergen administration in mice from FIG.

[0077] 13F. FIG. 13H shows PSA in mice from 6 months following initial immunization (n=4). Data shows mean ± SEM with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 obtained by 1-way or 2-way ANOVA with Tukey’s multiple comparison test.

[0078] FIGS. 14A-14H show that EndoFl-treated TNP-mlgE is cleared from mouse serum at a faster rate than BC-a-TNP-mlgE, and EndoFl-treated OVA-mlgE generates adaptive humoral immune response. FIG. 14A shows schematic route of administration of a-TNP-mlgE treated with EndoFl and its buffer (EndoFl-a-TNP-mlgE) or just its buffer (BC-a-TNP-mlgE). FIG. 14B shows serum level of BC-a-TNP-mlgE or EndoFl -a-TNP-mlgE at 2hrs, 6hrs and 12hrs following intraperitoneal administration of 6.5pg of the antibodies into to WT Balb / c mice or Balb / c mice with mouse FcsRIa knocked out (KO) (n = 4). FIG. 14C shows total serum IgM from against BC-a-OVA-mlgE in mice administered with PBS or BC-a-OVA-mlgE or EndoFl -a-OVA-mlgE (n = 4). FIG. 14D shows total serum IgG from mice administered with PBS or BC-a-OVA-mlgE or EndoFl -a-OVA-mlgE (n = 4). FIGS.

[0079] 14E-14H show serum IgGl, IgG2a, IgG2b, IgG3 against EndoFl -a-OVA-mlgE (n = 4). Data shows mean ± SEM with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 obtained by 1-way ANOVA with Tukey’s multiple comparison test or 2-way ANOVA with Sidak’s multiple comparison test.

[0080] FIG. 15 shows exemplary IgE-based vaccine platforms.

[0081] DETAILED DESCRIPTION

[0082] Immunoglobulin E (IgE) antibodies mediate allergic diseases, which affect up to 40% of the global population. IgE binds with high affinity to FcsRI on mast cells and basophils and triggers cellular degranulation upon allergen exposure and IgE crosslinking, releasing histamine, leukotrienes, and prostaglandins. It is demonstrated herein that the N-linked oligomannose glycan at N394 in the Cs3 domain of IgE isAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0083] conserved across IgE from distinct conditions and essential for FcsRI binding and allergic effector function. Systematic removal of sugar moieties revealed the minimal glycan structure for FcsRI binding, mast cell sensitization, and anaphylaxis was a single mannose residue attached to two N-acetyl glucosamines, phenocopying oligomannosedeficient IgE. Negative-staining electron microscopy revealed a conformational collapse of the Fc region in glycan-deficient IgE, particularly in the Cs2 domain and the Cs3-Cs4 interface. Effector cell loading was shifted from FcsRE mast cells and basophils to FcsR.II B cells in oligomannose-deficient IgE.

[0084] Without wishing to be bound by theory, the removal of the oligomannose glycan converts IgE into an immunogenic molecule. For example, it is demonstrated herein that a single injection of oligomannose-deficient IgE induced robust anti-IgE IgG2a and IgG2b responses in the absence of adjuvant and conferred protection from anaphylaxis elicited following oligomannose-deficient IgE administration in vivo. These findings show methods for treating or preventing IgE-mediated disorders and / or anti-IgE immunogenic response (e.g., long-term immune protection against the IgE).

[0085] Certain terms employed within this document are collected here. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below to aid in describing particular embodiments.

[0086] Definitions

[0087] As used herein, the word “a” before a noun represents one or more of the particular noun. For example, the phrase “a mutation” encompasses “one or more mutations.”

[0088] As used herein, the term “about” means approximately, in the region of, roughly, or around. When used in conjunction with a numerical range, the term “about” modifies that range by extending the boundaries above and below the numerical values set forth. Unless otherwise defined, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%.

[0089] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated in the present disclosure. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old). In addition to humans, patients include but are not limited to mice, rats, hamsters,Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0090] guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, nonhuman primates (e.g., monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

[0091] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.

[0092] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0093] As used herein, the term “antibody” or “immunoglobulin” refers to any antigenbinding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody contains an Fc region of a human antibody. The term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.

[0094] As used herein, the term “human antibody” refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) present in a human. In some embodiments, a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line).

[0095] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences or Fc of antibodies) of mostly human sequences, which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipientAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0096] antibody) in which residues from a hypervariable region (also CDR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, “humanized antibodies” as used herein may also comprise residues which are found neither in the recipient antibody nor the donor antibody. These modifications are made to further refine and optimize antibody performance. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. BioL, 2:593-596 (1992).

[0097] Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen -binding activity and is still capable of crosslinking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the C HI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The region which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0098] Compositions comprising a modified IgE or an Fc fragment thereof Immunoglobulin E (IgE) is a mammal-specific immunoglobulin (Ig) isotype and exists as monomers consisting of two heavy chains (a chain) and two light chains, with the a chain comprising 4 Ig-like constant domains (Cal-Ca ).Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0099] IgE exerts its function through binding to two principal receptors: the high-affinity Fc epsilon receptor I (FcsRI) and the low-affinity receptor FcsRII, also known as CD23. FcsRI is constitutively expressed on mast cells and basophils and binds IgE with sub-nanomolar affinity in a 1 : 1 stoichiometry at the top of the Cs3 domain in the Fc (FIG. 1 A). Upon allergen exposure, multivalent allergen cross-links receptor-bound IgE, leading to immediate cellular degranulation and release of inflammatory mediators such as histamine, leukotrienes, and prostaglandins. These mediators are responsible for the hallmark symptoms of allergy, including pruritus, wheezing, vascular leakage, hypotension, and, in severe cases, life-threatening anaphylaxis.

[0100] In contrast, CD23 is a C-type lectin-like receptor expressed on B cells, dendritic cells, macrophages and epithelial cells, where it plays a role in IgE homeostasis and antigen presentation. CD23 binds at a 2: 1 ratio to a distinct site on the Fc region of IgE at the junction of Cs3 and Cs4 domains and has a markedly lower affinity in the micromolar range (10'6-10'5M). Importantly, CD23 is involved in the uptake and processing of IgE-bound antigens and facilitates their presentation to T cells, promoting adaptive immune responses. These two receptors, while structurally unrelated and functionally distinct, both interact with the Fc portion of IgE and thereby influence IgE’s immunological fate.

[0101] Structurally, the IgE antigen-binding fragment (Fab) is made up of light chains with variable and constant domains pairing with heavy chain variable and Cal domains (FIG. 1 A). The heavy chains extend forming the Fc region, which is comprised of three constant domains (Cs2, Ca3, and Cs4) and adopts dynamic conformations that dictate receptor binding. The Fc domain can transition between an “open” conformation favoring FcaRI binding and a “closed” conformation compatible with CD23 engagement. Crystallographic and biophysical studies suggest that receptor binding stabilizes one of these two conformations, thereby preventing simultaneous engagement of both receptors. This conformational flexibility has been proposed as a regulatory mechanism to control IgE effector function.

[0102] Human IgE is the most heavily glycosylated monomeric antibody isotype, containing seven conserved N-linked glycosylation sites on its heavy chain, and mouse IgE has nine (FIGS. 1A and 2A). These glycans play essential roles in regulating the functions of IgE. Glycan at N394 in human IgE (N384 in mouse IgE), located in the Cs3 domain, is uniquely decorated with oligomannose-type glycans (FIGS. 1A andAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0103] 2A). Five N-glycosylation sites harbor complex biantennary structures, and a single site (N383) is unoccupied (FIG. 1A). This site-specific enrichment of oligomannose at N394 indicates a distinct structural or functional role, possibly due to restricted access to glycan-processing enzymes in the Golgi during secretion. Enzymatic removal of this glycan, or site-directed mutagenesis of the N394 site, completely abrogates FcsRI binding, indicating a critical role for this structure in IgE effector function.

[0104] IgE oligomannose glycans can vary in composition, typically containing between five and nine mannose residues and oligomannose glycans with 5 to 9 mannose residues are detected on primary and recombinant IgE at N394. The studies herein assessed whether a minimal glycan motif is sufficient for FcsRI binding, and how progressive glycan trimming influences IgE structure and function. The studies herein also dissected the structural and functional contributions of the conserved N394 oligomannose glycan on human and mouse IgE. Using a panel of enzymatically modified IgE molecules with defined glycan compositions, the minimal oligomannose structure required for FcsRI binding and allergic effector function as a Man-1 glycan was identified. Furthermore, the impact of oligomannose on IgE conformation, receptor specificity, and immunogenicity was assessed. The findings herein establish the IgE N394 glycan as a critical determinant of Fc structure and function, critical for receptor binding, and point to glycan modification as a promising strategy for IgE-targeted allergy immunotherapy.

[0105] The modified IgE antibodies described herein can include an Fc fragment alone (e.g., Cs2, Cs3, and Cs4, or even only Cs3 and Cs4), optionally linked to an antigen binding domain (e.g., a Fab) that binds to an allergen, as described herein. The antigen binding domain (e.g., Fab) can also be absent. When a Fab is included (e.g., when a whole IgE antibody is used), the Fab can be specific for an allergen such as food allergens, environmental allergens (e.g., tree, grass, weed pollens, dust mites, pet saliva / fur, and mold, and / or seasonal allergies), venom allergens, and drug allergens. For example, the Fab can be specific for OVA, Derpl, and Arah2. In some embodiments, the modified IgE antibodies described herein include an Fc fragment alone (e.g., Cs2, Cs3, and Cs4, or even only Cs3 and Cs4) linked to an antigen of interest (e.g., as shown in FIG. 15), which can be used as an antigen delivery system.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0106] Deglycosylated IgE

[0107] Provided herein is a modified IgE antibody or an Fc fragment thereof comprising a deglycosylated N394, wherein the modified IgE antibody is a human antibody or a humanized antibody. In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises one or more deglycosylated sites (e.g., one or more deglycosylated asparagines). In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises a deglycosylated N383 and / or a deglycosylated N371.

[0108] Also provided herein is a modified IgE antibody or an Fc fragment thereof comprising a deglycosylated N394, a deglycosylated N383, and / or a deglycosylated N371.

[0109] In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a deglycosylated N394 and a deglycosylated N383. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a deglycosylated N394 and a deglycosylated N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a deglycosylated N394, a deglycosylated N383, and a deglycosylated N371.

[0110] Glycosylation, as used herein, refers to a reaction in which a carbohydrate, i.e., a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (e.g., a glycosyl acceptor). Glycosylation is a form of co-translational and post-translational modification. Glycans serve a variety of structural and functional roles in membrane and secreted proteins. The majority of proteins synthesized in the rough ER undergo glycosylation. It is an enzyme-directed site-specific process.

[0111] The term “deglycosylated” protein denotes a protein, such as an IgE protein or an Fc fragment thereof, that has one or more sugars removed as compared to the native or unmodified form (e.g., reduced glycosylation, and partially glycosylated) and / or absent, e.g., missing from and / or not added to the glycan structure of a fully glycosylated instance of the protein and in which the protein substantially retains its native conformation / folding. A “deglycosylated” protein includes a partially glycosylated protein in which, for example, the deglycosylation process leaves a monoglycosylation, a diglycosylation, or a triglycosylation at one or more glycosylation sites present on the glycoprotein, relative to the fully glycosylated instance of the protein.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0112] In some embodiments, the deglycosylated N394 lacks oligomannose. In some embodiments, the deglycosylated N394 is linked to N-acetylgalactosamine, galactose, and / or sialic acid. In some embodiments, the deglycosylated N394 lacks N-acetylgalactosamine, galactose, and / or sialic acid.

[0113] In some embodiments, the deglycosylated N383 lacks oligomannose. In some embodiments, the deglycosylated N383 is linked to N-acetylgalactosamine, galactose, and / or sialic acid. In some embodiments, the deglycosylated N383 lacks N-acetylgalactosamine, galactose, and / or sialic acid.

[0114] In some embodiments, the deglycosylated N371 lacks oligomannose. In some embodiments, the deglycosylated N371 is linked to N-acetylgalactosamine, galactose, and / or sialic acid. In some embodiments, the deglycosylated N371 lacks N-acetylgalactosamine, galactose, and / or sialic acid.

[0115] In some embodiments, the modified IgE antibody or an Fc fragment thereof is deglycosylated at the Cs3 domain.

[0116] In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises a deglycosylated N394 and one or more amino acid modifications (e.g., mutations or deletions) at one or more glycosylation sites on the modified IgE antibody or an Fc fragment thereof (e.g., at one or more glycosylation sites at the Cs3 domain). In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises one or more amino acid modifications (e.g., mutations or deletions). In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises one or more amino acid modifications (e.g., mutations or deletions) at N371, N383, and / or T396. In some embodiments, the mutation atN383 isN383Q, N383A, orN383D. In some embodiments, the mutation at N371 is N371Q, N371A, or N371D. In some embodiments, the mutation at N383 is N383Q. In some embodiments, the mutation at T396 is T396A or T396V.

[0117] Provided herein is a modified IgE antibody or an Fc fragment thereof comprising a deglycosylated N384, wherein the modified IgE antibody is a mouse antibody. In some embodiments, the modified mouse IgE antibody or an Fc fragment thereof further comprises one or more deglycosylated sites (e.g., one or more deglycosylated asparagines).Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0118] Amino Acid Modifications

[0119] Provided herein is a modified IgE antibody or an Fc fragment thereof comprising one or more amino acid modifications (e.g., mutations or deletions) at N394 and / or T396, wherein the modified IgE antibody is a human antibody or a humanized antibody.

[0120] Also provided herein is a modified IgE antibody or an Fc fragment thereof comprising an amino acid modification (e.g., mutation or deletion) atN394, wherein the modified IgE antibody is a human antibody or a humanized antibody. In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises one or more amino acid modifications (e.g., mutations or deletions). In some embodiments, the one or more amino acid modifications (e.g., mutations or deletions) are at T396, N383, and / or N371.

[0121] Also provided herein is a modified IgE antibody or an Fc fragment thereof comprising an amino acid modification (e.g., mutation or deletion) at T396, wherein the modified IgE antibody is a human antibody or a humanized antibody. In some embodiments, the modified IgE antibody or an Fc fragment thereof further comprises one or more amino acid modifications (e.g., mutations or deletions). In some embodiments, the one or more amino acid modifications (e.g., mutations or deletions) atN394, N383, and / or N371

[0122] Also provided herein is a modified IgE antibody or an Fc fragment thereof comprising one or more amino acid modifications (e.g., mutations or deletions) at N394, T396, N383, and / or N371, wherein the modified IgE antibody is a human antibody or a humanized antibody.

[0123] In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a mutation atN394. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a mutation at T396. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a mutation at N383. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a mutation atN371.

[0124] In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a deletion atN394. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a deletion at T396. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a deletion at N383. In someAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0125] embodiments, the modified IgE antibody or an Fc fragment thereof comprises a deletion atN371.

[0126] In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises mutations at N394 and T396. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises mutations at N394 and N383. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises mutations atN394 and N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises mutations at N394, T396, and N383. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises mutations atN394, T396, and N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises mutations at N394, N383, and N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises mutations atN394, T396, N383, andN371.

[0127] In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises deletions at N394 and T396. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises deletions at N394 and N383. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises deletions at N394 and N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises deletions at N394, T396, and N383. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises deletions at N394, T396, and N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises deletions atN394, N383, and N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises deletions at N394, T396, N383, and N371.

[0128] An “amino acid modification” refers to an amino acid mutation and / or deletion in a protein or peptide sequence. An “amino acid mutation” or “mutation” refers to replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. A mutation can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to theAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0129] same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. The following are examples of various groupings of amino acids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Amino acids with uncharged polar R groups: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): Lysine, Arginine, Histidine (at pH 6.0). Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine.

[0130] In some embodiments, the mutation at N394 is N394Q, N394A, or N394D. In some embodiments, the mutation at T396 is T396A or T396V. In some embodiments, the mutation atN383 isN383Q, N383A, orN383D. In some embodiments, the mutation at N371 is N371Q, N371A, or N371D. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises one or more amino acid modifications (e.g., mutations or deletions) atN394 and / or T396, and wherein one or more glycosylation sites on the modified IgE antibody or an Fc fragment thereof (e.g., one or more glycosylation sites at the Cs3 domain) are deglycosylated.

[0131] In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an amino acid modification (e.g., mutation or deletion) at N394, and a deglycosylated N383 and / or a deglycosylated N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an amino acid modification (e.g., mutation or deletion) at N394 and a deglycosylated N383. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an amino acid modification (e.g., mutation or deletion) at N394 and a deglycosylated N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an amino acid modification (e.g., mutation or deletion) at N394, and a deglycosylated N383 and a deglycosylated N371.

[0132] In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an amino acid modification (e.g., mutation or deletion) at T396, and a deglycosylated N394, a deglycosylated N383 and / or a deglycosylated N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an amino acid modification (e.g., mutation or deletion) atAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0133] T396 and a deglycosylated N394. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an amino acid modification (e.g., mutation or deletion) at T396 and a deglycosylated N383. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an amino acid modification (e.g., mutation or deletion) at T396 and a deglycosylated N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an amino acid modification (e.g., mutation or deletion) at T396, and a deglycosylated N394 and a deglycosylated N383. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an amino acid modification (e.g., mutation or deletion) at T396, and a deglycosylated N394 and a deglycosylated N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an amino acid modification (e.g., mutation or deletion) at T396, and a deglycosylated N383 and a deglycosylated N371. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an amino acid modification (e.g., mutation or deletion) at T396, and a deglycosylated N394, a deglycosylated N383, and a deglycosylated N371.

[0134] In some embodiments, the mutation at N383 is N383Q, N383A, or N383D. In some embodiments, the mutation at N371 is N371Q, N371A, or N371D. In some embodiments, the mutation at T396 is T396A or T396V.

[0135] Provided herein is a modified mouse IgE antibody or an Fc fragment thereof comprising one or more amino acid modifications (e.g., mutations or deletions) at N384 and / or T386, wherein the modified IgE antibody is a human antibody or a humanized antibody. In some embodiments, the mutation atN384 isN384Q, N384A, or N384D. In some embodiments, the mutation at T386 is T386A or T386V.

[0136] In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises or consists of a Fc Cs3 domain. In some embodiments, the Fc CE3 domain comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18 or a functional fragment thereof. In some embodiments, the Fc Cs3 domain comprises the sequence as set forth in SEQ ID NO: 18, 28, or 29 or a functional fragment thereof .

[0137] In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises the Fc CE2 domain and / or the Fc CE4 domain. In someAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0138] embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises or consists of the Fc CE2 domain, the Fc CE3 domain, and the Fc CE4 domain. In some embodiments, wherein the Fc Cs2 domain comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17 or a functional fragment thereof. In some embodiments, the Fc Cs4 domain comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 19 or a functional fragment thereof. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 or a functional fragment thereof. In some embodiments, the modified IgE antibody or an Fc fragment thereof comprises the sequence as set forth in SEQ ID NO: 25, 26, or 27 or a functional fragment thereof.

[0139] In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an antigen-binding fragment.

[0140] As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain). Non-limiting examples of antibody fragments include, e.g., Fab, Fab’, F(ab’)2, Fv, and scFv.

[0141] The "Fab" fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain. F(ab') 2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

[0142] “Fv” is the minimum antibody fragment which contains a complete antigenrecognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half ofAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0143] an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0144] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315 (1994).

[0145] In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises a heavy chain. In some embodiments, the heavy chain comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14, 23 or 24 or a functional fragment thereof. In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises a light chain. In some embodiments, the light chain comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20, 21, or 22 or a functional fragment thereof.

[0146] IgE mediates allergic reactions by binding to high-affinity (FcsRI) and low-affinity (CD23) receptors on immune cells. Unlike IgG, IgE lacks a flexible hinge region, possessing an additional constant domain pair (Cs2) that makes it highly flexible, allowing it to transition between acutely bent (closed) and extended (open) conformations. The heavy chains extend forming the Fc, which is comprised of three constant domains (Cs2, Cs3, and Cs4) and adopts dynamic conformations that dictate receptor binding. An “open” conformation of Immunoglobulin E (IgE) refers to a structural state of the IgE-Fc region where the domains are separated and pointed upward, exposing the binding sites for the high-affinity receptor FcsRI. A “closed” conformation of IgE refers to a compact, folded shape of the antibody's Fc region where the Cs2 domain folds back onto the Cs3 and Cs4 domains, obscuring the binding site for the high-affinity receptor FcsRI. The Fc domain can transition between an “open” conformation favoring FcsRI binding and a “closed” conformation compatible with CD23 engagement. The majority conformation adopted by an unmodified or native IgEAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0147] antibody or an Fc fragment thereof (e.g., fully glycosylated and / or has glycosylated Cs3) is consistent with an open conformation that featured overlapping Cs2 domains and a well-defined pocket between Cs3 and Cs4 domains, facilitating binding to FcsRI and the minority conformation of an unmodified or native IgE antibody or an Fc fragment thereof (e.g., fully glycosylated and / or has glycosylated Cs3) can have extended Cs2 domains, and a slightly oblong Cs3 and Cs4 domain pocket, consistent with the closed, CD23 binding conformation (e.g., FIG. 10). In some examples, although the modified IgE antibody or an Fc fragment thereof disclosed herein can form majority and minority conformations, both may have extended Cs2 domains that are more consistent with the minority CD23-binding conformation of an unmodified or native IgE antibody or an Fc fragment thereof (e.g., fully glycosylated and / or has glycosylated Cs3). In some embodiments, the reduced intramolecular interactions within the Cs2 domain can facilitate exposure of immunogenic epitopes and induce immune protection (e.g., long-term immune protection) against IgE, mitigating symptoms of an IgE-mediated disorder (e.g., by lowering IgE titers) and / or reducing the risk of developing an IgE-mediated disorder.

[0148] In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein comprises an Fc region not in an open conformation (e.g., in a closed conformation). In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein does not bind to FcsRI or binds to FcsRI with an affinity lower at least 100 times (e.g., at least 1000, 5000, 10,000, 50,000, 100,000, or 1,000,000 times) lower than a reference control (e.g., an IgE with unmodified Fc region). In some embodiments, the modified IgE antibody or an Fc fragment thereof disclosed herein does not bind to FcsRI or binds to FcsRI with an affinity lower at least 100,000 times lower than a reference control (e.g., an IgE with unmodified Fc region). In some embodiments, the modified IgE antibody or an Fc fragment thereof binds to an FcsRI with a dissociation constant KD of at least 100 times (e.g., at least 1000, 5000, 10,000, 50,000, 100,000, or 1,000,000 times) higher than a reference control (e.g., an IgE with unmodified Fc region). In some embodiments, the modified IgE antibody or an Fc fragment thereof binds to an FcsRI with a dissociation constant KD of at least 10'2M (e.g., at least 2*10'2M, 5*10'2M, or 10-1M). In some embodiments, the modified modified IgE antibody or an Fc fragment thereof binds to an FcsRI with a dissociation constant KD of at least 10-1M. Affinity or binding can be quantified using knownAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0149] methods such as, Surface Plasmon Resonance (SPR) (described in Scarano S, Mascini M, Turner A P, Minunni M. Surface plasmon resonance imaging for affinity-based biosensors. Biosens Bioelectron. 2010, 25: 957-66), and can be calculated using, e.g., a dissociation constant, KD, such that a lower KD reflects a higher affinity. The binding affinity and dissociation rate of the affinity ligand can be determined by any method known in the art. For example, the binding affinity can be measured by competitive ELISAs, RIAs, BIACORE™, or KINEXA™ technology. The dissociation rate also can be measured by BIACORE™ or KINEXA™ technology. The binding affinity and dissociation rate are measured by surface plasmon resonance using, e.g., a BIACORE™.

[0150] Additional modifications to the antibodies or antigen-binding fragments can be made. For example, a cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have any increased half-life in vitro and / or in vivo. Homodimeric antibodies with increased half-life in vitro and / or in vivo can also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al., "Monoclonal antibody homodimers: enhanced antitumor activity in nude mice." Cancer research 53.11 (1993): 2560-2565. Alternatively, an antibody can be engineered which has dual Fc regions.

[0151] In some embodiments, a covalent modification can be made to the modified immunoglobulin. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N- or C-terminal residues.

[0152] Pharmaceutical compositions

[0153] Pharmaceutical compositions comprising or consisting of the modified IgE antibody or an Fc fragment thereof described herein as an active ingredient are also provided herein. Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungalAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0154] agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0155] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Exemplary compositions containing the modified IgE antibody or an Fc fragment thereof described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage).

[0156] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0157] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such asAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0158] bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0159] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0160] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0161] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0162] Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0163] In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0164] Also provided herein are compositions comprising the modified IgE antibody or an Fc fragment thereof disclosed herein.

[0165] Also provided herein are vaccine compositions comprising the modified IgE antibody or an Fc fragment thereof disclosed herein. In some embodiments, the vaccine composition comprises an adjuvant.

[0166] Also provided herein are compositions comprising the modified IgE antibody or an Fc fragment thereof disclosed herein for use in manufacture of a medicament for inducing an anti-IgE immunogenic response in a subject, and / or treating or preventing an IgE-mediated disorder in a subject.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0167] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. Optionally, the compositions can be in a unit dose form, e.g., in a single-dose syringe or auto-injector.

[0168] Methods of Treatment

[0169] Provided herein are methods of treating or preventing an IgE-mediated disorder in a subject comprising administering to the subject an effective amount of a composition comprising a modified IgE antibody or an Fc fragment thereof disclosed herein.

[0170] Provided herein are methods of treating or preventing an IgE-mediated disorder in a subject comprising administering to the subject a composition comprising a modified IgE antibody or an Fc fragment thereof disclosed herein. Also provided herein are methods of treating an IgE-mediated disorder in a subject comprising administering to the subject a composition comprising a modified IgE antibody or an Fc fragment thereof disclosed herein.

[0171] Also provided herein are methods of inducing an anti-IgE immunogenic response in a subject, comprising administering to the subject an effective amount of a composition comprising a modified IgE antibody or an Fc fragment thereof disclosed herein.

[0172] Also provided herein are methods of inducing an anti-IgE immunogenic response in a subject, comprising administering to the subject a composition comprising a modified IgE antibody or an Fc fragment thereof disclosed herein.

[0173] As used herein, “preventing” means reducing the risk of developing, and need not completely eliminate risk 100%.

[0174] As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., an IgE-mediated disorder. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the therapeutic agent is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.

[0175] IgE mediated disorders includes atopic disorders, which are characterized by an propensity to respond immunologically to many common naturally occurring inhaled and ingested antigens and the continual production of IgE antibodies. Specific atopic disorders include allergic asthma, allergic rhinitis, atopic dermatitis and allergicAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0176] gastroenteropathy. Atopic patients often have multiple allergies, meaning that they have IgE antibodies to, and symptoms from, many environmental allergens, including pollens, fungi (e.g., molds), animal and insect debris and certain foods.

[0177] Disorders associated with elevated IgE levels are not limited to those with an atopic etiology. Other disorders associated with elevated IgE levels, which appear to be IgE-mediated and are treatable with the formulations described herein, include hypersensitivity (e.g., anaphylactic hypersensitivity), eczema, allergic bronchopulmonary aspergillosis, parasitic diseases, hyper-IgE syndrome (HIES), ataxia-telangiectasia, Wiskott-Aldrich syndrome, thymic alymphoplasia, IgE myeloma and graft-versus-host reaction urticaria (e.g., chronic spontaneous urticaria), systemic lupus erythematosus (SLE), nephritis caused by SLE, bullous pemphigoid, non-allergic asthma, and IgE-mediated gastroenteropathy.

[0178] An allergy is an inflammatory disorder caused by acquired hypersensitivity to a substance (allergen). Allergic conditions include eczema, allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions. A "subject having an allergy" is a subject that has had (or is at risk of developing based on family or personal history) an allergic reaction in response to an allergen. An "allergen" refers to a substance that can induce an allergic or asthmatic response in a susceptible subject. The term "atopic" as used herein refers to a state of atopy or allergy to an allergen or a state of hypersensitivity to an allergen. Typically, atopic refers to Type I hypersensitivity which results from release of mediators (e.g., histamine and / or leukotrienes) from IgE- sensitized basophils and mast cells after contact with an antigen (allergen). An example of an atopic disease is atopic asthma, which is allergic asthma and is characterized by an IgE response.

[0179] Allergens of interest include antigens found in food, such as strawberries, peanuts, milk polypeptides, egg whites, etc. Other allergens of interest include various airborne antigens, such as grass pollens, animal danders, house mite feces, etc.

[0180] Anaphylaxis is an acute, generalized IgE-mediated allergic reaction with simultaneous involvement of several organ systems, usually cardiovascular, respiratory, cutaneous and gastrointestinal. The reaction is immunologically mediated, and it occurs on exposure to an allergen to which the subject has been previously sensitized. It is caused suddenly by allergen induced, mast cell loaded IgE, resulting in profound and life-threatening alteration in the functioning of various vital organs.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0181] Vascular collapse, acute airway obstruction, cutaneous vasodilation and edema, and gastrointestinal and genitourinary muscle spasm occur almost simultaneously, although not always to the same degree.

[0182] Anaphylaxis can be characterized by urticaria and angioedema, which refer to the physical swelling, erythema and itching resulting from histamine stimulated receptor in superficial cutaneous blood vessels, and is the hallmark cutaneous feature of systemic anaphylaxis.

[0183] The presence of IgE autoantibodies has been observed in some autoimmune diseases along with the prevalence of autoreactive IgE in systemic lupus erythematosus (SLE). Hyper-IgE is a marker of immune dysregulation and facilitates the generation of immune complexes (ICs) that mediate lupus nephritis and SLE.

[0184] Allergic rhinitis, also known as allergic rhinoconjunctivitis or hay fever, is the most common manifestation of an atopic reaction to inhaled allergens, the severity and duration of which is often correlative with the intensity and length of exposure to the allergen. It is a chronic disease, which may first appear at any age, but the onset is usually during childhood or adolescence. A typical attack consists of profuse watery rhinorrhea, paroxysmal sneezing, nasal obstruction and itching of the nose and palate. Postnasal mucus drainage also causes sore throat, throat clearing and cough. There can also be symptoms of allergic blepharoconjunctivitis, with intense itching of the conjunctivae and eyelids, redness, tearing, and photophobia. Severe attacks are often accompanied by systemic malaise, weakness, fatigue, and sometime, muscle soreness after intense periods of sneezing.

[0185] Asthma, also known as reversible obstructive airway disease, is characterized by hyperresponsiveness of the tracheobronchial tree to respiratory irritants and bronchoconstrictor chemicals, producing attacks of wheezing, dyspnea, chest tightness, and cough that are reversible spontaneously or with treatment. It is a chronic disease involving the entire airway, but varies in severity from occasional mild transient episodes to severe, chronic, life-threatening bronchial obstruction. Asthma and atopy may coexist, but only about half of asthmatics are also atopic, and an even smaller percentage of atopic patients also have asthma. However, atopy and asthma are not entirely independent in that asthma occurs more frequently among atopic than amongst nonatopic individuals, especially during childhood. Asthma can be triggered by such things as exposure to an allergen (allergic asthma), or non-allergens (non-allergicAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0186] asthma) such as cold air, pollution (e.g., ozone), warm air, moist air, exercise or exertion, or emotional stress. In children, the most common triggers are viral illnesses such as those that cause the common cold (Zhao J., et. al., 2002, J Pediatr Allergy Immunol. 13 : 47- 50).

[0187] The present methods can include administration of a modified IgE antibody or Fc fragment thereof as described herein to a subject with an allergy, wherein the antibody comprises an antigen binding domain that binds to an antigen to which the subject is allergic. For example, if the subject has an allergy to peanuts, the antibody can include an antigen binding domain that binds to a peanut antigen.

[0188] Administration of the modified IgE antibody or Fc fragment thereof as described herein can result in long-term immune protection in a subject. For example, the methods can result in induction of antibodies and / or memory immune cells that are detectable in a biological sample obtained from the subject at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, two years, three years, four years, five years, ten years, or later post-administration.

[0189] Thus, the methods provided herein can further include measuring a level of long-term immune protection (e.g., levels of antibodies and / or memory immune cells) against the modified IgE antibody or an Fc fragment thereof in a biological sample obtained from the subject at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, two years, three years, four years, five years, ten years, or later post-administration. For example, the methods can include measuring levels of IgM, IgGl, IgG2, and / or IgG4 that specifically bind to the modified IgE antibody or an Fc fragment thereof. The level of long-term immune protection (indicated by levels of IgM, IgGl, IgG2, and / or IgG4 that specifically bind to the IgE antibody or an Fc fragment thereof) can be measured, e.g., by an assay selected from the group consisting of Enzyme-Linked Immunoassay (ELISA), surface plasmon resonance (SPR) / biolayer interferometry (BLI), chemiluminescent immunoassays (CLIA), multiplex bead-based immunoassays (e.g., Luminex), flow cytometry-based analysis, immunofluorescence assay (IF A), cytokine secretion assay, ELISpot, T cell proliferation assay, cytotoxicity assays, immunoprecipitation, Western Blot, and immunocytochemistry.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0190] Biological samples include, but are not limited to, scrapes (e.g., buccal scrapes), whole blood, plasma, serum, saliva, ascites, pleural fluid, or cerebrospinal fluid.

[0191] The compositions containing the modified IgE antibody or an Fc fragment thereof as described herein can be administered via parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration, optionally in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage, e.g., a single-dose syringe or auto-injector).

[0192] Methods of making modified immunoglobulins

[0193] The present disclosure also provides polynucleotide sequences encoding a modified IgE antibody or an Fc fragment thereof as described herein, e.g., comprising one or more mutations that affect glycosylation as described, recombinant vectors (e.g., expression vectors) that include a polynucleotide sequence encoding a modified IgE antibody or an Fc fragment thereof as described herein, host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and / or vector comprising the polynucleotide), and the production of modified immunoglobulins by recombinant techniques.

[0194] As used herein, a “vector” is any construct capable of delivering one or more polynucleotide(s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide(s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and / or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

[0195] A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to generateAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0196] recombinant virus), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0197] The expression vectors can include at least one selectable marker. Such markers include e.g., dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and human cells such as HEK 293 cell. Appropriate culture mediums and conditions for the host cells described herein are known in the art.

[0198] The modified IgE antibody or an Fc fragment thereof disclosed herein can be manufactured by treating a fully glycosylated protein (e.g., a native human IgE or an Fc fragment thereof (optionally produced recombinantly in mammalian or human host cells), having a native glycosylation pattern, e.g., as shown in FIG. 1 A) with an enzyme (e.g., Endo-beta-N-acetylglucosaminidase Fl (EndoFl) and / or Peptide N-glycosidase F (PNGaseF)) that removes oligomannose. Accordingly, provided herein is a method of manufacturing the modified IgE antibody or an Fc fragment thereof disclosed herein comprising treating a fully glycosylated IgE antibody or an Fc fragment thereof with EndoFl or PNGaseF.

[0199] Exemplary Sequences and Constructs

[0200] Also provided herein are nucleic acid sequences that encode a modified IgE or an Fc fragment thereof that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to a modified IgE or an Fc fragment thereof as described herein, and an amino acid sequence of a modified IgE or an Fc fragment thereof that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to an amino acid sequence of a modified IgE or an Fc fragment thereof as described herein. Also provided are nucleotide sequences encoding a modified IgE or an Fc fragment thereof, as well as amino acid sequences of a modified IgE or an Fc fragment thereof, that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids substituted or deleted as compared to a sequence provided herein.

[0201] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gapsAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0202] can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0203] SEQ ID NO: 1 -- WT-Anti-OVA-mlgE heavy chain with N384 and T386 in bold and underlined (N382 of SEQ ID NO: 1 is referred to herein as "N384" and T384 of SEQ ID NO: 1 is referred to herein as "T386") QVQLQQSGAELVRPGTSMKISCKASGYTFTNYWLGWINQRPGHGLEWIGDIYPGRGY TNYNEKFKDKATLTADTSSSTAYMQLTSLTSEDSAVYFCARKNYFGSTYFDFWGQGT TLTVSSAS IRNPQLYPLKPCKGTASMTLGCLVKDYFPGPVTVTWYSDSLNMSTVNFP ALGSELKVTTSQVTSWGKSAKNFTCHVTHPPSFNESRTILVRPVNITEPTLELLHSS CDPNAFHSTIQLYCFIYGHILNDVSVSWLMDDREITDTLAQTVLIKEEGKLASTCSK LNITEQQWMSESTFTCKVTSQGVDYLAHTRRCPDHEPRGVITYLIPPSPLDLYQNGA PKLTCLWDLESEKNVNVTWNQEKKTSVSASQWYTKHHNNATTSITSILPWAKDWI EGYGYQCIVDHPDFPKPIVRSITKTPGQRSAPEVYVFPPPEEESEDKRTLTCLIQNF FPEDISVQWLGDGKLISNSQHSTTTPLKSNGSNQGFFIFSRLEVAKTLWTQRKQFTC QVIHEALQKPRKLEKTISTSLGNTSLRPS

[0204] SEQ ID NO: 2 -- WT Anti -OVA mlgE Variable Region of Heavy Chain (VH) QVQLQQSGAELVRPGTSMKISCKASGYTFTNYWLGWINQRPGHGLEWIGDIYPGRGY TNYNEKFKDKATLTADTSSSTAYMQLTSLTSEDSAVYFCARKNYFGSTYFDFWGQGT TLTVSSAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0205] SEQ ID NO: 3 -- WT Anti -OVA mlgE First Constant Domain of Heavy Chain (CHel)

[0206] AS IRNPQLYPLKPCKGTASMTLGCLVKDYFPGPVTVTWYSDSLNMSTVNFPALGSEL KVTTSQVTSWGKSAKNFTCHVTHPPSFNESRTILVRPVNITEPT

[0207] SEQ ID NO: 4 -- WT Anti -OVA mlgE Second Constant Domain of Heavy Chain (CHe2) LELLHSSCDPNAFHSTIQLYCFIYGHILNDVSVSWLMDDREITDTLAQTVLIKEEGK LASTCSKLNITEQQWMSESTFTCKVTSQGVDYLAHTRRCPDHE

[0208] SEQ ID NO: 5 — WT Anti -OVA mlgE Third Constant Domain of Heavy Chain (CHe3) with N384 and T386 in bold and underlined PRGVITYLIPPSPLDLYQNGAPKLTCLWDLESEKNVNVTWNQEKKTSVSASQWYTK HHNNATTSITSILPWAKDWIEGYGYQCIVDHPDFPKPIVRSI

[0209] SEQ ID NO: 6 -- WT Anti -OVA mlgE Fourth Constant Domain of Heavy Chain (CHe4) TKTPGQRSAPEVYVFPPPEEESEDKRTLTCLIQNFFPEDISVQWLGDGKLISNSQHS TTTPLKSNGSNQGFFIFSRLEVAKTLWTQRKQFTCQVIHEALQKPRKLEKTISTSLG NTSLRPS

[0210] SEQ ID NO: 7 -- Anti-OVA-mlgE light chain DVLMTQTPLSLPVSLGDQAS ISCRSSQS IVHSNGNTYLEWYLQKPGQSPKFLI YKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKRA DAAPTVS I FPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0211] SEQ ID NO: 8 -- Anti -OVA mlgE Variable Region of Light Chain (VL)

[0212] DVLMTQTPLSLPVSLGDQAS ISCRSSQS IVHSNGNTYLEWYLQKPGQSPKFLI YKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKRAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0213] SEQ ID NO: 9 -- Anti -OVA mlgE First Constant Domain of Light Chain (CL)

[0214] ADAAPTVS I FPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTD QDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0215] SEQ ID NO: 10 -- Oligomannose Deficient (N384Q) Anti -OVA mlgE with N384Q in bold and underlined (Q382 of SEQ ID NO: 10 is referred to herein as "N384Q mutation") QVQLQQSGAELVRPGTSMKISCKASGYTFTNYWLGWINQRPGHGLEWIGDIYPGRGY TNYNEKFKDKATLTADTSSSTAYMQLTSLTSEDSAVYFCARKNYFGSTYFDFWGQGT TLTVSSAS IRNPQLYPLKPCKGTASMTLGCLVKDYFPGPVTVTWYSDSLNMSTVNFP ALGSELKVTTSQVTSWGKSAKNFTCHVTHPPSFNESRTILVRPVNITEPTLELLHSS CDPNAFHSTIQLYCFIYGHILNDVSVSWLMDDREITDTLAQTVLIKEEGKLASTCSK LNITEQQWMSESTFTCKVTSQGVDYLAHTRRCPDHEPRGVITYLIPPSPLDLYQNGA PKLTCLWDLESEKNVNVTWNQEKKTSVSASQWYTKHHNQATTSITSILPWAKDWI EGYGYQCIVDHPDFPKPIVRSITKTPGQRSAPEVYVFPPPEEESEDKRTLTCLIQNF FPEDISVQWLGDGKLISNSQHSTTTPLKSNGSNQGFFIFSRLEVAKTLWTQRKQFTC QVIHEALQKPRKLEKTISTSLGNTSLRPS

[0216] SEQ ID NO: 11 - WT Anti -OVA mlgE CHe2 - CHe3 - CHe4 with N384 in bold and underlined LELLHSSCDPNAFHSTIQLYCFIYGHILNDVSVSWLMDDREITDTLAQTVLIKEEGK LASTCSKLNITEQQWMSESTFTCKVTSQGVDYLAHTRRCPDHEPRGVITYLIPPSPL DLYQNGAPKLTCLWDLESEKNVNVTWNQEKKTSVSASQWYTKHHNNATTSITSILP WAKDWIEGYGYQCIVDHPDFPKPIVRSITKTPGQRSAPEVYVFPPPEEESEDKRTL TCLIQNFFPEDISVQWLGDGKLISNSQHSTTTPLKSNGSNQGFFIFSRLEVAKTLWT QRKQFTCQVIHEALQKPRKLEKTISTSLGNTSLRPS

[0217] SEQ ID NO: 12 - Oligomannose Deficient (N384Q) Anti -OVA mlgE CHe2 - CHe3 - CHe4 with N384Q in bold and underlined LELLHSSCDPNAFHSTIQLYCFIYGHILNDVSVSWLMDDREITDTLAQTVLIKEEGK LASTCSKLNITEQQWMSESTFTCKVTSQGVDYLAHTRRCPDHEPRGVITYLIPPSPL DLYQNGAPKLTCLWDLESEKNVNVTWNQEKKTSVSASQWYTKHHNQATTSITSILP WAKDWIEGYGYQCIVDHPDFPKPIVRSITKTPGQRSAPEVYVFPPPEEESEDKRTLAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0218] TCLIQNFFPEDISVQWLGDGKLISNSQHSTTTPLKSNGSNQGFFIFSRLEVAKTLWT QRKQFTCQVIHEALQKPRKLEKTISTSLGNTSLRPS

[0219] SEQ ID NO: 13 - Oligomannose Deficient (N384Q) Anti -OVA mlgE Third Constant Domain of Heavy Chain (CHe3) with N384Q in bold and underlined PRGVITYLIPPSPLDLYQNGAPKLTCLWDLESEKNVNVTWNQEKKTSVSASQWYTK HHNQATTSITSILPWAKDWIEGYGYQCIVDHPDFPKPIVRSI

[0220] SEQ ID NO: 14 - WT-Anti-OVA hlgE heavy chain with N371, N383, N394, and T396 in bold and underlined (N369 of SEQ ID NO: 14 is referred to herein as "N371", N381 of SEQ ID NO: 14 is referred to herein as "N383", N392 of SEQ ID NO: 14 is referred to herein as "N394", and T394 of SEQ ID NO: 14 is referred to herein as "T396") QVQLQQSGAELVRPGTSMKISCKASGYTFTNYWLGWINQRPGHGLEWIGDIYPGRGY TNYNEKFKDKATLTADTSSSTAYMQLTSLTSEDSAVYFCARKNYFGSTYFDFWGQGT TLTVSSGAPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMT LPATTLTLSGHYATISLLTVSGAWAKQMFTCRVAHTPSSTDWVDNKTFSVCSRDFTP PTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQE GELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPS PFDLFIRKSPTITCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTS TLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRD KRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAE WEQKDE FI CRAVHEAAS PSQTVQRAVSVNPGKHHHH

[0221] SEQ ID NO: 15 - WT Anti -OVA hlgE Variable Region of Heavy Chain (VH) QVQLQQSGAELVRPGTSMKISCKASGYTFTNYWLGWINQRPGHGLEWIGDIYPGRGY TNYNEKFKDKATLTADTSSSTAYMQLTSLTSEDSAVYFCARKNYFGSTYFDFWGQGT TLTVSSAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0222] SEQ ID NO: 16 - WT Anti -OVA hlgE First Constant Domain of Heavy Chain (CHel) GAPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMTLPATTL TLSGHYATISLLTVSGAWAKQMFTCRVAHTPSSTDWVDNKTFS

[0223] SEQ ID NO: 17 - WT Anti -OVA hlgE Second Constant Domain of Heavy Chain (CHe2) VCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDL STASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCA

[0224] SEQ ID NO: 18 - WT Anti -OVA hlgE Third Constant Domain of Heavy Chain (CHe3) with N371, N383, N394, and T396 in bold and underlined DSNPRGVSAYLSRPSPFDLFIRKSPTITCLWDLAPSKGTVNLTWSRASGKPVNHST RKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTS

[0225] SEQ ID NO: 19 - WT Anti -OVA hlgE Fourth Constant Domain of Heavy Chain (CHe4) GPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQ PRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGKHHHH

[0226] SEQ ID NO: 20 - Anti-OVA-hlgE light chain DVLMTQTPLSLPVSLGDQAS ISCRSSQS IVHSNGNTYLEWYLQKPGQSPKFLI YKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKRT VAAPSVEI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0227] SEQ ID NO: 21 - Anti -OVA hlgE Variable Region of Light Chain (VL)

[0228] DVLMTQTPLSLPVSLGDQAS ISCRSSQS IVHSNGNTYLEWYLQKPGQSPKFLI YKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKRAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0229] SEQ ID NO: 22 - Anti -OVA hlgE First Constant Domain of Light Chain (CL)

[0230] TVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0231] SEQ ID NO: 23 - Oligomannose Deficient (N394Q) Anti -OVA hlgE Heavy Chain with N394Q in bold and underlined (Q392 of SEQ ID NO: 23 is referred to herein as "N394Q mutation") QVQLQQSGAELVRPGTSMKISCKASGYTFTNYWLGWINQRPGHGLEWIGDIYPGRGY TNYNEKFKDKATLTADTSSSTAYMQLTSLTSEDSAVYFCARKNYFGSTYFDFWGQGT TLTVSSGAPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMT LPATTLTLSGHYATISLLTVSGAWAKQMFTCRVAHTPSSTDWVDNKTFSVCSRDFTP PTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQE GELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPS PFDLFIRKSPTITCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRQGTLTVTS TLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRD KRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAE WEQKDE FI CRAVHEAAS PSQTVQRAVSVNPGKHHHH

[0232] SEQ ID NO: 24 - T396A mutated Anti -OVA hlgE Heavy Chain with T396A in bold and underlined (A394 of SEQ ID NO: 24 is referred to herein as "T396A mutation") QVQLQQSGAELVRPGTSMKISCKASGYTFTNYWLGWINQRPGHGLEWIGDIYPGRGY TNYNEKFKDKATLTADTSSSTAYMQLTSLTSEDSAVYFCARKNYFGSTYFDFWGQGT TLTVSSGAPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMT LPATTLTLSGHYATISLLTVSGAWAKQMFTCRVAHTPSSTDWVDNKTFSVCSRDFTP PTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQE GELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPS PFDLFIRKSPTITCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGALTVTS TLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRD KRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAE WEQKDE FI CRAVHEAAS PSQTVQRAVSVNPGKHHHHAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0233] SEQ ID NO: 25 - WT Anti -OVA hlgE CHe2 - CHe3 - CHe4 with N371, N383, N394, and T396 in bold and underlined VCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDL STASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGV SAYLSRPSPFDLFIRKSPTITCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQR NGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFAT PEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFS RLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGKHHHH

[0234] SEQ ID NO: 26 - Oligomannose Deficient (N394Q) Anti -OVA hlgE CHe2 - CHe3 - CHe4 with N394Q in bold and underlined VCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDL STASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGV SAYLSRPSPFDLFIRKSPTITCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQR QGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFAT PEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFS RLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGKHHHH

[0235] SEQ ID NO: 27 - T396A mutated Anti -OVA hlgE CHe2 - CHe3 -CHe4 with T396A in bold and underlined VCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDL STASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGV SAYLSRPSPFDLFIRKSPTITCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQR NGALTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFAT PEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFS RLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGKHHHH

[0236] SEQ ID NO: 28 - Oligomannose Deficient (N394Q) Anti -OVA hlgE Third Constant Domain of Heavy Chain (CHe3) with N394Q in bold and underlined DSNPRGVSAYLSRPSPFDLFIRKSPTITCLWDLAPSKGTVNLTWSRASGKPVNHST RKEEKQRQGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0237] SEQ ID NO: 29 - T396A mutated Anti -OVA hlgE Third Constant Domain of Heavy Chain (CHe3) with T396A in bold and underlined DSNPRGVSAYLSRPSPFDLFIRKSPTITCLWDLAPSKGTVNLTWSRASGKPVNHST RKEEKQRQGALTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTS

[0238] EXAMPLES

[0239] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0240] Materials and Methods

[0241] The following materials and methods were used in the following examples. All human samples were collected under Institutional Review Board (IRB)-approved protocols, including informed consent obtained in accordance with relevant ethical regulations. Arah2+peanut allergic and non-allergic serum samples and were obtained from individuals subjected to oral food challenge before treatment. Non-atopic adults were recruited based on self-identification as non-allergic donors (Research Blood Components). Lupus nephritis patients were treated and recruited at the BIDMC Lupus Center (2006P000298, Tsokos). Helminth infected samples diagnosed with Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus, Loa loa, or other parasitic worms. The human JK-IgE was prepared from a subject with an IgE monoclonal gammopathy of uncertain significance. A small amount of the well characterized human PS- IgE, prepared from a subject with IgE myeloma, was used in this study but limited amounts prevented further work with this reagent.

[0242] IgE Antibodies and Oligomannose Engineering: Hybridoma-produced human IgE (hlgE) or (a-TNP -mouse IgE (a-TNP-mlgE) were purchased from Abeam and BioLegend respectively. Recombinant a-OVA-mouse-IgE (a-OVA-mlgE) or a-OVA-human-IgE (a-OVA-hlgE) was produced. IgE preparations were digested with glycosidases, including a (l-2,3)-Mannosidase (New England Biolabs) to generate Man-3-IgE, a (l-2,3,6)-Mannosidase (New England Biolabs) to generate Man-l-IgE, or Endoglycosidase Fl (EndoFl, Sigma and QA-Bio) to produce EndoFl -IgE. Some IgE was sequentially treated with a (l-2,3,6)-Mannosidase and P(1 -4)-Mannosidase (P-Mannosidase, Sigma and Megazyme) to yield Man-O-IgE. Briefly, lOOpg of theAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0243] antibodies were treated with lOO .1 of the mannosidase enzymes or 33.3 .1 of Endo Fl along with the respective buffers and co-factors or only buffers and co-factors (buffer control, BC) for 4 days at 37°C. 1-Man-IgE reactions were then buffer exchanged into PBS using lOOKda minicon (Millipore) and was further digested with P-Mannosidase (Sigma and Megazyme) at pH 5.5 such that Ipg of the antibody was treated with 1.325 pl of the enzyme and its buffer or the buffer for 4 days at 37°C to produce the 0-Mannose IgE glycoform (Man-O-IgE) or its BC (Man-l-BC-IgE), respectively. After 4 days, all the reactions were run on SDS-PAGE gel to check for IgE integrity and were then buffer exchanged into PBS using lOOKDa minicons (Millipore). Following every PBS buffer exchange step, the antibodies were quantified using ELISA as outlined below. lOpg of the engineered Man-3-IgE or Man-l-IgE antibodies were then digested with EndoFl, while Man-O-IgE antibodies were digested with PNGaseF for another 2 days at 37°C to release the residual glycans from IgE and the released glycan was then assessed by HPLC.

[0244] Oligomannose HPLC Analysis: Engineered IgE oligomannose glycans were released using EndoFl as described above. The reaction mix was then initially labelled with 2 aminobenzamide (2AB) and then with octanal. The sugar containing aqueous phase was carefully aspirated. The sugars were resolved on Agilent 1260 INFINITY Quaternary LC system having the AdvanceBio Glycan Mapping column 2.1 x 150mm, 2.7pm and a fluorescent detector. The intensity of the resulting sugar peaks was analyzed by OpenLAB software (Agilent) and area under the curve values were used to calculate percentage removal of mannose from the oligomannose structure by the different mannosidases or its respective buffers.

[0245] ELISA: Sandwich ELISA was conducted to quantify IgEs following digestion by mannosidases or Endo Fl or its respective buffer controls. Initially, 96-well high binding plates were coated with lOOpl of 1 pg / ml of anti-human IgE light chain antibody (Bethyl Laboratories) or lOOpl of lOOpg / ml of OVA or lOOpl of 1 pg / ml of TNP-OVA (Lucigen Corporation) and were kept overnight in the fridge. Human FcsRII (hCD23, R&D Systems) binding to a-OVA-hlgE following treatment with Endo Fl and its buffer, or its corresponding BC was assayed following manufacturer protocol (R&D Systems). The next day the wells were washed with PBS having 0.05% Tween 20 (PBST) five times and then blocked using 250pl of ELISA blocking buffer (PBST having 0.01% BSA (w / v)) on a rocker at room temperature for 2 hours. The wells wereAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0246] again washed for five times with PBST. Serial dilutions of lOOpl of hlgE a-OVA-hlgE or a-OVA-mlgE or a-TNP-mlgE at appropriate concentrations used as standards were added to the wells along with serial dilutions of mannosidase or Endo Fl digested IgE or its respective BC IgEs or mouse serum and incubated on the rocker at room temperature for 1 hour. For checking concentration of mouse serum IgG, the plates were coated with lOOpl of Ipg / ml of EndoFl -a-OVA-mlgE or BC-a-OVA-mlgE or anti-mouse IgG-Fc and the plates were kept in the fridge overnight. The next day the wells were washed with PBST five times and then blocked in 250pl of ELISA blocking buffer at room temperature on a rocker for 2hrs. The plates were washed again and then lOOpl of 1:2,000 starting serum dilution was added to the respective wells and incubated for 1 hour at room temperature on the rocker. After 1 hour incubation, the wells were washed with PBST for five times and incubated with lOOpl of 1:10,000 dilution of HRP -labelled a-mIgG2a, or a-mIgG2b or a-mIgG3 or total mouse IgG or 1 : 120,000 dilution of HRP-labelled a-mlgGl (Bethyl Laboratories, Sigma- Aldrich, BD BioSciences) for Ihour on the rocker. The wells were washed again with PBST for five times and then incubated lOOpl of 1 : 1 mixture of 3,3',5,5'-Tetramethylbenzidine (TMB, Thermo Fisher Scientific) for 5-20 minutes. The reactions were stopped by adding lOOpl of IM phosphoric acid and the absorbance of the samples were read at 450nm. The absorbance values were used to calculate antibody concentrations.

[0247] ELISA was also employed to study binding of the different mannosidase digested a-OVA-mlgE glycoforms or its buffer control to OVA. Briefly, the wells of the plates were coated with lOOpl of lOOpg / ml of OVA (Sigma) and the plates were incubated overnight in the fridge. Following washing and blocking of the wells, the wells were incubated with lOOpl of equal concentrations of serially diluted mannosidase digested or their respective buffer controls of mouse IgE for Ihour on a room temperature rocker. The plates were again washed and incubated with appropriate 1:30,000 dilution of HRP-conjugated anti-mouse-IgE secondary antibodies and then with TMB and phosphoric acid as mentioned above. The absorbance read out at 450nm was plotted against concentrations of IgEs to understand antigen binding with IgEs.

[0248] Glycopeptide Mass Spectroscopy: Primary human IgE was purified from patient serum samples. The IgE was subjected to an initial denaturation step by 6M guanidine HC1 treatment and was followed up by a reduction reaction with 25mM dithiothreitol and alkylation with iodoacetamide treatment. The samples were thenAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0249] dialyzed into 25mM ammonium bicarbonate pH 7.8. The polypeptide chain on IgE was then proteolytically digested with trypsin for quantification of glycosylation at N218, N371 andN394 sites or chymotrypsin to quantify sugars atN140, N168 andN265 sites. For digestion with trypsin, the protein was incubated with trypsin (Trypsin Gold Promega) at a ratio of 1:50 overnight at 37°C. Chymotrypsin digestion was done at 1 : 100 enzyme to protein ratio for 4 hours at 25°C. Both the reactions were stopped by adding 2% w / w to the reaction tubes. The digested peptides were separated by using Thermo EASY-Spray™ C18 nLC column with 0.75pMx50cm specifications with phase A and B being water and acetonitrile with 0.1% formic acid respectively. A linear gradient of mobile phase B from 1% to 35% was run over 75 minutes. Mass spectra were recorded on a Thermo Q Exactive™ mass spectrometer operated in positive mode using data independent acquisition (DIA) targeting the masses shown in Shade KC et al. Sialylation of immunoglobulin E is a determinant of allergic pathogenicity. Nature.

[0250] 2020 Jun;582(7811):265-270 (which incorporated by reference in its entirety). Quantification of the glycopeptides were based on the extracted ion area of the Y 1 ion. The relative abundance of the ions covering all the specific glycosylation sites were calculated. Myeloma IgE (Sigma- Aldrich AG30P) was run before paired sample sets for monitoring shifts in retention time and to check for consistency across runs on the analytical samples. The value of relative abundance of glycan at each site was utilized to calculate the percentage glycan occupancy.

[0251] LAD2 mast cells degranulation and IgE loading: The LAD2 human mast cells were cultured in StemPro™-34 SFM™ media (STEMCELL Technologies) that was supplemented with 10% penicillin, streptomycin and amphotericin B (Gibco), 2mM L-glutamine (Gibco) and lOOng / ml of recombinant human stem cell factor (PreproTech) at a density of 2* 105- 5* 105cells / ml in an incubator maintained at 37°C with 5% CO2.

[0252] Degranulation assays were performed on LAD2 cells to study effector functions of the engineered human IgE as described earlier. Briefly, 104cells were sensitized with 40ng of antibody or PBS overnight in 96-well V-bottom cell culture plates. The next day the plates were washed 3 times with HEPES buffer (lOmM HEPES, 137mM NaCl, 2.7mM KC1, 0.4mM Na2HPO4.7H2O, 5.6mM glucose, 1.8mM CaCl2.2H2O, 1.3mM BSA) and then challenged with anti-human IgE light chain antibody at a final concentration of lOpg / ml in HEPES buffer or HEPES buffer alone for 20 minutes at 37Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0253] °C to crosslink the IgE bound to the cells. Subsequently, the plates were centrifuged, and half of the supernatant was collected. Mast cell activation was quantified by assaying for the percentage release of P-hexosaminidase into the supernatant from the total P-hexosaminidase present within the mast cell granules by using p -nitrophenyl -N-acetyl-P-D-glucosamide (PNAG) as the P-hexosaminidase substrate in a colorimetric assay by reading the absorbance of the assay at 405nm.

[0254] To study the effect of oligomannose engineered human IgEs on loading of IgEs onto LAD2 cells, 105cells were sensitized with 400ng of the different antibodies or PBS overnight in 96-well V-bottom cell culture plates. The following day, the cells were washed three times with ice-cold FACS buffer (10% heat deactivated FBS contained in PBS having 1% sodium azide and 0.005M sodium ethylene diamino tetra acetic acid) to remove loosely bound antibodies. Then the cells were stained with 1 : 100 dilutions of PE-anti-human c-kit antibody, APC-anti-human IgE, antibody and BV-605-anti-human FcsRIa antibody for 30 minutes. Loosely bound antibodies were removed by two rounds of FACS buffer wash and then fixed in 2% formaldehyde solution for 20 minutes at room temperature. Fluorescent assisted cell sorting technique on CytoFLEX S was employed to study percentage human IgE loaded LAD2 cells.

[0255] Biolayer Interferometry: Biolayer Interferometry assay using the Octet® K2 system was conducted to study the affinity of the different mannose glyco-variants of human IgE or its respective buffer control to the human IgE high affinity receptor (hFcsRIa) and hCD23. The histidine tagged hFcsRIa (ACROBiosystems) diluted in Octet® Buffer (PBS having 0.1% BSA and 0.2% TWEEN® 20) to a concentration of 25pg / ml was initially loaded on anti-penta-HIS (HIS IK) biosensors (Molecular Devices) or hCD23 (R&D Systems) diluted in Octet® Buffer to a concentration of 20pg / ml was loaded onto arginine biosensors (Molecular Devices). The loaded sensors were then dipped into 2-fold serially diluted concentrations of different human IgEs starting at 30pg / ml or 18 pg / ml (for analyzing affinity to hFcsRIa) and at lOOpg / ml or 217pg / ml (for analyzing affinity to hCD23) or octet buffer and then removed to set up and break apart IgE-FcsRIa or IgE-CD23 interactions. The association and dissociation traces were used on the Octet Data Analysis Software 12.2 to calculate dissociation constant (KD) between the IgE molecules to FcsRIa or CD23.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0256] Mice: Female BALB / c mice within the age group 8-12 weeks were purchased from The Jackson Laboratory and were housed in the animal facility until they were used for experiments. Four to six mice were used for every treatment conditions.

[0257] For passive cutaneous anaphylaxis (PCA), age and sex matched BALB / c mice were intradermally sensitized on their ears for overnight with lOpl of 2ng / pl of the different enzyme digested mouse IgEs or PBS. The next day the mice were intravenously challenged with 125pg of OVA dissolved in PBS and 2% Evans Blue dye (Sigma). After 45 minutes, the mice were sacrificed, images of ears were taken, the ears were cut, minced and then incubated in 150pl of N, N-dimethyl-formamide (EMD Millipore) at 55°C for 3 hours for extracting the blue dye into the solvent. The degree of anaphylaxis was evaluated by measuring the absorbance of the dye (650nm) that had leaked into the ear tissues during the OVA challenge.

[0258] Some mice were systemically administered lOOpl of PBS or lOOpl of 0.5mg / ml EndoFl -a-OVA-mlgE or BC-a-OVA-mlgE. After fourteen days, these mice were cheek bled to quantify the BC-a-OVA-mlgE or EndoFl -a-OVA-mlgE response by ELISA. After another four days mice were systemically sensitized with 20pg a-OVA-mlgE. Three days later, the mice were challenged intravenously with Img of OVA in PBS, and core body temperature was recorded at 10-minute intervals in a model of passive systemic anaphylaxis (PSA).

[0259] For in vivo cell loading, mlgE preparations were fluorescently labeled with Alexa Flour® 647 per manufacturers’ protocol (Thermo Scientific). 6.5 pg of Alexa Flour® 647 labeled EndoFl -mlgE or BC-mlgE was administered to mice intraperitoneally. After 2 hours, the mice were sacrificed, and peritoneal cells were harvested, and the collected cells were stained with 1:200 dilution of FITC-labelled anti-mouse CD 19 antibody, Brilliant Violet 605™ (BV605)-conjugated anti -mouse c-kit antibody along with 1:2000 dilution of zombie violet for live-dead staining. Separately, the peritoneal cells from naive mice were stained with 1:200 dilution of APC-Fire-conjugated mouse CD23, FITC-conjugated anti-mouse CD19, PE-conjugated anti-mouse FcsRIa and BV-605-conjugated anti-mouse c-kit and 1:2000 dilution of zombie violet. The stained cells were then fixed in 2% formaldehyde. Unstained cells, zombie violet single stained cells, only Alexa Flour® 647 labeled peritoneal cells and the antibodies used to stain the samples were again used to stain beads for compensation on Cytek® Aurora™. The fixed cells were then run on Cytek®Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0260] Aurora™ to check for CD23 and FcsRIa expression and BC-a-TNP-mlgE and EndoFl -a-TNP-mlgE loading on mouse peritoneal mast cells and B cells.

[0261] IgE Structure Analysis: Following completion of EndoFl digestion or its buffer treatment of a-OVA-hlgE, the reactions were passed over OVA beads to remove the residual enzymes. The IgE eluted from the beads were buffer exchanged. Five microliters of samples at ~0.1 pM were applied to carbon-coated copper grids (CF300-CU; Electron Microscopy Sciences, Hatfield, PA) that had been glow-discharged in air for 30 s. Grids were rinsed twice with deionized water and stained with 1% (w / v) uranyl acetate (Electron Microscopy Sciences, Hatfield, PA) for 30 s. Images were collected on a CM10 transmission electron microscope (Philips Electron Optics / FEI, Eindhoven, The Netherlands) operated at 100 kV, at a nominal magnification of 64,000*, under minimal electron-dose conditions. Image processing was performed using the RELION suite. Particles were manually picked from CTF-corrected micrographs, and reference-free 2D class averages were generated. Selected 2D classes were used for initial 3D model generation, and the resulting models were further refined. The refined 3D volumes were used for rigid-body model docking in UCSF Chimera. Crystal structures of IgE-Fc (PDB ID: 1O0V), FcsRIa (PDB ID: 2Y7Q), and CD23 (PDB ID: 5LGK) were docked into the corresponding density regions using the Fit in Map function, based on cross-correlation optimization. The resulting fits were visually inspected and adjusted where necessary to ensure consistency with the density.

[0262] IgE Serum Quantification: Four age-matched BALB / c mice or age-matched BALB / c FcsRIa knockout mice were intraperitoneally sensitized with 6.5pg of EndoFl buffer treated or buffer and Endo Fl -treated a-TNP-mlgE. The mice were cheek bled at 2 hours, 6 hours and 12 hours. Blood was allowed to clot and then centrifuged to collect serum. To quantify a-TNP-mlgE concentration in these serum samples, ELISA plates were initially coated with Ipg / ml of TNP-OVA (Lucigen Corporation) and then assayed for quantification as described in the ELISA section.

[0263] Human PBMC IgE loading: All experiments using primary human samples were done under MGH IRB approval and following individual consenting. Peripheral blood was drawn from 3 human consenting donors volunteers, processed to get PBMCs and the cells were stripped with lactic acid. The stripped PBMCs were diluted at 1 million cells / ml in BAT buffer (RPMI having ImM CaCh and 0.5% BSA) and the cells were sensitized with Ipg of EndoFl buffer-treated human IgE or human IgE treatedAttorney Docket No. 29539-0864WO1 / MGH 2025-213

[0264] with the buffer and EndoFlfor Ihr at 37°C. The cells were then centrifuged at 750*g for 10 mins at 4°C and later the dead cells were stained with zombie aqua at 1:2000 dilution in PBS on ice for 20 mins. Subsequently, the cells were stained with a combination of antibodies at 1:200 dilution in FACS buffer with the mixture having PE-conjugated anti-human CD 123, BV-605-conjugated anti-human ckit, APC-labelled anti-human IgE, Brilliant Violet™ 421 (BV421)-tagged anti-human CD 19, PerCP - Cy5.5-conjugated ant-human CD14, PE / Dazzle™-labelled CDllc, PE-Cy7- conjugated anti-human CD23 and 10pg / ml of OVA-FITC for 30 mins on ice. Eventually, the stained cells were fixed with 2% formaldehyde. The same antibodies bound to compensation beads at the same dilution and 20pg / ml of OVA-FITC bound to 2 pg IgE sensitized PBMCs were used as single stain controls for compensation. The formaldehyde fixed cells were then analyzed for human IgE loading on them on Cytek® Aurora™.

[0265] Table 1. Reagent and Resource

[0266] REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies

[0267] Anti-mouse IgG-Fc-HRP Bethyl Laboratories Cat#: A90-131A Human IgE protein (Azide Free) Abeam Cat#: AB65866 Goat anti-human IgE-HRP Bethyl Laboratories Cat#: A80-108P Rabbit anti-human IgE Bethyl Laboratories Cat#: A80-109A Goat anti-mouse IgE-HRP Bethyl Laboratories Cat#: A90-115P Rat anti-mouse IgGl-HRP BD Biosciences Cat#: 559626 Rat anti-mouse IgG2a-HRP BD Biosciences Cat#: 553391 Goat anti-mouse IgG2a-HRP Bethyl Laboratories Cat#: A90-107P Rabbit anti-mouse IgG2b-HRP Sigma Aldrich Cat#: SAB3701185 Goat anti-mouse IgG2b-HRP Bethyl Laboratories Cat#: A90-109P Rabbit anti-mouse IgG3-HRP Sigma Aldrich Cat#: SAB5600002 Anti-TNP-mouse IgE (Purified Biolegend Cat#: 401701 Mouse IgE, K Isotype Control)

[0268] PE-anti-human CD117 (c-kit), Biolegend Cat#: 313204 Clone: 104D2

[0269] FITC-anti-human FcsRIa, Clone: Biolegend Cat#: 334608 AER-37 (CRA-1)

[0270] BV605-anti-human FcsRIa, Clone: Biolegend Cat#: 334628 AER-37 (CRA-1)

[0271] APC-anti-human IgE, Clone: Biolegend Cat#: 325508 MHE-18

[0272] PE-anti-human CD123, Clone: 6H6

[0273]

[0274] Biolegend Cat#: 306006Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0275] REAGENT or RESOURCE SOURCE IDENTIFIER BV421-anti-human CD19, Clone: Biolegend Cat#: 363018 SJ25C1

[0276] PE / Cy anine7-anti -human CD23 , Biolegend Cat#: 338516 Clone: EBVCS-5

[0277] PE / Dazzle™ 594 anti-human Biolegend Cat#: 337228 CDllc, Clone: Bui 5

[0278] PerCP / Cyanine 5.5 anti-human Biolegend Cat#: 367109 CD 14, Clone: 63D3

[0279] Human TruStainFcX™ Biologend Cat#: 422302 BV605-anti-mouse CD117 (c-kit), Biolegend Cat#: 135122 Clone: ACK2

[0280] FITC-anti-mouse CD 19, Clone: Biolegend Cat#: 152404 1D3 / CD19

[0281] Mouse TruStainFcX™ Biolegend Cat#: 101320 Alexa Flour 647 Antibody Labeling Invitrogen Cat#: REF A88068 Kit

[0282] Chemicals, Peptides, and Recombinant Proteins

[0283] Sodium citrate Millipore Sigma Cat#: 6132-04-3 Citric acid Millipore Sigma Cat#: 77-92-9 Sodium chloride Millipore Sigma Cat#: 7647-14-5 Sodium bicarbonate Thermo Scientific Cat#: 424270010 0.5M Ethanolamine

[0284] Sodium Acetate Research Products Cat#: S22040-1000.0

[0285] International

[0286] B upHTM Carbonate-Bi carbonate Thermo Scientific Cat#: REF28382 Buffer Packs

[0287] Sodium phosphate dibasic Fisher Cat#: CAS 7782-85-6 Heptahydrate

[0288] Calcium Dihydrate Fisher Cat#: CAS 10035-04- 8

[0289] HEPES Fisher Cat#: CAS 7365-45-9 Glucose Sigma Cat#: G6152-500G Potassium chloride Millipore Sigma Cat#: 7447-40-7 Sodium bicarbonate Millipore Sigma Cat#: 144-55-8 Magnesium Sulfate Ward’s Science Cat#: 10034-99-8 DL-Dithiothreitol Sigma Aldrich Cat#: D5545-5G lodoacetamide Sigma Aldrich Cat#: I1149-25G Glycine Research Products Cat#: G36050-1000.0

[0290]

[0291] InternationalAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0292] REAGENT or RESOURCE SOURCE IDENTIFIER Acetonitrile Sigma Aldrich Cat#: SHBS5995 Ammonium Formate Sigma Aldrich Cat#: 09735-250G 4-(2-Hy droxy ethyl) piperazine- 1 - Millipore Sigma Cat#: 7365-45-9 ethanesulfonic acid, N-(2- Hydroxy ethyl) piperazine-N'-(2- ethanesulfonic acid)

[0293] Magnesium chloride hexahydrate Millipore Sigma Cat#: 7791-18-6 Phosphate Buffered Saline

[0294] Tris-HCl Research Products Cat#: T60050- 1000.0

[0295] International

[0296] Geneticin Sulfate (G418) Fisher Scientific Cat#: G-1033-50GM Tris Borate Saline Research Products Cat#: T60040-1000.0

[0297] International

[0298] pcDNA™3.4 TOPO™ TA Cloning Thermo Fisher Cat#: A14697 Kit

[0299] Expi293™ Expression System Kit Thermo Fisher Cat#: A14635 Alpha-( 1 -2, 3 )-Mannosidase New England Biolabs Cat#: P0729S Alpha-( 1 -2,3 ,6)-Mannosidase New England Biolabs Cat#: P0768S B eta-Mannosi dase Sigma Aldrich Cat#: M7819 B eta-Mannosi dase Megazyme Cat#: 700004199 Endoglycosidase Fl Sigma Cat#: E9762 Endoglycosidase Fl Fisher Scientific Cat#: 502236644 PNGase F New England Biolab Cat#: P0704L Trypsin Thermo Fisher Cat#: A40009 Chymotyrpsin Promega Cat#: 76201-268 Anthranilamide (2-AB) Sigma-Aldrich Cat#: A89804 TMB Substrate Biolegend Cat#: 421101 PageRuler™ Prestained Protein Thermo Fisher Cat#: 26617 Ladder, 10 to 180 kDa

[0300] NuPAGE™ 3-8% Tris- Acetate Gel Invitrogen Cat#: REF EA0375BOX Tris-Acetate SDS Running Buffer Novex Cat#: REF LA0041 Aquastain Bulldog Bio Cat#: AS001000 Tween 20 Research Products Cat#: P20370-0.5 International

[0301] 2-Methylpyridine borane complex Millipore Sigma Cat#: 654213-5G Sodium cyanoborohydride Millipore Sigma Cat#: L56159-10G Bovine Serum Albumin Sigma Aldrich Cat#: SLCP1156 Albumin from Chicken Egg White Sigma Aldrich Cat#: A5503-25G Ficoll-Plaque™PLUS Cytiva Cat#: 17144002

[0302]

[0303] OVA-FITC Thermo Fisher Cat#: 023020Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0304] REAGENT or RESOURCE SOURCE IDENTIFIER TNP-OVA Lucigen Corporation Cat#: T-5051-100 Compensation Beads Biolegend Cat#: 424602 Recombinant Human CD23 / Fc R&D Systems Cat#: 123-FE-050 epsilon RII Protein, CF

[0305] Human Fc epsilon RI alpha Protein, Aero Biosystems Cat#: FCA-H5228 His Tag (MALS verified)

[0306] Critical Commercial Assays

[0307] CellTiter-Glo® 2.0 Cell Viability Promega Cat#: G9241 Assay

[0308] Experimental Models: Cell Lines

[0309] Expi293F™ Cells Thermo Fisher RRID:CVCL_D615

[0310] Cat#: A14527 LAD2 Cells Kind donation from NIH, Bethesda, MD NIAD

[0311] Experimental Models: Organisms / Strains

[0312] Mouse: Balb / cJ The Jackson Strain #:000651

[0313] Laboratory RRID:IMSR_JAX:000

[0314] 651

[0315] Software and Algorithms

[0316] GraphP ad Prism 10 GraphPad Software,

[0317] Inc.

[0318] OpenLAB software Agilent

[0319] Adobe Illustrator Creative cloud

[0320] FlowJo FlowJo LLC

[0321] Octet Analysis Studio Sartorius

[0322] Biorender Biorender

[0323] Other

[0324] Agilent AdvanceBio Glycan Agilent Cat#: 683775-913 Mapping column (120A, 2.1 * 150

[0325] mm, 2.7 pm)

[0326] Supelclean™ ENVI-Carb™ SPE Millipore Sigma Cat#: 57088 Tube

[0327] AdvanceBio SEC 300A, 7.8 x 300 Agilent Cat#: PL1180-5301 mm, 2.7 um, HPLC column

[0328] Octet® AR2G Biosensors Sartorius Cat#: 18-5092 Octet® HIS IK Biosensors Sartorius Cat#: 18-5120 Octet® Amine Coupling Second Sartorius Cat#: 18-5095 Generation Reagent Kit

[0329] PierceTM Concentrator, PES, Thermo Scientific Cat#: REF 88512

[0330]

[0331] 3KMWC0, 0.5mlAttorney Docket No. 29539-0864WO1 / MGH 2025-213

[0332] REAGENT or RESOURCE SOURCE IDENTIFIER PierceTM Concentrator, PES, Thermo Scientific Cat#: REF ZH391063 30KMWCO, 2-6ml

[0333] Amicon R Ultra, 100KMWCO, Millipore Cat#: REF

[0334] 0.5ml UFC510096 StemPro R-34 SFM (IX) Gib co Cat#: REF 10640-019 Expi293TM Gib co Cat#: REF 14351-01 RPMI Medium 1640 (IX) Gib co Cat#: REF 11875-085 Human Stem Cell Factor Peprotech Cat#: REF 300-07 Human Interleukin-3 R&D Systems Cat#: 203-IL-050 / CF Heat-Deactivated FBS Cytiva Cat#: SH30071.03 0.5M EDTApH 8.0 KD Medicals Cat#: RGE3130 Evan’ SIGMA Aldrich

[0335]

[0336] s Blue Cat#: E2129-10G EXAMPLE 1. IgE Site-Specific Glycan Structures

[0337] Human IgE glycosylation was studied but the sources of the IgE have been limited to multiple myeloma, hyper IgE, recombinant IgE produced in HEK293T, total IgE from peanut allergic sera (Ara h2+Allergic), and total IgE from non-atopic sera. In this study, total primary IgE was isolated from several additional conditions, helminth infected sera, multiple myeloma sera, immunodominant peanut antigen Ara h2+non- atopic sera (Ara h2+non-allergic), systemic lupus erythematosus nephritis (lupus nephritis), and recombinant Expi293T cell and hybridoma-produced IgE (Table 2). The isolated IgE was digested with trypsin and chymotrypsin proteases and subjected to glycopeptide mass spectrometry to determine the glycan type across all N-linked sites.

[0338] Table 2. Patient Demographic Data

[0339] Sample # Diagnosis

[0340] Details stated on Non-Allergic

[0341] Reference # 26 Arah2+non-allergic

[0342] Arah2+Allergic

[0343] L072 Systemic Lupus Erythematosus Nephritis (SLE) L078 Systemic Lupus Erythematosus Nephritis (SLE) L137 Systemic Lupus Erythematosus Nephritis (SLE) L140 Systemic Lupus Erythematosus Nephritis (SLE)

[0344] Multiple Myeloma

[0345] Helminth Infected

[0346] Helminth Infected

[0347]

[0348] Helminth Infected

[0349] The identified glycans from these primary IgE samples were compared with IgE from Ara h2+Allergic sera, non-atopic sera, and recombinant HEK293TAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0350] produced IgE, revealing conservation of N-linked glycan structures across the IgE heavy chains. Biantennary glycans were detected exclusively on N140, N168, N218, and N371 on primary IgE, except for a minor proportion of oligomannose structures at the sites on recombinant IgE. N383 was found to be unoccupied in primary samples (FIG. IB). Oligomannose structures were found exclusively atN394 on all hlgE, and atN384 on mlgE (FIG. 2B, arrow pointed). Taken together, these results suggest that the N-linked glycan structures, particularly the N394 oligomannose, on IgE are conserved.

[0351] EXAMPLE 2. Oligomannose Requirements of IgE-Receptor Binding and Anaphylaxis

[0352] The oligomannose glycan at N394 and N384 on mouse and human IgE (mlgE, hlgE, respectively) is critical for FcsRI binding, mast cell loading, and anaphylaxis. To identify the minimal oligomannose structure required for IgE function, mannose residues were enzymatically trimmed using a(l-2,3)-mannosidase, generating IgE with a Man-3 glycan (Man-3 -hlgE) (FIG. 3 A). Control IgE (WT) was treated with enzyme buffer alone (BC), and Endo Fl -treated IgE (EndoFl -hlgE) served as an oligomannosedeficient control. Non-denaturing SDS-PAGE revealed similar migration profiles for WT (hlgE), BC-hlgE and Man-3-hIgE (FIG. 2B). HPLC analysis confirmed >88% of glycoforms were Man-3 in the enzyme-treated sample, while buffer-treated IgE retained Man5-9 structures (FIGS. 3C and 3D).

[0353] LAD2 human mast cells were then sensitized with PBS, hlgE, BC-hlgE, Man-3-hIgE, or EndoFl-hlgE in vitro (FIGS. 4A and 4B). The sensitized LAD2 mast cells were stimulated by anti-hlgE crosslinking antibody, and degranulation was evaluated by quantifying P-hexosaminidase release. Indeed, robust mast cell degranulation was observed in hlgE, BC-hlgE and Man-3-hIgE treated cells, but significantly attenuated in PBS and EndoFl-hlgE cells (FIG. 4A). Next, IgE-mast cell surface loading was evaluated by flow cytometry. Man-3-hIgE displayed comparable surface IgE levels to hlgE and BC-hlgE, while no IgE was detected on PBS or EndoFl-hlgE treated cells (FIG. 4B, FIG. 3E). These findings were extended using OVA-specific mouse IgE (mlgE) treated with a(l-2,3)-mannosidase, BC, or EndoFl. In a passive cutaneous anaphylaxis (PCA) model, the panel of OVA-specific mlgE with defined oligomannose glycans were injected intradermally in the ears of mice. The following day, the mice were challenged systemically with the allergen, OVA, in Evan’s blue dye. The amountAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0354] of blue coloration of the ears was measured to quantify anaphylaxis. mlgE, BC-mlgE, and Man-3 -mlgE induced robust blue extravasation, while EndoFl -mlgE failed to elicit significant responses (FIG. 4C). ELISAs examining antigen binding confirmed BC-mlgE and Man-3 -mlgE bound OVA identically (FIG. 4D). Finally, biolayer interferometry (BLI) real time binding experiments examining interactions of hlgE with its receptors showed that Man-3 -hlgE retained binding to both FcsRI and CD23 similarly to BC-hlgE (FIGS. 4E-4H). Together, these results demonstrate Man-3 -IgE can bind to its allergen, receptors, and trigger anaphylaxis.

[0355] To further define the minimal IgE oligomannose structure required functionally, IgE with a single terminal mannose (Man-1) was generated via a(l-2,3,6)-mannosidase digestion or buffer only (BC) (FIG. 5A). SDS-PAGE and HPLC confirmed efficient conversion to Man-1 (94%), while control IgE (BC-hlgE) retained Man5-9 structures (FIGS. 3B-3D). LAD2 mast cell degranulation following IgE-crosslinking was similar between BC-hlgE and Man- 1 -hlgE, while degranulation by EndoFl -hlgE resembled PBS-treated condition (FIG. 6A). LAD2 cells sensitized with BC-hlgE or Man-l-hlgE, showed comparable surface loading by flow cytometry, while EndoFl -hlgE was not detected on the cell surface (FIG. 6B, FIG. 5E). In PCA experiments, BC-mlgE and Man- 1 -mlgE induced robust reactions, in contrast to EndoFl -mlgE and PBS-treatment (FIG. 6C). Antigen binding was similar-between BC-mlgE and Man- 1 -mlgE as determined by ELISA (FIG. 6D). Also, BLI experiments revealed similar binding kinetics for Man- 1 -hlgE and BC-hlgE with FcsRI (KD -10 * M) and FcsRII (KD -IO5M) (FIGS. 6E-6H). Slightly reduced binding affinities compared to Man-3 hlgE preparations are likely a consequence of glycosidase reaction conditions, as both buffer control and enzyme-treated IgE were impacted.

[0356] To generate IgE lacking all mannose residues, Man- 1 -IgE was further digested with P-mannosidase, yielding Man-O-IgE, or buffer control (Man- 1 -BC-hlgE) (FIG.

[0357] 7 A). SDS-PAGE and HILIC HPLC analysis confirmed a 4-fold reduction in Man-1 structure yielding Man-0 following treatment with P-mannosidase (FIGS. 7A-7D). Man-O-hlgE showed significantly reduced degranulation in LAD2 cells compared to Man- 1 -BC-hlgE, resembling EndoFl-hlgE (FIG. 7A). Similarly, Man-O-hlgE was minimally detected on mast cell surface at significantly reduced levels compared to Man- 1 -BC-hlgE (FIG. 7B, FIG. 8E). In a PCA assay, mice sensitized with Man-O-mlgE or EndoFl -mlgE showed no detectable anaphylaxis, whereas Man- 1 -BC-mlgE inducedAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0358] robust reactions (FIG. 7C). Antigen binding to Man-O-mlgE or Man-l-BC-mlgE remained intact as determined by ELISA (FIG. 7D). BLI analysis showed Man-O-hlgE did not bind FcsRI, but retained detectable interaction with FcsRII, albeit with ~ 10-fold lower affinity than Man-l-BC-hlgE (FIGS. 7E-7H). Taken together, these findings demonstrate that the terminal mannose linked via 3(1—4) to GlcNAc is both necessary and sufficient for FcsRI-mediated allergic activity.

[0359] EXAMPLE 3. IgE Oligomannose Maintains Fc Conformation Required for FcsRI Binding

[0360] Studies using circular dichroism (CD) spectroscopy indicated that oligomannose-deficient IgE exhibits altered secondary structure compared to WT IgE. To explore the structural changes in greater detail, a-OVA-hlgE was treated with only EndoFl buffer (BC-a-OVA-hlgE) or buffer and EndoFl (EndoFl -a-OVA-hlgE) (FIGS. 9A-9D). The IgE molecules were then purified by FPLC which indicated no multimerization (FIG. 9E). Subsequently, negative-staining electron microscopy (nsEM) was performed on EndoFl -a-OVA-hlgE or BC-a-OVA-hlgE. Collected nsEM-images were used for reference-free two-dimensional class averaging, and appropriate two-dimensional classes were selected to reconstruct three-dimensional density maps display in forward and side views (FIG. 10, FIG. 9F). Consistent with previous reports, BC-OVA-hlgE exhibited two conformations, consistent with open and closed Fc conformations (FIGS. 9A, 9B, 9E, and 9F). The majority conformation adopted by -80% of BC-a-OVA-hlgE was consistent with an open conformation that featured overlapping Cs2 domains and a well-defined pocket between Cs3 and Cs4 domains, facilitating FcsRI binding (FIGS. 10A and 10B). The BC-a-OVA-hlgE minority conformation had extended Cs2 domains, and a slightly oblong Cs3 and Cs4 domain pocket, consistent with the closed, CD23 binding conformation (FIGS. 10E and 10F). Although EndoFl -a-OVA-hlgE also formed majority and minority conformations, both had extended Cs2 domains, which were more consistent with BC-a-OVA-hlgE minority CD23 binding conformation (FIGS. 10B, 10C, 10F, and 10G).

[0361] The refined three-dimensional maps were used to model IgE docking on receptors (FIGS. 11A-11F). These revealed the major open conformation of BC-a-OVA-hlgE interacting with FcsRI (FIG. 11 A), while the Cs2 conformation in both major and minority conformations of EndoFl -a-OVA-hlgE would sterically hinderAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0362] FcsRI engagement (FIGS. 11B and 11C). The minority, closed BC-a-OVA-hlgE conformation was modeled to interact with two CD23 molecules at the juncture of Cs3 and Cs4 domains (FIG. 1 ID). Interesting, both major and minor conformations of EndoFl -a-OVA-hlgE were modeled to interact with CD23, with minimal contortion of the Cs3 and Cs4 domain interface (FIGS. 1 IE and 1 IF).

[0363] Binding of the IgE preparations with IgE receptors was examined in real time binding assay. BLI experiments confirmed that BC-hlgE bound FcsRI with high affinity, whereas Endo Fl -treated hlgE showed no measurable binding (FIGS. 11G, 11H). However, both IgEs retained binding to FcsRII, with the EndoFl -a-OVA-hlgE form displaying reduced but detectable binding affinity (FIGS. 111- 11 J). ELISA-based steady-state binding assays corroborated these findings, where both control EndoFl -treated IgE bound CD23 similarly (FIG. 12 A).

[0364] EXAMPLE 4. Endo Fl-Treated IgE Induces Anti-IgE Antibodies and Confers Protection from Anaphylaxis

[0365] The structural and binding studies above indicate that the conserved IgE oligomannose is essential for FcsRI binding, but less important for CD23 interactions. To assess whether the receptor binding bias remained in cellular context, human peripheral blood mononuclear cells (PBMCs) were incubated with EndoFl buffer treated WT (BC-a-OVA-hlgE) or oligomannose-deficient hlgE (EndoFl -a-OVA-hlgE), and cell loading assessed by flow cytometry (FIG. 1 IK, FIG. 12B). BC-a-OVA-hlgE was detected in a dose-dependent manner on both FcsR.1 basophils and CD23+B cells, whereas EndoFl -a-OVA-hlgE was found only CD23+B cells. To extend these findings in vivo, mice were administered TNP specific BC-mlgE or Endo Fl-mlgE and loading onto peritoneal cells was assessed after 2 hours. BC-mlgE was detected predominantly on FcsRI mast cells, however EndoFl -mlgE surface staining was significantly reduced (FIG. 11L, FIGS. 12C-12E). Consistently, little BC-mlgE was observed on B cells, the highest FcsRII expressing cells, while significant enrichment of EndoFl -mlgE was observed on the B cells (Figure 1 IL, FIGS. 12C-12E). These data are consistent with the BLI studies and confirm that removal of the IgE oligomannose glycan shifts receptor preference from FcsRI to CD23 both in vitro and in vivo.

[0366] CD23 has been reported to play a role in antigen opsonization and presentation. The studies described herein suggest that antigen-presentation of oligomannose-deficient IgE would be more efficient than control IgE. Mice were systemicallyAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0367] administered PBS, OVA-specific BC- or EndoFl-mlgE, and serum was collected 14 days later (FIG. 13 A). Although no anti-EndoFl-mlgE IgGs was detected in mice administered with PBS or BC-mlgE, significantly increased total IgG, and anti-EndoFl-mlgE specific IgM, IgGl, IgG2a, and IgG2b, but not IgG3 were observed in the sera of EndoFl -mlgE treated mice, which tracked with reduced serum half-life (FIGS. 14A-14H). The next study determined whether the mice mounted a response against BC-mlgE. Indeed, mice sensitized with PBS or BC-mlgE did not have detectable anti-IgE antibodies (FIGS. 13B-13E). Surprisingly, administration of EndoFl -mlgE induced increased titers of anti -BC-mlgE IgGl, IgG2a, and IgG2b, but notIgG3 (FIGS. 13B-13E).

[0368] The next experiment examined whether the anti-BC-mlgE response resulting from a single administration of EndoFl -mlgE conferred protection from anaphylaxis. Therefore, mice were administered PBS, OVA-specific BC-mlgE, or EndoFl-mlgE.

[0369] 18 days following administration the mice were sensitized with WT OVA-specific mlgE, and 3 days later challenged by systemic OVA administration (FIG. 13F). Core body temperatures were monitored at regular intervals to assess passive systemic anaphylaxis (PSA). PBS and BC-mlgE-treated mice all elicited robust anaphylaxis responses and dropped approximately 2°C 20 minutes following OVA-challenge (FIG.

[0370] 13G). In contrast, mice that received EndoFl-mlgE did not exhibit any temperature drop upon allergen administration (FIG. 13G). Indeed, the observed protection from PSA was intact 6 months following initial administration of oligomannose-deficient mlgE, but not in BC-mlgE-treated animals (FIG. 13H).

[0371] Conclusion

[0372] While IgE is required for atopic diseases, several nuances have emerged over the last decade that implicate non-allergic functionality. Indeed, IgE is protective against toxins and venoms. Allergen-specific IgE are detected in non-allergic individuals. IgE-based immunotherapy has had some initial success in treatment of solid tumors. Anti-nuclear IgE has been implicated in the pathology of lupus nephritis. It is unclear whether glycosylation influences IgE biology across these divergent conditions, although the importance of glycosylation in allergic disease is increasingly appreciated. The results described herein reveal a previously unappreciated structural and functional role for the conserved oligomannose glycan at N394 on human IgE, andAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0373] N384 in murine IgE. Specifically, it is demonstrated herein that this single glycan is essential for maintaining the conformation of the IgE Fc required for high-affinity binding to FcsRI. Removal of this glycan disrupts FcsRI binding, prevents mast cell loading, and abrogates anaphylaxis, despite intact antigen specificity and CD23 binding. These results underscore the critical importance of Fc glycosylation in shaping IgE effector function.

[0374] The function of this glycan is reminiscent of the well-characterized N297 glycan on IgG, which is required for Fey receptor binding and effector function. Analogous to the IgG N297Q mutation that disables FcyR interactions, the absence of oligomannose at IgE N394 results in loss of FcsRI engagement. Given that this glycosylation site is conserved across mammalian species, its role appears to be evolutionarily fixed and functionally indispensable.

[0375] Interestingly, although complete removal of the oligomannose glycan prevents FcsRI binding, stepwise enzymatic trimming revealed that a single terminal mannose (Man-1) is sufficient to preserve IgE function. Man-1 IgE bound both FcsRI and CD23 with similar kinetics to wild-type IgE and retained full anaphylactic potential in vitro and in vivo. In contrast, Man-0 IgE, lacking all mannose residues, failed to bind FcsRI or trigger mast cell degranulation, despite retaining low-affinity CD23 binding. These findings pinpoint the P(l-4)-linked mannose residue on the core GlcNAc as both necessary and sufficient for IgE’s effector function.

[0376] Structural analyses support this mechanism. Negative staining microscopy revealed that oligomannose-deficient IgE adopts a conformation with extended Cs2 domains and a partially collapsed Cs3-Cs4 interface, predicted to hinder FcsRI engagement, with lesser impact on CD23 binding. This conformational shift is consistent with biophysical and binding data showing a loss of FcsRI interaction but preserved CD23 binding. These results indicate that the oligomannose glycan stabilizes the Fc in a conformation permissive to FcsRI binding, analogous to the conformational switch seen between the open (FcsRI-compatible) and closed (CD23 -compatible) states of IgE.

[0377] Remarkably, the removal of the oligomannose glycan not only disables allergic effector function but also converts IgE into an immunogenic molecule. A single injection of Endo Fl -treated IgE induced robust anti-IgE IgG2a and IgG2b responses in the absence of adjuvant. These antibodies conferred complete protection againstAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0378] subsequent passive systemic anaphylaxis triggered by wild-type IgE of the same specificity. However, this protection was antigen-specific; with mice challenged with a distinct IgE specificity (a-TNP-IgE) remained susceptible. These results suggest that the removal of the oligomannose glycan may expose cryptic epitopes or generate neoepitopes that break tolerance to IgE.

[0379] These findings have significant therapeutic implications. First, they indicate that targeted modification of the IgE N394 glycan, via enzymatic or genetic approaches, can be leveraged to prevent FcsRI-mediated allergic responses. Second, oligomannosedeficient IgE can serve as a novel immunogen to induce a potent immune response against IgE, offering a strategy for long-term desensitization. Given that the immune response occurred in the absence of adjuvant and following a single dose, this approach can represent a powerful, antigen-specific immunotherapy for allergic disease. Consistently, immunization with IgE Fc molecules engineered to maintain a closed conformation results in protective anti-IgE responses.

[0380] Finally, the results disclosed herein raise intriguing questions about how glycan composition at N394 is regulated. Unlike the other six N-linked glycosylation sites on IgE, which carry complex biantennary glycans, N394 exclusively carries oligomannose glycan structures. This suggests restricted access to glycan processing enzymes during transit through the Golgi, potentially due to protein conformation or chaperone interactions. This conservation of IgE glycan structure was held across IgE from multiple indications. Understanding the molecular basis of this selective glycosylation may reveal broader principles governing glycoprotein folding and receptor engagement.

[0381] In summary, the studies herein determine the conserved oligomannose glycan at IgE N394 as a structural gatekeeper of allergic effector function. Its selective removal abrogates FcsRI binding, shifts receptor preference to CD23, and elicits anti-IgE immunity, offering a foundation for new classes of allergy therapeutics.

[0382] Reference:

[0383] 1. Broide, D.H. (2001). Molecular and cellular mechanisms of allergic disease. The Journal of allergy and clinical immunology 108, S65-71.

[0384] 2. Gould, H.J., and Sutton, B.J. (2008). IgE in allergy and asthma today. Nature reviews. Immunology 8, 205-217. 10.1038 / nri2273.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0385] 3. Spiegelberg, H.L. (1989). Biological role of different antibody classes. International archives of allergy and applied immunology 90 Suppl 7, 22-27.

[0386] 4. Bruhns, P, Fremont, S., and Daeron, M. (2005). Regulation of allergy by Fc receptors. Current opinion in immunology 77, 662-669. S0952-7915(05)00161-5 [pii] 10.1016 / j.coi.2005.09.012.

[0387] 5. Gould, H.J., Sutton, B.J., Beavil, A.J., Beavil, R.L., McCloskey, N., Coker, H.A., Fear, D., and Smurthwaite, L. (2003). The biology of IGE and the basis of allergic disease. Annual review of immunology 27, 579-628.

[0388] 10.1146 / annurev. immunol .21.120601.141103.

[0389] 6. Shade, K.T., Conroy, M.E., and Anthony, R.M. (2019). IgE Glycosylation in Health and Disease. Curr Top Microbiol Immunol 423, 77-93. 10.1007 / 82 2019 151.

[0390] 7. Shade, K.T., Platzer, B., Washburn, N., Mani, V, Bartsch, Y.C., Conroy, M., Pagan, J.D., Bosques, C., Mempel, T.R., Fiebiger, E., and Anthony, R.M. (2015). A single glycan on IgE is indispensable for initiation of anaphylaxis. The Journal of experimental medicine 272, 457-467. 10.1084 / jem.20142182.

[0391] 8. Arnold, J.N., Radcliffe, C.M., Wormaid, M.R., Royle, L., Harvey, D.J., Crispin, M., Dwek, R.A., Sim, R.B., and Rudd, PM. (2004). The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin. Journal of immunology 173, 6831-6840.

[0392] 9. Plomp, R., Hensbergen, P.J., Rombouts, Y, Zauner, G., Dragan, I., Koeleman, C.A., Deelder, A.M., and Wuhrer, M. (2013). Site-Specific N-Glycosylation Analysis of Human Immunoglobulin E. Journal of proteome research. 10.1021 / pr400714w. 10. Gyorgypal, A., Banerjee, S., Conroy, M.E., and Anthony, R.M. (2025). Glycobiology of IgE. Immunological reviews 331, e70032. 10.11 ll / imr.70032.

[0393] 11. Dhaliwal, B., Yuan, D., Pang, M.O., Henry, A. J., Cain, K., Oxbrow, A., Fabiane, S.M., Beavil, A.J., McDonnell, J.M., Gould, H.J., and Sutton, B.J. (2012). Crystal structure of IgE bound to its B-cell receptor CD23 reveals a mechanism of reciprocal allosteric inhibition with high affinity receptor FcepsilonRI. Proceedings of the National Academy of Sciences of the United States of America 109, 12686-12691.

[0394] 10.1073 / pnas.1207278109.

[0395] 12. Yokota, A., Kikutani, H., Tanaka, T., Sato, R., Barsumian, E.L., Suemura, M., and Kishimoto, T. (1988). Two species of human Fc epsilon receptor II (Fc epsilon RII / CD23): tissue-specific and IL-4-specific regulation of gene expression. Cell 55, 611-618.

[0396] 13. Yukawa, K., Kikutani, H., Owaki, H., Yamasaki, K., Yokota, A., Nakamura, H., Barsumian, E.L., Hardy, R.R., Suemura, M., and Kishimoto, T. (1987). AB cell-specific differentiation antigen, CD23, is a receptor for IgE (Fc epsilon R) on lymphocytes. Journal of immunology 138, 2576-2580.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0397] 14. Bach, J.F. (2002). The effect of infections on susceptibility to autoimmune and allergic diseases. The New England journal of medicine 347, 911-920.

[0398] 10.1056 / NEJMra020100.

[0399] 15. Asthma, G.B.D., and Allergic Diseases, C. (2025). Global, regional, and national burden of asthma and atopic dermatitis, 1990-2021, and projections to 2050: a systematic analysis of the Global Burden of Disease Study 2021. Lancet Respir Med 13, 425-446. 10.1016 / S2213-2600(25)00003-7.

[0400] 16. Umetsu, D.T., and DeKruyff, R.H. (2006). The regulation of allergy and asthma. Immunological reviews 212, 238-255.

[0401] 17. Kemter, A.M., and Nagler, C.R. (2019). Influences on allergic mechanisms through gut, lung, and skin microbiome exposures. The Journal of clinical investigation 129, 1483-1492. 10.1172 / JCI124610.

[0402] 18. McDonnell, J.M., Dhaliwal, B., Sutton, B.J., and Gould, H.J. (2023). IgE, IgE Receptors and Anti-IgE Biologies: Protein Structures and Mechanisms of Action. Annual review of immunology 41, ISS-llS. 10.1146 / annurev-immunol-061020-053712.

[0403] 19. Borthakur, S., Hibbert, R.G., Pang, M.O., Yahya, N., Bax, H.J., Kao, M.W., Cooper, A.M., Beavil, A.J., Sutton, B.J., Gould, H.J., and McDonnell, J.M. (2012). Mapping of the CD23 binding site on immunoglobulin E (IgE) and allosteric control of the IgE-Fc epsilonRI interaction. The Journal of biological chemistry 287, 31457-31461. 10.1074 / jbc.C112.397059.

[0404] 20. Karagiannis, S.N., Warrack, J.K., Jennings, K.H., Murdock, PR., Christie, G., Moulder, K., Sutton, B.J., and Gould, H.J. (2001). Endocytosis and recycling of the complex between CD23 and HLA-DR in human B cells. Immunology 103, 319-331.

[0405] 21. Sutton, B.J., and Davies, A.M. (2015). Structure and dynamics of IgE-receptor interactions: FcepsilonRI and CD23 / FcepsilonRII. Immunological reviews 268, 222-235. 10.1111 / imr.12340.

[0406] 22. Eggel, A., and Jardetzky, T.S. (2025). Structural and Functional Insights Into IgE Receptor Interactions and Disruptive Inhibition. Immunological reviews 331, e70031. 10.1111 / imr.70031.

[0407] 23. Holdom, M.D., Davies, A.M., Nettleship, J.E., Bagby, S.C., Dhaliwal, B., Girardi, E., Hunt, J., Gould, H.J., Beavil, A. J., McDonnell, J.M., et al. (2011). Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcvarepsilonRI. Nat Struct Mol Biol 18, 571-576. 10.1038 / nsmb.2044.

[0408] 24. Glycosylation of Therapeutic IgGs (2009). In Therapeutic Monoclonal Antibodies: From Bench to Clinic, Z. An, ed. (John Wiley and Sons).

[0409] 25. Jefferis, R. (2009). Glycosylation as a strategy to improve antibody -based therapeutics. Nat Rev Drug Discov 8, 226-234. nrd2804 [pii] 10.1038 / nrd2804.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0410] 26. Wang, T.T., and Ravetch, J.V. (2019). Functional diversification of IgGs through Fc glycosylation. The Journal of clinical investigation 729, 3492-3498.

[0411] 10.1172 / JCI130029.

[0412] 27. Arnold, J.N., Wormaid, M.R., Sim, R.B., Rudd, P.M., and Dwek, R.A. (2007). The impact of glycosylation on the biological function and structure of human immunoglobulins. Annual review of immunology 25, 21-50.

[0413] 28. Shade, K.C., Conroy, M.E., Washburn, N., Kitaoka, M., Huynh, D.J., Laprise, E., Patil, S.U., Shreffler, W.G., and Anthony, R.M. (2020). Sialylation of immunoglobulin E is a determinant of allergic pathogenicity. Nature 582, 265-270.

[0414] 10.1038 / s41586-020-2311-z.

[0415] 29. Lecoanet-Henchoz, S., Gauchat, J.F., Aubry, J.P, Graber, P., Life, P., Paul-Eugene, N., Ferrua, B., Corbi, A.L., Dugas, B., Plater-Zyberk, C., and et al. (1995). CD23 regulates monocyte activation through a novel interaction with the adhesion molecules CDllb-CD18 and CDllc-CD18. Immunity 3, 119-125.

[0416] 30. Ding, Z., Dahlin, J.S., Xu, H., and Heyman, B. (2016). IgE-mediated enhancement of CD4(+) T cell responses requires antigen presentation by CD8alpha(-) conventional dendritic cells. Sci Rep 6, 28290. 10.1038 / srep28290.

[0417] 31. Engeroff, P., Caviezel, E, Mueller, D., Thoms, E, Bachmann, M.F., and Vogel, M. (2020). CD23 provides a noninflammatory pathway for IgE-allergen complexes. The Journal of allergy and clinical immunology 145, 301-311 e304.

[0418] 10.1016 / j .jaci.2019.07.045.

[0419] 32. Engeroff, P, Plattner, K., Storni, E, Thoms, E, Frias Boligan, K., Muemer, L., Eggel, A., von Gunten, S., Bachmann, M.F., and Vogel, M. (2021). Glycan-specific IgG anti-IgE autoantibodies are protective against allergic anaphylaxis in a murine model. The Journal of allergy and clinical immunology 147, 1430-1441.

[0420] 10.1016 / j.jaci.2020.11.031.

[0421] 33. Villazala-Merino, S., Rodriguez-Dominguez, A., Stanek, V, Campion, N.J., Gattinger, P, Hofer, G., Froeschl, R., Fae, I., Lupinek, C., Vrtala, S., et al. (2020). Allergen-specific IgE levels and the ability of IgE-allergen complexes to cross-link determine the extent of CD23-mediated T-cell activation. The Journal of allergy and clinical immunology 145, 958-967 e955. 10.1016 / j .jaci.2019.11.019.

[0422] 34. Marichal, T., Starkl, P, Reber, L.L., Kalesnikoff, J., Oettgen, H.C., Tsai, M., Metz, M., and Galli, S.J. (2013). Abeneficial role for immunoglobulin E in host defense against honeybee venom. Immunity 39, 963-975. 10.1016 / j. immuni.2013.10.005. 35. Mukai, K., Tsai, M., Starkl, P, Marichal, T., and Galli, S.J. (2016). IgE and mast cells in host defense against parasites and venoms. Semin Immunopathol 38, 581-603.

[0423] 10.1007 / s00281-016-0565-l.

[0424] 36. Palm, N.W., Rosenstein, R.K., Yu, S., Schenten, D.D., Florsheim, E., and Medzhitov, R. (2013). Bee venom phospholipase A2 induces a primary type 2 response that is dependent on the receptor ST2 and confers protective immunity. Immunity 39, 976-985. 10.1016 / j.immuni.2013.10.006.Atorney Docket No. 29539-0864WO1 / MGH 2025-213

[0425] 37. Kartasamita, C.B., Rosmayudi, O., and Demedts, M. (1994). Total serum IgE and eosinophil count in children with and without a history of asthma, wheezing, or atopy in an urban community in Indonesia. The Respiratory Disease Working Group. The Journal of allergy and clinical immunology 94, 981-988.

[0426] 38. Bird, J.A., Crain, M., and Varshney, P. (2015). Food allergen panel testing often results in misdiagnosis of food allergy. J Pediatr 166, 97-100.

[0427] 10.1016 / j.jpeds.2014.07.062.

[0428] 39. Josephs, D.H., Spicer, J.F., Karagiannis, P, Gould, H.J., and Karagiannis, S.N. (2014). IgE immunotherapy: a novel concept with promise for the treatment of cancer. MAbs 6, 54-72. 10.4161 / mabs.27029.

[0429] 40. Karagiannis, P, Singer, J., Hunt, J., Gan, S.K., Rudman, S.M., Mechtcheriakova, D., Knittelfelder, R., Daniels, T.R., Hobson, P.S., Beavil, A.J., et al. (2009). Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2 / neu-positive tumour cells. Cancer Immunol Immunother 58, 915-930. 10.1007 / s00262-008-0607-l.

[0430] 41. Karagiannis, S.N., Josephs, D.H., Karagiannis, P, Gilbert, A.E., Saul, L., Rudman, S.M., Dodev, T., Koers, A., Blower, P.J., Corrigan, C., et al. (2012). Recombinant IgE antibodies for passive immunotherapy of solid tumours: from concept towards clinical application. Cancer Immunol Immunother 61, 1547-1564.

[0431] 10.1007 / s00262-011-1162-8.

[0432] 42. Leoh, L.S., Daniels-Wells, T.R., andPenichet, M.L. (2015). IgE immunotherapy against cancer. Curr Top Microbiol Immunol 388, 109-149. 10.1007 / 978-3-319-13725-4_6.

[0433] 43. Singer, J., and Jensen- Jarolim, E. (2014). IgE-based immunotherapy of cancer: challenges and chances. Allergy 69, 137-149. 10. Ill 1 / all.12276.

[0434] 44. Josephs, D.H., Nakamura, M., Bax, H.J., Dodev, T.S., Muirhead, G., Saul, L., Karagiannis, P, Ilieva, K.M., Crescioli, S., Gazinska, P, et al. (2018). An immunologically relevant rodent model demonstrates safety of therapy using a tumourspecific IgE. Allergy 73, 2328-2341. 10.1111 / all.13455.

[0435] 45. Osborn, G., Lopez-Abente, J., Adams, R., Laddach, R., Grandits, M., Bax, H.J., Chauhan, J., Pellizzari, G., Nakamura, M., Stavraka, C., et al. (2025). Hyperinflammatory repolarisation of ovarian cancer patient macrophages by antitumour IgE antibody, M0vl8, restricts an immunosuppressive macrophage :Treg cell interaction. Nat Commun 16, 2903. 10.1038 / s41467-025-57870-y.

[0436] 46. Dema, B., Pellefigues, C., Hasni, S., Gault, N., Jiang, C., Ricks, T.K., Bonelli, M.M., Scheffel, J., Sacre, K., Jablonski, M., et al. (2014). Autoreactive IgE is prevalent in systemic lupus erythematosus and is associated with increased disease activity and nephritis. PloS one 9, e90424. 10.1371 / joumal. pone.0090424.

[0437] 47. Henault, J., Riggs, J.M., Karnell, J.L., Liarski, V.M., Li, J., Shirinian, L., Xu, L., Casey, K.A., Smith, M.A., Khatry, D.B., et al. (2016). Self-reactive IgE exacerbatesAtorney Docket No. 29539-0864WO1 / MGH 2025-213

[0438] interferon responses associated with autoimmunity. Nature immunology 77, 196-203.

[0439] 10.1038 / ni.3326.

[0440] 48. Nagpal, S., Sriramarao, P, Krishnaswamy, PR., Metcalfe, D.D., and Rao, PV (1990). Demonstration of IgE antibodies to nucleic acid antigens in patients with SLE. Autoimmunity 8, 59-64.

[0441] 49. Dema, B., Charles, N., Pellefigues, C., Ricks, T.K., Suzuki, R., Jiang, C., Scheffel, J., Hasni, S., Hoffman, V, Jablonski, M., et al. (2014). Immunoglobulin E plays an immunoregulatory role in lupus. The Journal of experimental medicine 277, 2159-2168. 10.1084 / jem.20140066.

[0442] 50. Charles, N., Hardwick, D., Daugas, E., Illei, G.G., and Rivera, J. (2010). Basophils and the T helper 2 environment can promote the development of lupus nephritis. Nature medicine 76, 701-707. 10.1038 / nm.2159.

[0443] 51. Li, P, Wang, X., Zeng, M., and Liu, Z. (2025). Protein Glycosylation: An Emerging Regulator of Allergic Diseases. Allergy. 10.1111 / all.70171.

[0444] 52. Wurzburg, B.A., Garman, S.C., and Jardetzky, T.S. (2000). Structure of the human IgE-Fc C epsilon 3-C epsilon 4 reveals conformational flexibility in the antibody effector domains. Immunity 13, 375-385.

[0445] 53. Conde, E., Lamanna, E., Mougel, A., Kamphuis, J.B.J., Loste, A., Stackowicz, J., Godon, O., Maure, E., Pecalvel, C., Worrall, W.P.M., et al. (2025). A vaccine targeting human IgE induces long-term protection against anaphylaxis in humanized mice. Science translational medicine 77, eads0982. 10.1126 / scitranslmed.ads0982.

[0446] OTHER EMBODIMENTS

[0447] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 29539-0864WO1 / MGH 2025-213WHAT IS CLAIMED IS:

1. A method of inducing an anti-IgE immunogenic response in a subject, comprising administering to the subject an effective amount of a composition comprising a modified IgE antibody or an Fc fragment thereof comprising a deglycosylated N394, wherein the modified IgE antibody is a human antibody or a humanized antibody.

2. A method of treating or preventing an IgE-mediated disorder in a subject, comprising administering to the subject an effective amount of a composition comprising a modified IgE antibody or an Fc fragment thereof comprising a deglycosylated N394, wherein the modified IgE antibody is a human antibody or a humanized antibody.

3. The method of claim 1 or 2, wherein the deglycosylated N394 lacks oligomannose, optionally as a result of treatment of a native human IgE antibody or Fc fragment thereof with EndoFl or PNGaseF.

4. The method of any one of claims 1-3, wherein:the deglycosylated N394 is linked to N-acetylgalactosamine, galactose, and / or sialic acid; orthe deglycosylated N394 lacks N-acetylgalactosamine, galactose, and / or sialic acid.

5. The method of any one of claims 1-4, wherein the modified IgE antibody or an Fc fragment thereof further comprises a deglycosylated N383 and / or a deglycosylated N371.

6. The method of claim 5, wherein the deglycosylated N383 and / or the deglycosylated N371 lack oligomannose.

7. The method of claim 5 or 6, wherein:the deglycosylated N383 and / or the deglycosylated N371 are linked to N- acetylgalactosamine, galactose, and / or sialic acid; orAttorney Docket No. 29539-0864WO1 / MGH 2025-213the deglycosylated N383 and / or the deglycosylated N371 lack N- acetylgalactosamine, galactose, and / or sialic acid.

8. The method of any one of claims 1-7, wherein the modified IgE antibody or an Fc fragment thereof further comprises one or more amino acid modifications at N371, N383, and / or T396.

9. The method of claim 8, wherein the one or more amino acid modifications at N371, N383, and / or T396 comprise one or more mutations or deletions, optionally wherein:the mutation at N383 isN383Q, N383A, orN383D;the mutation at N371 isN371Q, N371A, orN371D; and / orthe mutation at T396 is T396A or T396V.

10. A method of inducing an anti-IgE immunogenic response in a subject, comprising administering to the subject an effective amount of a composition comprising a modified IgE antibody or an Fc fragment thereof comprising one or more amino acid modifications at N394 and / or T396, wherein the modified IgE antibody is a human antibody or a humanized antibody.

11. A method of treating or preventing an IgE-mediated disorder in a subject, comprising administering to the subject an effective amount of a composition comprising a modified IgE antibody or an Fc fragment thereof comprising one or more amino acid modifications at N394 and / or T396, wherein the modified IgE antibody is a human antibody or a humanized antibody.

12. The method of claim 10 or 11, wherein the one or more amino acid modifications at N394 and / or T396 comprise one or more mutations or deletions, optionally wherein:the mutation at N394 is N394Q, N394A, or N394D; and / orthe mutation at T396 is T396A or T396V.Attorney Docket No. 29539-0864WO1 / MGH 2025-21313. The method of any one of claims 10-12, wherein the modified IgE antibody or an Fc fragment thereof further comprises one or more amino acid modifications at N383 and / or N371.

14. The method of claim 13, wherein the one or more amino acid modifications at N383 and / or N371 comprise one or more mutations or deletions, optionally wherein:the mutation at N383 isN383Q, N383A, orN383D; and / orthe mutation atN371 is N371Q, N371A, or N371D.

15. The method of any one of claims 10-14, wherein the modified IgE antibody or an Fc fragment thereof comprises a deglycosylated N394, a deglycosylated N383, and / or a deglycosylated N371.

16. The method of claim 15, wherein the deglycosylated N394, the deglycosylated N383, and / or the deglycosylated N371 lack oligomannose.

17. The method of claim 15 or 16, wherein:the deglycosylated N394, the deglycosylated N383, and / or the deglycosylated N371 are linked to N-acetylgalactosamine, galactose, and / or sialic acid; or the deglycosylated N394, the deglycosylated N383, and / or the deglycosylated N371 lack N-acetylgalactosamine, galactose, and / or sialic acid.

18. The method of any one of claims 1-17, wherein the modified IgE antibody or an Fc fragment thereof comprises the Fc Cs3 domain.

19. The method of claim 18, wherein the Fc Cs3 domain comprises a sequence at least 80% identical to SEQ ID NO: 18, optionally wherein the Fc Cs3 domain comprises the sequence as set forth in SEQ ID NO: 18, 28, or 29.

20. The method of claim 18 or 19, wherein the modified IgE antibody or an Fc fragment thereof further comprises an Fc Cs2 domain and / or an Fc Cs4 domain, optionally wherein the Fc Cs2 domain comprises a sequence at least 80% identical to SEQ ID NO: 17, and the Fc Cs4 domain comprises a sequence at least 80% identicalAttorney Docket No. 29539-0864WO1 / MGH 2025-213to SEQ ID NO: 19, optionally wherein the modified IgE antibody or an Fc fragment thereof comprises a sequence at least 80% identical to SEQ ID NO: 25, optionally wherein the modified IgE antibody or an Fc fragment thereof comprises the sequence as set forth in SEQ ID NO:25, 26, or 27.

21. The method of any one of claims 1-20, wherein the modified IgE antibody or an Fc fragment thereof comprises an antigen-binding fragment.

22. The method of claim 21, wherein the antigen-binding fragment comprises a VHH, a Fab, or an scFv.

23. The method of any one of claims 1-22, wherein the modified IgE antibody or an Fc fragment thereof comprises a heavy chain.

24. The method of claim 23, wherein the heavy chain comprises a sequence at least 90% identical to SEQ ID NO: 14, 23 or 24.

25. The method of any one of claims 1-24, wherein the modified IgE antibody or an Fc fragment thereof comprises a light chain.

26. The method of claim 25, wherein the light chain comprises a sequence at least 90% identical to SEQ ID NO: 20.

27. The method of any one of claims 1-26, wherein the modified IgE antibody or an Fc fragment thereof comprises an Fc region not in an open conformation and / or in a conformation that precludes FcsRI binding.

28. The method of any one of claims 1-27, wherein the modified IgE antibody or an Fc fragment thereof does not bind to FcsRI or binds to FcsRI with an affinity lower at least 100 times (e.g., at least 1000, 5000, 10000, 50000, or 100000 times) lower than a reference control (e.g., an IgE with unmodified Fc region).Attorney Docket No. 29539-0864WO1 / MGH 2025-21329. The method of any one of claims 1-28, wherein the modified IgE antibody or an Fc fragment thereof binds to an FcsRI with a dissociation constant KD of at least 10-1M.

30. The method of any one of claims 1-29, wherein the composition is a vaccine composition, optionally the vaccine composition further comprises an adjuvant.

31. The method of any one of claim 1-30, wherein the subject is not in anaphylaxis.

32. The method of any one of claims 1-31, wherein the IgE-mediated disorder is selected from the group consisting of urticaria (e.g., chronic spontaneous urticaria), systemic lupus erythematosus (SLE), nephritis caused by SLE, bullous pemphigoid, allergic rhinitis, asthma, allergic asthma, non-allergic asthma, atopic dermatitis, gastroenteropathy, and hyper-IgE syndrome (HIES).

33. The method of any one of claims 1-32, wherein the IgE-mediated disorder is an IgE-mediated allergy.

34. The method of claim 33, wherein IgE-mediated allergy is selected from the group consisting of food allergy, environmental allergy (e.g., allergies to tree, grass, weed pollens, dust mites, pet saliva / fur, and mold, and / or seasonal allergies), venom allergy, and drug allergy.

35. The method of any one of claims 1-34, wherein administration of the modified IgE antibody or an Fc fragment thereof results in long-term immune protection (e.g., induction of antibodies and / or memory immune cells that are detectable at 1 month, 3 months, 6 months, 9 months, and / or 12 months post-administration) against the IgE antibody or an Fc fragment thereof in the subject.

36. The method of any one of claims 1-35, further comprises measuring a level of long-term immune protection by measuring levels of antibodies and / or memoryAttorney Docket No. 29539-0864WO1 / MGH 2025-213immune cells that bind to IgE antibody or an Fc fragment thereof at 1 month, 3 months, 6 months, 9 months, and / or 12 months post-administration.

37. The method of claim 36, comprising measuring levels of IgM, IgGl, IgG2, and / or IgG4 that specifically bind to IgE antibody or an Fc fragment thereof.

38. The method of claim 36 or 37, wherein the level of long-term immune protection is measured by an assay selected from the group consisting of Enzyme-Linked Immunoassay (ELISA), surface plasmon resonance (SPR) / biolayer interferometry (BLI), chemiluminescent immunoassays (CLIA), multiplex bead-based immunoassays (e.g., Luminex), flow cytometry-based analysis, immunofluorescence assay (IF A), cytokine secretion assay, ELISpot, T cell proliferation assay, cytotoxicity assays, immunoprecipitation, Western Blot, and immunocytochemistry.

39. A composition comprising a modified IgE antibody or an Fc fragment thereof comprising a deglycosylated N394, wherein the modified IgE antibody is a human antibody or a humanized antibody.

40. The composition of claim 25, wherein the deglycosylated N394 lacks oligomannose.

41. The composition of claim 39 or 40, whereinthe deglycosylated N394 is linked to N-acetylgalactosamine, galactose, and / or sialic acid; orthe deglycosylated N394 lacks N-acetylgalactosamine, galactose, and / or sialic acid.

42. The composition of any one of claims 39-41, wherein the modified IgE antibody or an Fc fragment thereof further comprises a deglycosylated N383 and / or a deglycosylated N371.

43. The composition of claim 42, wherein the deglycosylated N383 and / or the deglycosylated N371 lack oligomannose.Attorney Docket No. 29539-0864WO1 / MGH 2025-21344. The composition of claim 42, whereinthe deglycosylated N383 and / or the deglycosylated N371 are linked to N- acetylgalactosamine, galactose, and / or sialic acid; orthe deglycosylated N383 and / or the deglycosylated N371 lack N- acetylgalactosamine, galactose, and / or sialic acid.

45. The composition any one of claims 39-44, wherein the modified IgE antibody or an Fc fragment thereof further comprises one or more amino acid modifications at N371, N383, and / or T396.

46. The composition of claim 45, wherein the one or more amino acid modifications atN371, N383, and / or T396 comprise one or more mutations or deletions, optionally wherein:the mutation at N383 isN383Q, N383A, orN383D;the mutation at N371 isN371Q, N371A, orN371D; and / orthe mutation at T396 is T396A or T396V.

47. A composition comprising a modified IgE antibody or an Fc fragment thereof comprising one or more amino acid modifications at N394 and / or T396, wherein the modified IgE antibody is a human antibody or a humanized antibody.

48. The composition of claim 47, wherein the one or more amino acid modifications at N394 and / or T396 comprise one or more mutations or deletions, optionally whereinthe mutation at N394 is N394Q, N394A, or N394D; and / orthe mutation at T396 is T396A or T396V.

49. The composition of claim 47 or 48, wherein the modified IgE antibody or an Fc fragment thereof further comprises one or more amino acid modifications at N383 and / or N371.Attorney Docket No. 29539-0864WO1 / MGH 2025-21350. The composition of claim 49, wherein the one or more amino acid modifications at N383 and / or N371 comprise one or more mutations or deletions, optionally wherein:the mutation at N383 isN383Q, N383A, orN383D; and / orthe mutation at N371 is N371Q, N371A, or N371D.

51. The composition of any one of claims 47-50, wherein the modified IgE antibody or an Fc fragment thereof comprises a deglycosylated N394, a deglycosylated N383, and / or a deglycosylated N371.

52. The composition of claim 51, wherein the deglycosylated N394, the deglycosylated N383, and / or the deglycosylated N371 lack oligomannose.

53. The composition of claim 51, wherein:the deglycosylated N394, the deglycosylated N383, and / or the deglycosylated N371 are linked to N-acetylgalactosamine, galactose, and / or sialic acid; or the deglycosylated N394, the deglycosylated N383, and / or the deglycosylated N371 lack N-acetylgalactosamine, galactose, and / or sialic acid.

54. The composition of any one of claims 39-54, wherein the modified IgE antibody or an Fc fragment thereof comprises the Fc Cs3 domain.

55. The composition of claim 54, wherein the Fc Cs3 domain comprises a sequence at least 80% identical to SEQ ID NO: 18, optionally wherein the Fc Cs3 domain comprises the sequence as set forth in SEQ ID NO: 18, 28, or 29.

56. The composition of claim 54 or 55, wherein the modified IgE antibody or an Fc fragment thereof comprises the Fc Cs2 domain and / or the Fc Cs4 domain, optionally wherein the Fc Cs2 domain comprises a sequence at least 80% identical to SEQ ID NO: 17, and the Fc Cs4 domain comprises a sequence at least 80% identical to SEQ ID NO: 19, optionally wherein the modified IgE antibody or an Fc fragment thereof comprises a sequence at least 80% identical to SEQ ID NO: 25, optionallyAttorney Docket No. 29539-0864WO1 / MGH 2025-213wherein the modified IgE antibody or an Fc fragment thereof comprises the sequence as set forth in SEQ ID NO: 25, 26, or 27.

57. The composition of any one of claims 39-56, wherein the modified IgE antibody or an Fc fragment thereof comprises an antigen-binding fragment.

58. The composition of claim 57, wherein the antigen-binding fragment comprises a VHH, a Fab, or an scFv.

59. The composition of any one of claims 39-58, wherein the modified IgE antibody or an Fc fragment thereof comprises a heavy chain.

60. The composition of claim 59, wherein the heavy chain comprises a sequence at least 80% identical to SEQ ID NO: 14, 23 or 24.

61. The composition of any one of claims 39-60, the modified IgE antibody or Fc fragment thereof comprises a light chain.

62. The composition of claim 61, wherein the light chain comprises a sequence at least 80% identical to SEQ ID NO: 20.

63. The composition of any one of claims 39-62, wherein the modified IgE antibody or an Fc fragment thereof comprises an Fc region not in an open conformation and / or in a conformation that precludes FcsRI binding.

64. The composition of any one of claims 39-63, wherein the modified IgE antibody or an Fc fragment thereof does not bind to FcsRI or binds to FcsRI with an affinity lower at least 100 times (e.g., at least 1000, 5000, 10000, 50000, or 100000 times) lower than a reference control (e.g., an IgE with unmodified Fc region).

65. The composition of any one of claims 39-64, wherein the modified IgE antibody or an Fc fragment thereof binds to an FcsRI with a dissociation constant KD of at least 10-1M.Attorney Docket No. 29539-0864WO1 / MGH 2025-21366. The composition of any one of claims 39-65, wherein the composition is a vaccine composition, optionally the vaccine composition further comprises an adjuvant.

67. A composition comprising a modified IgE antibody, wherein the modified IgE antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a sequence at least 80% identical to SEQ ID NO: 14, 23 or 24 and the light chain comprises a sequence at least 80% identical to SEQ ID NO: 20, wherein the modified IgE antibody is a human antibody or a humanized antibody.

68. The composition of claim 67, wherein the modified IgE antibody comprises a deglycosylated N394, a deglycosylated N383, and / or a deglycosylated N371.

69. The composition of claim 67 or 68, wherein the heavy chain comprises a sequence at least 80% identical to SEQ ID NO: 14 and the light chain comprises a sequence at least 80% identical to SEQ ID NO: 20.

70. The composition of claim 69, wherein the heavy chain comprises a sequence as set forth in SEQ ID NO: 14 and the light chain comprises a sequence as set forth in SEQ ID NO: 20.

71. The composition of claim 67 or 68, wherein the heavy chain comprises a sequence at least 80% identical to SEQ ID NO: 23 and the light chain comprises a sequence at least 80% identical to SEQ ID NO: 20.

72. The composition of claim 71, wherein the heavy chain comprises a sequence as set forth in SEQ ID NO: 23 and the light chain comprises a sequence as set forth in SEQ ID NO: 20.

73. The composition of claim 67 or 68, wherein the heavy chain comprises a sequence at least 80% identical to SEQ ID NO: 24 and the light chain comprises a sequence at least 80% identical to SEQ ID NO: 20.Attorney Docket No. 29539-0864WO1 / MGH 2025-21374. The composition of claim 73, wherein the heavy chain comprises a sequence as set forth in SEQ ID NO: 24 and the light chain comprises a sequence as set forth in SEQ ID NO: 20.

75. The composition of any one of claims 39-74 for use in inducing an anti-IgE immunogenic response in a subject, or treating or preventing an IgE-mediated disorder an IgE-mediated disorder in a subject.

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