Patent Application
20220105179
This disclosure relates to glycoengineering immunoglobulin E (IgE), and methods of utilizing glycoengineering for treating various diseases or disorders. Also provided herein are methods for diagnosing allergies.
Allergic disease is a global health burden affecting almost one in three individuals worldwide. Mechanistically, IgE antibodies bind to the surface of mast cells or basophils that express the IgE high affinity receptor, FcεRI3. Subsequent exposure to allergen crosslinks cell-bound IgE, leading to cellular activation and release of allergic mediators including histamine, prostaglandins, and leukotrienes3. This cascade culminates in the canonical symptoms of allergic disease, the most severe of which is anaphylaxis. While IgE that recognizes otherwise innocuous allergens is well established as the causative agent of most allergic diseases1,3, testing for allergic disease remains relatively inaccurate4-6, and curative therapies, including oral immunotherapy, are cumbersome, and only partially effective8-10. Further, allergen-specific IgE is detected in many people who do not express allergic symptoms11. Thus, while IgE is absolutely necessary for triggering the allergic cascade, it is not clear how IgE causes allergic disease in some circumstances and not others.
Approximately one-third of the world's population suffers from allergies1,2. Allergen exposure crosslinks mast cell- and basophil-bound immunoglobulin E (IgE), triggering the release of inflammatory mediators, including histamine3. Although IgE is absolutely required for allergies, it is not understood why total and allergen-specific IgE concentrations do not reproducibly correlate with allergic disease4-6. It is well-established that glycosylation of IgG dictates its effector function and has disease-specific patterns. However, whether IgE glycans differ in disease states or impact biological activity is completely unknown. We therefore unbiasedly examined glycosylation patterns of total IgE from peanut-allergic and non-atopic individuals. This revealed an increase in sialic acid content on total IgE from peanut allergic individuals compared to non-atopic subjects. Sialic acid removal from IgE attenuated effector cell degranulation and anaphylaxis in multiple functional models of allergic disease. Therapeutic interventions, including sialic acid removal from cell-bound IgE with a FIERI targeted-neuraminidase, or administration of asialylated IgE, markedly reduced anaphylaxis. Together, these results reveal a role for IgE glycosylation, and specifically sialylation, in regulating allergy and anaphylaxis, and establish IgE sialylation as a biomarker and therapeutic target for allergies.
Thus, provided herein are fusion polypeptides comprising: an Immunolobulin E (IgE) or IgG antibody Fc domain region; and a sialidase or a functional portion thereof, preferably wherein the sialidase or a functional portion thereof can hydrolyze alpha-(2->3)-, alpha-(2->6)-, alpha-(2->8)-glycosidic linkages of terminal sialic residues on IgE. In some embodiments, the sialidase is NEU1, NEU2, NEU3, NEU4, or Vibrio cholerae serotype O1 sialidase. In some embodiments, the sialidase is a human sialidase. In some embodiments, the fusion polypeptide comprises an IgE CH2 region, an IgE CH3 region, and/or an IgE CH4 region; or an IgG CH2 and CH3 region.
Also provided herein are polynucleotides encoding the fusion polypeptides described herein, vectors comprising polynucleotides encoding the fusion polypeptides, and cells comprising the vectors, and optionally expressing the fusion polypeptides described herein.
Further, provided herein are methods for treating a subject having an IgE-mediated disorder. The methods include administering to the subject an effective amount of a composition comprising a fusion protein described herein. In some embodiments, the IgE-mediated disorder is an allergic disorder. In some embodiments, the allergic disorder is an anaphylactic allergy. In some embodiments, the allergic disorder is asthma, atopic dermatitis. allergic rhinitis, allergic conjunctivitis, eczema, or urticaria.
Additionally, provided herein are methods for preparing glycoengineered IgE, e.g., a composition comprising glycoengineered IgE, the method comprising: providing a composition comprising IgE, preferably human IgE, obtained from a plurality of subjects, contacting the IgE with a sialidase under conditions and for a time sufficient to remove sialylation, e.g., a desired amount of sialylation, from the IgE; thereby preparing glycoengineered IgE. In some embodiments, the method further comprises formulating the glycoengineered IgE for intravenous administration. In addition, provided herein are compositions comprising the glycoengineered IgE prepared by a method described herein, and a pharmaceutically acceptable carrier. In some embodiments, the compositions are formulated for intravenous administration.
Also provided herein are methods for treating a subject having an IgE-mediated disorder. The methods include administering to the subject an effective amount of a composition comprising glycoengineered IgE as described herein.
Further, provided herein are fusion proteins, glycoengineered IgE, and compositions comprising a fusion polypeptide and/or glycoengineered IgE as described herein, optionally with a pharmaceutically acceptable carrier, and the use of these compositions, fusion proteins, glycoengineered IgE and in treating a subject having an IgE-mediated disorder.
In some embodiments, the IgE-mediated disorder is an allergic disorder, e.g., an anaphylactic allergy. In some embodiments, the allergic disorder is asthma, atopic dermatitis. allergic rhinitis, allergic conjunctivitis, eczema, or urticaria.
This disclosure relates to glycoengineering, and methods of utilizing glycoengineering for treating various diseases or disorders (e.g., IgE-mediated disorders).
In another aspect, the disclosure relates to a fusion polypeptide having an antibody heavy chain CH2 region; an antibody heavy chain CH3 region; and a catalytic domain of sialidase, wherein the catalytic domain of sialidase removes sialic acid from a glycoprotein.
In some embodiments, the sialidase is NEU1, NEU2, NEU3, NEU4, or Vibrio cholerae serotype O1 sialidase. In some embodiments, the sialidase is a human sialidase.
In some embodiments, the fusion polypeptide has an IgG CH2 region, and an IgG CH3 region.
In some embodiments, the fusion polypeptide has an IgE CH2 region, an IgE CH3 region, and an IgE CH4 region.
In another aspect, the disclosure provides a polynucleotide encoding the fusion polypeptide as described herein.
In another aspect, the disclosure also relates to a vector having a polynucleotide sequence encoding the fusion polypeptide as described herein.
In one aspect, the disclosure relates to a cell having the vector as described herein, and the vector optionally expresses the fusion polypeptide as described herein.
In one aspect, the disclosure relates to a heteromultimer that has a first fusion polypeptide having an antibody heavy chain CH2 region, an antibody heavy chain CH3 region, and a catalytic domain of mannosidase, wherein the catalytic domain of mannosidase removes mannose from a glycoprotein; and a second fusion polypeptide having an antibody heavy chain CH2 region, an antibody heavy chain CH3 region, and a catalytic domain of sialidase, wherein the catalytic domain of sialidase removes sialic acid from a glycoprotein.
In some embodiments, the heteromultimer is a heterodimer, and the first fusion polypeptide associates with the second fusion polypeptide, thereby forming the heterodimer.
In some embodiments, the mannosidase is MAN1B1 or MAN2A1.
In some embodiments, the sialidase is NEU1, NEU2, NEU3, NEU4, or Vibrio cholerae serotype O1 sialidase.
In some embodiments, the first fusion polypeptide and the second polypeptide each has a human IgE CH2 region, a human IgE CH3 region, and a human IgE CH4 region.
In some embodiments, the first fusion polypeptide and the second polypeptide each has a human IgG CH2 region, and a human IgG CH3 region.
In another aspect, the disclosure also relates to methods of treating a subject having an IgE-mediated disorder. The methods involve administering to the subject an effective amount of a composition having the heteromultimer as described herein.
In some embodiments, the IgE-mediated disorder is an allergic disorder.
In some embodiments, the IgE-mediated disorder is an autoimmune disease.
In some embodiments, the IgE-mediated disorder is anaphylaxis.
In some embodiments, the allergic disorder is asthma. In some embodiments, the allergic disorder is atopic dermatitis. In some embodiments, the allergic disorder is allergic rhinitis, allergic conjunctivitis, eczema, or urticaria.
In one aspect, the disclosure relates to methods of treating a subject having an IgE-mediated disorder. The methods involve administering to the subject an effective amount of one or both of the following:
(a) a first polypeptide having a catalytic domain of mannosidase; and
(b) a second polypeptide having a catalytic domain of sialidase,
wherein the catalytic domain of the mannosidase removes mannose from a glycoprotein, and the catalytic domain of sialidase removes sialic acid from a glycoprotein.
In some embodiments, the first polypeptide further has a human IgE CH2 region, a human IgE CH3 region, and a human IgE CH4 region.
In some embodiments, the first polypeptide further has a human IgG CH2 region, and a human IgG CH3 region.
In some embodiments, the second polypeptide further has a human IgE CH2 region, a human IgE CH3 region, and a human IgE CH4 region.
In some embodiments, the second polypeptide further has a human IgG CH2 region, and a human IgG CH3 region.
In some embodiments, the IgE-mediated disorder is an allergic disorder. In some embodiments, the IgE-mediated disorder is an autoimmune disease. In some embodiments, the IgE-mediated disorder is anaphylaxis.
In some embodiments, the allergic disorder is asthma, atopic dermatitis, allergic rhinitis, allergic conjunctivitis, eczema, or urticaria.
In one aspect, the disclosure provides a heteromultimer that has a first fusion polypeptide having a collagen trimerizing domain and a catalytic domain of mannosidase; a second fusion polypeptide having a collagen trimerizing domain and a catalytic domain of sialidase; and a third fusion polypeptide having a collagen trimerizing domain, wherein the first fusion polypeptide, the second fusion polypeptide, and the third fusion polypeptide bind to each other, forming the heteromultimer.
In some embodiments, the third fusion polypeptide further has a catalytic domain of sialidase. In some embodiments, the third fusion polypeptide further has a catalytic domain of mannosidase.
In another aspect, the disclosure relates to a heteromultimer that has a tetramer having four streptavidin polypeptides; and four polypeptides, wherein each of the four polypeptides is linked with biotin, and one or more of the four polypeptides has a catalytic domain of mannosidase or a catalytic domain of sialidase, wherein each of the four polypeptides binds to the tetramer having the four streptavidin polypeptides.
In some embodiments, each of the four polypeptides has a catalytic domain of mannosidase or a catalytic domain of sialidase. In some embodiments, each of the four polypeptides has a catalytic domain of mannosidase. In some embodiments, each of the four polypeptides has a catalytic domain of sialidase.
In some embodiments, two of the four polypeptides each has a catalytic domain of mannosidase, and two of the four polypeptides each has a catalytic domain of sialidase.
In one aspect, the disclosure also relates to a heteromultimer that has an antibody or antibody fragment thereof; a catalytic domain of mannosidase; and/or a catalytic domain of sialidase, wherein the catalytic domain of mannosidase and the catalytic domain of sialidase each is linked to the antibody or antibody fragment thereof.
In some embodiments, the heteromultimer has an antibody, and the antibody has two antibody heavy chains, and two antibody light chains.
In some embodiments, the catalytic domain of mannosidase is linked to C-terminus of the antibody heavy chain. In some embodiments, the catalytic domain of mannosidase is linked to C-terminus of the antibody light chain.
In some embodiments, the catalytic domain of sialidase is linked to C-terminus of the antibody heavy chain. In some embodiments, the catalytic domain of sialidase is linked to C-terminus of the antibody light chain.
As used herein, the term “multimer” refers to a protein having two or more polypeptides or a polypeptide complex formed by two or more polypeptides. The polypeptides can associate with each other, forming a quaternary structure.
As used herein, the term “heteromultimer” refers to a multimer having more than one type of polypeptides.
As used herein, the term “homodimer” refers to a multimer having two identical polypeptides.
As used herein, the term “heterodimer” refers to a multimer having two polypeptides, and the two polypeptides are different.
As used herein, the term “luminal domain” or “enzymatic luminal domain” refers to the portion of a glycosylation enzyme that is located within the lumen of the Golgi apparatus in its native state. The enzymatic luminal domain of a glycosyltransferase is usually the soluble portion of the glycosylation enzyme.
As used herein, the term “soluble portion” or “soluble domain” refers to the portion of glycosylation enzyme that is soluble. For trans-Golgi glycosylation enzymes, the soluble portions are often the enzymatic luminal domains of the glycosylation enzymes. For non-trans-Golgi glycosylation enzymes, the entire glycosylation enzymes can be soluble. Thus, in some embodiments, the soluble portion can be the entire glycosylation enzyme or part of the glycosylation enzyme.
As used herein, the term “catalytic domain” refers to a portion of a protein that has a catalytic activity.
As used herein, the term “antibody-mediated disorder” refers to a disorder caused by or characterized by an increased level or an increased activity of an antibody.
As used herein, the term “IgE-mediated disorder” refers to a disorder caused by or characterized by an increased level or an increased activity of IgE.
As used herein, the term “linked” refers to being covalently or non-covalently associated, e.g., by a chemical bond (e.g., a peptide bond, or a carbon-carbon bond), by hydrophobic interaction, by Van der Waals interaction, and/or by electrostatic interaction.
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.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
IgE-mediated allergic diseases are multifactorial, with a broad range of clinical presentations. While the presence of peanut-specific IgE associates with peanut allergy, there is a high rate of false positive allergy test results4,6,9,35. Many non-mutually exclusive mechanisms for this discrepancy exist, including differences in IgE affinity or epitope diversity for allergens, mast cell numbers, FcεRI expression levels, Syk signaling, allergen-specific IgG antibodies, anti-IgE antibodies, and regulatory T cells numbers36,37. While IgE from primary allergic samples is severely limited because of its low serum concentrations, recent studies have identified and sequenced B cells that produce peanut-specific antibodies IgE9,38. However, the role of post-translation modifications of the IgE constant chains, including glycosylation, in regulating allergic disease has not been considered. As demonstrated herein, sialic acid content on total IgE distinguishes peanut-allergic and non-atopic IgE. Further, IgE-mediated allergic reactions are attenuated through removal of sialic acid from IgE or administration of asialylated glycoproteins. The sialic acid content and its role in regulating IgE in other atopies and non-atopic conditions is not known39-41. Glycoengineering has been applied to tailor therapeutic IgGs with desirable pro- and anti-inflammatory functions18,20. The present studies revealed that modulating IgE sialic acid content can attenuate anaphylaxis and affirms the application of glycoengineering to allergic disease. Thus, the sialic acid content on IgE can be used as a biomarker for allergic disease, and modulating the IgE sialylation axis presents a powerful means to attenuate allergies and anaphylaxis.
The present disclosure shows engineered glycosylation enzymes can modulate antibody effector function by engineering antibody glycans in vivo for various therapeutic effects. As shown herein, sialic acid in IgE glycans are important for IgE functions; the present disclosure further provides engineered glycosylation enzymes for modulating IgE, e.g., by removing sialic acid (neuraminidase or sialidase) from IgE Fcs, and thus inhibiting IgE pro-allergic function or activity.
Thus, the present disclosure relates to methods and compositions comprising a fusion peptide comprising a catalytic domain of a deglycosylation enzyme (e.g., neuraminidase or sialidase) fused to Fc (e.g., IgG Fc or IgE Fc). The methods and compositions described herein can be used to modulate IgE effector function for various therapeutic effects.
The proteins and cells that make up the human body are decorated by sugars often referred to as glycans (Varki, A. Glycobiology 3, 97-130 (1993)). Glycans can be linked to many types of biological molecule to form glycoconjugates. The enzymatic process that links sugars/saccharides to themselves and to other molecules is known as glycosylation. Glycoproteins, proteoglycans, and glycolipids are the most abundant glycoconjugates found in mammalian cells.
Glycans have an important role in the function of many proteins. Glycans are saccharides (i.e., a plurality of monosaccharides linked glycosidically) that form the carbohydrate portion of glycoconjugates (e.g., glycoproteins, glycopeptides, peptidoglycans, glycolipids, glycosides and lipopolysaccharides). They can be added to proteins in the endoplasmic reticulum, and further modified as proteins travel through the Golgi apparatus. Precursor glycan structures can be attached to asparagine (N-linked), serine or threonine (O-linked), phospholipids (GPI), tryptophan (C-linked), or by phosphodiester bonds (phosphoglycosylation).
Immunoglobulin E (IgE) has two heavy chains (ε chain) and two light chains, with the E chain containing 4 Ig-like constant domains (Cε1, Cε2, Cε3, Cε4; also referred to as CH1, CH2, CH3, CH4). IgE antibodies are primary mediators of allergic disease, and are heavily glycosylated with 7 N-linked glyclosylation sites distributed across its four constant regions (Cε1-Cε4). The distinct glycans on IgE play important and divergent roles in allergic inflammation. Removal of the conserved oligomannose in the constant domains (e.g., Cε1, Cε2, Cε3, Cε4) prevents binding to the high affinity receptor FcεRI on FcεRI-expressing cells (e.g., mast cells and basophils), therefore can inhibit the function or activity of IgE.
Analysis of the glycosylation of human serum IgE indicated that oligomannose structures are present on IgE. In fact, IgE is the most heavily glycosylated monomeric immunoglobulin in mammals. There are six complex-type biantennary (N140, N168, N218, N265, N371, N383) and one oligomannose-type (N394) conserved N-linked glycosylation sites on the constant region of each heavy chain of IgE. The total glycan weight on E heavy chains contributes to ˜12% of the molecular weight of IgE.
The composition of the single N-linked glycan on IgG antibodies profoundly influences its biological activity, and impacts the outcome of many diseases, including Dengue hemorrhagic fever12, Mycobacterium tuberculosis latency13, Influenza vaccination14, Rheumatoid Arthritis7,15, and Granulomatosis with polyangiitis16,17. For example, IgG with afucosylated glycans gain affinity to the activating Fc receptor, FcγRIIIA, 50-fold, making IgG markedly more cytotoxic in vivo18. Conversely, terminal sialylation of the IgG glycan converts IgG into anti-inflammatory mediators, and is thought to be responsible for the immunomodulatory activity of high dose intravenous immunoglobulin19,20. IgE is the most heavily glycosylated monomeric immunoglobulin with seven asparagine (N)-linked glycosylation sites distributed across the heavy chains of human IgE (hIgE)7,21. However, whether particular IgE glycans are associated with allergic disease, or impact IgE function, is completely unknown. IgE is the least abundant antibody class in circulation, and, as such, analysis of hIgE glycosylation has been restricted to samples from subjects with myelomas, hyper IgE syndromes, hyperimmune syndromes pooled from multiple donors, or recombinant IgE21-24. These studies revealed a single N-linked oligomannose glycan at N394 on IgE, N383 is unoccupied, and the remaining five sites are occupied by complex antennary glycans (
IgE Fc glycans can be removed by enzymatic treatment with mannosidase, neuraminidase, Endo F, and/or PNGase F. The enzymatic treatment can inhibit binding of IgE molecules or IgE-Fc fragments to FcεRI. Mutagenesis of the conserved N394 site, which corresponds to N297 on IgG Fc, also reduces the binding to FcεRI.
A detailed description regarding glycans on IgE and the functions thereof can be found, e.g., in Arnold, et al., “The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin.” The Journal of Immunology 173.11 (2004): 6831-6840; Shade, et al., “A single glycan on IgE is indispensable for initiation of anaphylaxis.” Journal of Experimental Medicine 212.4 (2015): 457-467; Shade, et al., “Antibody glycosylation and inflammation.” Antibodies 2.3 (2013): 392-414; and Plomp, et al., “Site-specific N-glycosylation analysis of human immunoglobulin E.” Journal of proteome research 13.2 (2013): 536-546; each of which is incorporated herein by reference in its entirety.
Glycosylation enzymes are responsible for the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor, e.g., proteins, lipids, and glycans). There are many different kinds of glycosylation enzymes, e.g., α-2,6 sialyltransferase (ST6GAL1), β-1,4-galactosyltransferase 1 (B4GALT1), mannosyl-oligosaccharide 1,2-alpha-mannosidase (MAN1B1), alpha-mannosidase 2 (MAN2A1), human sialidase-1 (NEU1), human sialidase-2 (NEU2), human sialidase-3 (NEU3), human sialidase-4 (NEU4), Vibrio cholerae serotype O1 sialidase, Elizabethkingia meningoseptica Endo F1, endo-beta-N-acetylglucosaminidase (Endo S), etc. As shown herein, sialic acid removal attenuated IgE effector functions. Thus, provided herein are fusion proteins in which soluble portions (or the enzymatic luminal domains) or the catalytic domains of sialidases can be fused with Fc (e.g., IgG Fc or IgE Fc), or other appropriate peptides to form multimers, and can be used in any methods described herein.
Sialidases (also known as neuraminidases) hydrolyze alpha-(2->3)-, alpha-(2->6)-, alpha-(2->8)-glycosidic linkages of terminal sialic residues in oligosaccharides, glycoproteins, glycolipids, colominic acid and synthetic substrates. There are four types of human sialidases. They are classified according to their major intracellular location as intralysomal (NEU1), cytosolic (NEU2), plasma membrane (NEU3) and lysosomal or mitochondrial membrane (NEU4) associated sialidases. These human isoforms are distinct from each other in their enzymatic properties as well as their substrate specificity. The sequences for NEU1 (SEQ ID NO: 1), NEU2 (SEQ ID NO: 2), NEU3 (SEQ ID NO: 3) and NEU4 (SEQ ID NO: 4) are shown in
Sialidases can also be found in bacteria, e.g., Vibrio cholerae. Vibrio cholerae is a Gram-negative, comma-shaped bacterium. Some strains of V. cholerae can cause cholera. Vibrio cholerae serotype O1 sialidase has been suggested to be a pathogenic factor in microbial infections. It facilitates cholera toxin binding to host intestinal epithelial cells by converting cell surface polysialogangliosides to GM1 monogangliosides. The sequence for Vibrio cholerae serotype O1 sialidase is shown in
Thus, exemplary neuraminidases useful in the methods and compositions described herein include human NEU1, NEU2, NEU3, and NEU4; and Vibrio cholerae serotype O1 sialidase. See, e.g.,
NEU1 can include, e.g., human NEU1, e.g., the full length soluble NEU1 (SEQ ID NO: 1) or an active portion thereof comprising the luminal domain of human NEU1 (amino acids: 48-415 of SEQ ID NO: 1) and/or the catalytic domain residues of human NEU1 (including catalytic amino acid residues: R78, R97, D103, D135, S156, E264, R280, Q282, R342, Y370, and E394 of SEQ ID NO: 1).
NEU2 can include, e.g., human NEU2, e.g., the full length, soluble NEU2 (SEQ ID NO: 2) or an active portion thereof comprising the active site residues of human NEU2 (amino acids: R21, D46, M85, E111, Y179, Y181, L217, R237, R283, S288, and Y377 of SEQ ID NO: 2).
NEU3 can include, e.g., human NEU3, e.g., the full length human NEU3 (SEQ ID NO: 3) or an active portion thereof comprising the putative catalytic active sites of human NEU3 (amino acids: R25, R45, D50, M87, N88, R108, Q115, A160, E225, R235, R340, Y370, and E387 of SEQ ID NO: 3).
NEU4 can include, e.g., human NEU4, e.g., the full length NEU4 (SEQ ID NO: 4) or an active portion thereof comprising the catalytic active sites of human NEU4 (amino acids: R35, R55, D59, N88, V117, E234, R254, P256, R381, Y431, and E452 of SEQ ID NO: 4).
Vibrio cholerae serotype O1 sialidase can include, e.g., the full length sialidase (SEQ ID NO: 5) or an active portion thereof comprising the catalytic active sites of sialidase (AA25-781, as the first 24AA correspond to the signal peptide).
The active portions retain the ability of the full-length proteins to hydrolyze alpha-(2->3)-, alpha-(2->6)-, alpha-(2->8)-glycosidic linkages of terminal sialic residues on IgE.
The enzymes, the soluble portions thereof (or the luminal domains), the catalytic domains thereof, active sites, and catalytic amino acid residues of these glycosylation enzymes are described, e.g., in Seyrantepe, Volkan, et al. “Neu4, a novel human lysosomal lumen sialidase, confers normal phenotype to sialidosis and galactosialidosis cells.” Journal of Biological Chemistry 279.35 (2004): 37021-37029; Chavas, Leonard M G, et al. “Crystal Structure of the Human Cytosolic Sialidase Neu2—Evidence For The Dynamic Nature Of Substrate Recognition.” Journal of Biological Chemistry 280.1 (2005): 469-475; MONTI, Eugenio, et al. “Identification and expression of NEU3, a novel human sialidase associated to the plasma membrane.” Biochemical Journal 349.1 (2000): 343-351; and Seyrantepe, Volkan, et al. “Molecular pathology of NEU1 gene in sialidosis.” Human mutation 22.5 (2003): 343-352; each of which is incorporated by reference herein in its entirety.
In some embodiments, the sialidase used in the present methods is not receptor destroying enzyme (RDE) (II). Yamazaki et al., J Biol Chem. 2019 Apr. 26; 294(17):6659-6669. Epub 2019 Mar. 4.
This disclosure provides various nucleic acid sequences and amino acid sequences.
In some embodiments, the nucleic acid sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleic acid sequences disclosed herein. In some embodiments, the nucleic acid sequence is identical to any of the sequences described in this disclosure.
In some embodiments, the amino acid sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the amino acid sequences disclosed herein. In some embodiments, the amino acid sequence is identical to any of the sequences described in this disclosure.
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., gaps 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 length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%. 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 purposes of the present disclosure, 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.
Described herein are fusion proteins comprising the Fc region of IgE, preferably human IgE, fused at the N or C terminus to a neuraminidase, referred to herein as Neu-IgE Fc Fusion Proteins. Exemplary sequences of Neu-IgE Fc Fusion Proteins are shown in
The neuraminidases, e.g., full proteins or active portions thereof can be fused to IgE, or a part thereof. The neuraminidases can be fused to IgG Fc. Fc fusions have a number of advantageous: the soluble protein will have an extended serum half-life (e.g., more than 5 days, 10 days, 14 days, or 20 days), and also will form a dimer. In some embodiments, these fusion polypeptides can form homodimers or heterodimers, depending on the glycosylation target.
The IgE Fc can be the Fc region of any IgE known in the art. For example, the IgE Fc can be a human IgE-Fc (e.g., comprising SEQ ID NO: 6), a mouse IgE-Fc (e.g., comprising SEQ ID NO: 7), a canine IgE Fc (e.g., comprising SEQ ID NO: 8), or a feline IgE Fc (e.g., comprising SEQ ID NO: 9). See
In some embodiments, the peptides comprise an IgE antibody epsilon chain CE2, CE3, and/or CE4 region, and an enzymatic luminal domains or a catalytic domain of neuraminidase (e.g., NEU1, NEU2, NEU3, NEU4, Vibrio cholerae serotype O1 sialidase). In some embodiments, the peptide has the amino acid sequence that is set forth in SEQ ID NOS: 1, 2, 3, 4, or 5, e.g., amino acids 48-415 of SEQ ID NO: 1, amino acids 1-380 (full length) of SEQ ID NO:2, amino acids 1-428 (full length) of SEQ ID NO:3, amino acids 1-484 (full length) of SEQ ID NO:4, amino acids 25-781 of SEQ ID NO: 5, or amino acids 557-747 of SEQ ID NO: 5.
Although fusion proteins comprising IgE Fc are exemplified herein, fusion proteins comprising IgG Fc are also described herein. Thus, in some embodiments the neuraminidase can be fused to IgG (e.g., IgG1, IgG2, IgG3, IgG4) or a part thereof. In some embodiments, the neuraminidase can be fused to the Fc portion of an IgG (e.g., IgG1, IgG2, IgG3, IgG4). Fc fusions have a number of advantageous: the soluble protein will have an extended serum half-life (e.g., more than 5 days, 10 days, 14 days, or 20 days), and also will form a dimer. In some embodiments, these fusion polypeptides can form homodimers or heterodimers, depending on the glycosylation target.
The IgG Fc can be the Fc region of any IgG known in the art. For example, the IgG Fc can be a human IgG1-Fc (e.g., comprising SEQ ID NO: 10), a human IgG2-Fc (e.g., comprising SEQ ID NO:11), a human IgG3-Fc (e.g., comprising SEQ ID NO: 12), a human IgG4-Fc (e.g., comprising SEQ ID NO: 13), a mouse IgG1-Fc (e.g., comprising SEQ ID NO: 14), a mouse IgG2a-Fc (e.g., comprising SEQ ID NO: 15), a mouse IgG2b-Fc (e.g., comprising SEQ ID NO: 16), a mouse IgG3-Fc (e.g., comprising SEQ ID NO: 17), a canine IgG-A Fc (e.g., comprising SEQ ID NO: 18), or a feline IgG1 Fc (e.g., comprising SEQ ID NO: 19). See, e.g.,
In some embodiments, these polypeptides can form a homodimer. The homodimer can have two enzymatic luminal domains (or catalytic domains) of mannosidase. In some other cases, the homodimer can have two enzymatic luminal domains (or catalytic domains) of sialidase or neuraminidase. In some embodiments, these polypeptides can form a heterodimer. In some embodiments, the heterodimer can have one enzymatic luminal domain (or catalytic domain) of mannosidase and one enzymatic luminal domain (or catalytic domain) of sialidase or neuraminidase.
In some embodiments, the peptides comprise an enzymatic luminal domain or a catalytic domain of sialidase or neuraminidase (e.g., NEU1, NEU2, NEU3, NEU4, Vibrio cholerae serotype O1 sialidase), and an IgE antibody heavy chain CH2 region, an IgE antibody heavy chain CH3 region, and/or an IgE antibody heavy chain CH3 region.
In some embodiments, the peptide can comprise IgE antibody heavy chain constant regions (e.g., CH1, CH2, CH3 and/or CH4) and/or glycosylation enzymes derived from non-human animals (e.g., dog, cat, cow, or horse; see
In some embodiments, these peptides can additionally include signal sequences, e.g., IL2-signal sequence (e.g., MYRMQLLSCIALSLALVTNS, SEQ ID NO: 32), a secretion signal (e.g., MPLLLLLPLLWAGALA, SEQ ID NO:33), or κ-signal sequence (e.g., METDTLLLWVLLLWVPGSTGDAAQPARRAVRSLVPSSDP, SEQ ID NO: 34). These signal sequences usually present at the N-terminus of the peptides.
In some embodiments, the fusion proteins also include one or more flexible linkers. The linkers can be used to attach the separate parts of the fusion protein together. In some embodiments, the linker is a peptide linker. Peptide linkers can be from about 2-100, 10-50, or 15-30 amino acids long. In some embodiments, peptide linkers may be at least 2, 4, 5, 6, 10, 15, or at least 20 amino acids long and/or up to 20, 25, 35, 40, 60, 80, 90, or no more than 100 amino acids long. In some embodiments, the linker is a peptide linker comprising one or more glycines and/or serines, e.g., a single or repeating GGGGS (SEQ ID NO: 35), GGGS (SEQ ID NO: 36), GS, GGGGGG (SEQ ID NO: 37), GSGGS (SEQ ID NO: 38), GGSG (SEQ ID NO: 39), GGSGG (SEQ ID NO: 40), GSGSG (SEQ ID NO: 41), GSGGG (SEQ ID NO: 42), GGGSG (SEQ ID NO: 43), and/or GSSSG (SEQ ID NO: 44) sequence(s). Other linkers are known in the art. Intact antibodies with desired specificity can also be fused to glycosylation enzymes, enabling specific targeting of the enzymes. Further, similar protein fusions can be generated using dog/cat/horse/cow equivalent/homologous antibodies or glycosylation enzymes, enabling treatment of non-human animals (e.g., pets and livestock).
Glycoengineered Intravenous IgE (gIVIE)
Also provided herein are glycoengineered intravenous IgE (gIVIE) compositions. Analogous to the intravenous immunoglobulin (IVIg) compositions presently in clinical use, the compositions can comprise normal polyspecific obtained from large numbers of healthy donors. The compositions can be polyclonal natural antibodies synthesized, in response to immune stimuli (antigens and T cells), by plasma B cells. Methods for the production of therapeutic IVIG compositions are known in the art (see, e.g., Afonso and Joao, Biomolecules. 2016 March; 6(1): 15 and references cited therein) and can be adapted for production of IVIE, e.g., as shown in
IgE is known to mediate allergic responses and is produced by B cells in both membrane-bound and secretory form. IgE binds to B-cells through its Fc region to a low affinity IgE receptor, known as FcεRII. Upon exposure to an allergen, B-cells bearing a surface-bound IgE molecule specific for the allergen are activated and further develop into IgE-secreting plasma cells. The secreted IgE molecules, which are specific for the allergen, circulate through the bloodstream and become bound to the surface of mast cells in tissue and basophils in bloodstream through the high affinity receptor, known as FcεRI. This binding by allergen-specific IgE, sensitizes the mast cells and basophils for the allergen. Subsequent exposure to the allergen causes cross-linking of FcεRI on basophils and mast cells, leading to up-regulation of the granular molecule CD63 and the release of a number of factors, such as histamine, platelet activating factors, eosinophil and neutrophil chemotactic factors, and cytokines such as IL-3, IL-4, IL-5 and GM-CSF.
As used herein, the term “IgE-mediated response” refers to responses of IgE receptor expressing cells (e.g., basophils and mast cells) induced directly or indirectly by IgE. In some embodiments, the response can be observed (e.g., degranulation) and/or measured by up-regulation of the granular molecule CD63, or the release of one or more of histamine, platelet activating factors, eosinophil and neutrophil chemotactic factors, and cytokines such as IL-3, IL-4, IL-5 and GM-CSF. In some embodiments, IgE-mediated responses include e.g., degranulation, up-regulation of the granular molecule CD63, and/or the release of histamine from basophils. In some embodiments, IgE-mediated responses can cause allergic reactions.
As used herein, the term “attenuating an IgE-mediated response” refers to the extent, occurrence and/or frequency of an IgE-mediated response that is reduced by the methods as described herein, e.g., by administering an agent as described herein as compared to without administering the agent. The extent of reduction can be statistically significant and in certain embodiments, by at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or greater.
The IgE-mediated disorder is characterized by abnormal responses mediated by IgE. In some embodiments, the abnormal responses mediated by IgE are due to overproduction of IgE and/or hypersensitivity of basophils or mast cells to IgE. Thus, IgE-mediated disorders include, e.g., (1) allergic disorders (e.g., asthma, atopic dermatitis, allergic rhinitis, allergic conjunctivitis, eczema, urticaria, food allergy and seasonal allergy, as well as anaphylactic shock); (2) autoimmune disorders (e.g., lupus, rheumatoid arthritis, psoriasis); and (3) anaphylaxis, etc. A detailed description regarding IgE-mediated disorder and IgE-mediated response can be found, e.g., in U.S. Pat. No. 8,828,394 B2, which is incorporated herein by reference in its entirety.
IgE that can specifically recognize an allergen has a unique long-lived interaction with its high-affinity receptor FcεRI so that basophils and mast cells, capable of mediating inflammatory reactions, become “primed”, ready to release chemicals like histamine, leukotrienes, and certain interleukins. These chemicals cause many of the symptoms associated with allergy, such as airway constriction in asthma, local inflammation in eczema, increased mucus secretion in allergic rhinitis, and increased vascular permeability, which allow other immune cells to gain access to tissues, but which can lead to a potentially fatal drop in blood pressure as in anaphylaxis.
Anaphylaxis is a serious allergic reaction that is rapid in onset and may cause death. It typically causes e.g., an itchy rash, throat or tongue swelling, shortness of breath, vomiting, lightheadedness, and low blood pressure. These symptoms typically come on over minutes to hours. When anaphylaxis occurs, IgE binds to the antigen. The antigen-bound IgE then activates FcεRI receptors on mast cells and basophils. This leads to the release of inflammatory mediators such as histamine. These mediators subsequently increase the contraction of bronchial smooth muscles, trigger vasodilation, increase the leakage of fluid from blood vessels, and cause heart muscle depression.
As histamine is central to the pathogenesis of allergic disorders, e.g., asthma and atopic dermatitis, by attenuating IgE-mediated responses such as histamine release, the present method is also effective in treating allergic disorders.
Thus in some embodiments, the fusion proteins described herein can be used to target FcεRI-expressing cells.
The methods described herein include methods for treating IgE-mediated disorders, e.g., allergies, e.g., anaphylactic allergies, and methods for attenuating IgE-mediated responses. Generally, the methods include administering a therapeutically effective amount of compositions comprising Neu-IgE Fc fusion proteins or gIVIE as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. In some embodiments, the subject can be allergic to a food antigen, e.g., eggs, milk, peanuts, soy, fish, shellfish, tree nuts, and/or wheat, or to an environmental allergen, e.g., dust mite excretions, pollen, pet dander, or royal jelly, inter alia. See, e.g., Valenta et al., Gastroenterology. 2015 May; 148(6): 1120-1131.e4.
As used in this context, to “treat” means to ameliorate at least one symptom of the disorders or the diseases. Often, the treatment results in an improvement in the symptoms. In some embodiments, the treatment can result in a reduction of histamine release. In some embodiments, one or more of the clinical symptoms are ameliorated or reduced, the duration being shortened, the frequency of the occurrence of the symptoms is reduced, or the clinical symptoms are prevented from manifesting.
As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, e.g., a mammal, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. 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, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human 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. Thus, in some embodiments, the glycosylation enzymes, the antibodies, or the parts thereof (e.g., Fc regions of the antibodies or the catalytic domain of the glycosylation enzymes) as described herein can also derive from these non-human animals. The present disclosure further provides the amino acid sequences of the glycosylation enzymes, and the antibodies or the parts thereof that derive from some of these non-human animals. For example,
In some embodiments, the subject is a human (e.g., male human or female human) with an age over 6 months old, 12 months old, 2 years old, 5 years old, 6 years old, 10 years old, 12 years old, 16 years old, 18 years old, 25 years old, 30 years old, 40 years old, 50 years old, 60 years old, 70 years old, or 80 years old.
As used herein, the terms “therapeutically effective” and “effective amount”, used interchangeably, applied to a dose or amount refers to a quantity of a composition, compound or pharmaceutical formulation that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “therapeutically effective” refers to that the composition, compound or pharmaceutical formulation, in a sufficient amount, can reduce or eliminate at least one symptom or one condition of the disorders as described herein, delay or reduce risk or frequency of symptoms, or delay or reduce risk of progression.
To use the fusion proteins or peptides as described herein, it may be desirable to express them from a nucleic acid that encodes them. This can be performed in a variety of ways. For example, the nucleic acid encoding the fusion proteins or peptides can be cloned into an intermediate vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression. Intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the fusion proteins or peptides for production. The nucleic acid encoding the fusion proteins or peptides can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoan cell.
To obtain expression, a sequence encoding a fusion protein or peptide is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 2010). Bacterial expression systems for expressing the engineered protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., 1983, Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. In some embodiments, the fusion proteins and peptides are expressed by transfection of HEK-293T cells, Expi293 cells, or CHO cells with vectors comprising the polynucleotides encoding fusion proteins and peptides as described in this disclosure.
The promoter used to direct expression of a nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of fusion proteins. In contrast, when a vector encoding the fusion protein or peptide is to be administered in vivo, either a constitutive or an inducible promoter can be used, depending on the particular need. In some embodiments, the promoter for administration of the vector encoding the fusion protein or peptide can be a weak promoter, such as HSV TK or a promoter having similar activity. The promoter can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, Gene Ther., 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat. Biotechnol., 16:757-761).
In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding the fusion protein or peptide, and any signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals.
The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and commercially available tag-fusion expression systems such as GST and LacZ.
Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
The vectors for expressing the fusion protein or peptide can include RNA Pol III promoters to drive expression of the guide RNAs, e.g., the H1, U6 or 7SK promoters. These human promoters allow for expression of fusion protein or peptide in mammalian cells following plasmid transfection.
Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with the encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.
Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., 1989, J. Biol. Chem., 264:17619-22; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, 1977, J. Bacteriol. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).
Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the fusion protein or peptide.
The present disclosure also includes the vectors and cells comprising the vectors, as well as kits comprising the proteins and nucleic acids described herein, e.g., for use in various methods as described herein.
An “effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms.
An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of polypeptides, multimers, or compositions (i.e., an effective dosage) depends on the polypeptides, multimers, or compositions that are selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the polypeptides, multimers, or compositions described herein can include a single treatment or a series of treatments.
Dosage, toxicity and therapeutic efficacy of the polypeptides, multimers, or compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Polypeptides, multimers, or compositions which exhibit high therapeutic indices are preferred. While polypeptides, multimers, or compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets polypeptides, multimers, or compositions to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of polypeptides, multimers, or compositions lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any polypeptides, multimers, or compositions used in the methods as described in this disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test polypeptide, multimer, or composition which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
The methods described herein include the use of pharmaceutical compositions comprising fusion proteins as described in this disclosure as an active ingredient as well as the compositions themselves.
Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
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.
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, N.Y.). 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.
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, N.J.) 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 as 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.
Sterile injectable solutions can be prepared by incorporating polypeptides, multimers, or compositions as described in this disclosure 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 polypeptides, multimers, or compositions 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.
Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active agents 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 agents 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.
For administration by inhalation, the composition 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. Pat. No. 6,468,798.
In some embodiments, the polypeptides or multimers are prepared with carriers that will protect the polypeptides or multimers 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. Pat. No. 4,522,811.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Included herein are methods for diagnosing allergy. The methods rely on detection of a sialylation levels on IgE. The methods include obtaining a sample from a subject, and evaluating the presence and/or level of sialylation on IgE, e.g., on total IgE, or on allegen-specific IgE (i.e., IgE that binds specifically to a selected allergen) in the sample, and comparing the presence and/or level with one or more references, e.g., a control reference that represents a normal level of sialylation on IgE e.g., a level in an unaffected (non-allergic) subject, and/or a disease reference that represents a level of the proteins associated with allergy e.g., a level in a subject having an allergy, e.g., an anaphylactic allergy. Suitable reference values can include those shown in
As used herein the term “sample”, when referring to the material to be tested for the presence of a biological marker using the method of the invention, includes whole blood, plasma, or serum. The type of sample used may vary depending upon the clinical situation in which the method is used. Various methods are well known within the art for the identification and/or isolation and/or purification of IgE from a sample. In some embodiments, the methods include isolating antigen-specific IgE, e.g., by purifying total IgE, and then enriching antigen/allergen-specific IgE using antigen/allergen-coupled beads.
The presence and/or level of sialylation on IgE can be evaluated using methods known in the art, e.g., using standard electrophoretic and quantitative immunoassay methods, including but not limited to, Western blot; enzyme linked immunosorbent assay (ELISA); radio-immunoassay; immunohistochemistry (IHC); or mass spectrometry (Kim (2010) Am J Clin Pathol 134:157-162; Yasun (2012) Anal Chem 84(14):6008-6015; Brody (2010) Expert Rev Mol Diagn 10(8):1013-1022; Philips (2014) PLOS One 9(3):e90226; Pfaffe (2011) Clin Chem 57(5): 675-687).
In some embodiments, an ELISA method may be used, wherein the wells of a mictrotiter plate are coated with an antibody against which the protein is to be tested. The sample containing or suspected of containing the biological marker is then applied to the wells. After a sufficient amount of time, during which antibody-antigen complexes would have formed, the plate is washed to remove any unbound moieties, and a detectably labelled molecule is added. Again, after a sufficient period of incubation, the plate is washed to remove any excess, unbound molecules, and the presence of the labeled molecule is determined using methods known in the art. Variations of the ELISA method, such as the competitive ELISA or competition assay, and sandwich ELISA, may also be used, as these are well-known to those skilled in the art.
Mass spectrometry, and particularly matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS), is useful for the detection of biomarkers of this invention. (See U.S. Pat. Nos. 5,118,937; 5,045,694; 5,719,060; 6,225,047)
In some embodiments, the presence and/or level of sialylation on IgE is comparable to the presence and/or level of the protein(s) in the disease reference, and the subject has or has had one or more symptoms associated with an allergic reaction, then the subject can be diagnosed with an allergy, e.g., an anaphylactic allergy. In some embodiments, the subject has no overt signs or symptoms of allergy or allergic reaction, but the presence and/or level of sialylation on IgE is comparable to the presence and/or level of the protein(s) in the disease reference, then the subject has an increased risk of developing an allergy, e.g., an anaphylactic allergy. In some embodiments, once it has been determined that a person has an allergy, e.g., an anaphylactic allergy, or has an increased risk of developing an allergy, e.g., an anaphylactic allergy, then a treatment, e.g., as known in the art or as described herein, can be administered.
Suitable reference values can be determined using methods known in the art, e.g., using standard clinical trial methodology and statistical analysis. The reference values can have any relevant form. In some cases, the reference comprises a predetermined value for a meaningful level of sialylation on IgE, e.g., a control reference level that represents a normal level of sialylation on IgE, e.g., a level in an unaffected subject or a subject who is not at risk of developing an allergy as described herein, and/or a disease reference that represents a level of the proteins associated with conditions associated with allergy or anaphylactic allergy, e.g., a level in a subject having an allergy (e.g., an anaphylactic allergy).
The predetermined level can be a single cut-off (threshold) value, such as a median or mean, or a level that defines the boundaries of an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from the other segments. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where association with risk of developing disease or presence of disease in one defined group is a fold higher, or lower, (e.g., approximately 2-fold, 4-fold, 8-fold, 16-fold or more) than the risk or presence of disease in another defined group. It can be a range, for example, where a population of subjects (e.g., control subjects) is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group, or into quartiles, the lowest quartile being subjects with the lowest risk and the highest quartile being subjects with the highest risk, or into n-quantiles (i.e., n regularly spaced intervals) the lowest of the n-quantiles being subjects with the lowest risk and the highest of the n-quantiles being subjects with the highest risk.
In some embodiments, the predetermined level is a level or occurrence in the same subject, e.g., at a different time point, e.g., an earlier time point.
Subjects associated with predetermined values are typically referred to as reference subjects. For example, in some embodiments, a control reference subject does not have a disorder described herein (e.g. an allergy, e.g., an anaphylactic allergy). In some cases it may be desirable that the control subject is non-allergic, and in other cases it may be desirable that a control subject has an allergy, e.g., to a different allergen, or a non-anaphylactic allergy.
A disease reference subject is one who has (or has an increased risk of developing) an allergy, e.g., an anaphylactic allergy. An increased risk is defined as a risk above the risk of subjects in the general population.
Thus, in some cases the level of sialylation on IgE in a subject being less than or equal to a reference level of sialylation on IgE is indicative of a clinical status (e.g., indicative of a disorder as described herein, e.g., an allergy, e.g., an anaphylactic allergy. In other cases the level of sialylation on IgE in a subject being greater than or equal to the reference level of sialylation on IgE is indicative of the absence of disease or normal risk of the disease. In some embodiments, the amount by which the level in the subject is the less than the reference level is sufficient to distinguish a subject from a control subject, and optionally is a statistically significantly less than the level in a control subject. In cases where the level of sialylation on IgE in a subject being equal to the reference level of sialylation on IgE the “being equal” refers to being approximately equal (e.g., not statistically different).
The predetermined value can depend upon the particular population of subjects (e.g., human subjects) selected. For example, an apparently healthy non-allergic population may have a different ‘normal’ range of levels of sialylation on IgE than will a population of subjects which have, are likely to have, or are at greater risk to have, an allergy, e.g., an anaphylactic allergy. Accordingly, the predetermined values selected may take into account the category (e.g., sex, age, health, risk, presence of other diseases) in which a subject (e.g., human subject) falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.
In characterizing likelihood, or risk, numerous predetermined values can be established.
Upon diagnosis with an allergy, e.g., an anaphylactic allergy, the subject can be administered or prescribed a treatment, e.g., avoidance of the allergen, immunotherapy (e.g., oral, sublingual, or subcutaneous immunotherapy, e.g., Sublingual immunotherapy (SLIT)), and/or a pharmacological treatment, e.g., a chronically administered treatment (e.g., corticosteroids, antihistamines, Leukotriene receptor antagonists (LTRAs), Anti-IgE antibody) or an acutely-administered treatment (e.g., epinephrine or a rapid-acting bronchodilator). See, e.g., Min, Allergy Asthma Immunol Res. 2010 April; 2(2): 65-76.
In some embodiments, the methods rely on the observation that Human lgE has seven N-linked glycosylation sites, 5 of which are occupied by complex biantennary glycans. One site is occupied by an oligomannose glycan, and one site is unoccupied. On complex biantennary glycans, sialic acid is attached to galactose. Thus provided herein is an in vitro assay method to determine the pathogenicity of circulating lgEs in allergic humans. The method can include measuring the levels of terminal sialic acid sugar residues or terminal galactose residues on sera lgEs, isolated from said humans, wherein higher levels of sialylation (higher sialylation correlates with less terminal galactose, and vice versa) predict susceptibility to a pathogenic reaction (e.g., anaphylaxis) in said allergic humans. In some embodiments, the in vitro assay is an ELISA in which total lgE is captured, and sialylation levels are quantified by the ratio of the amount of lgE-bound labelled lectin, specific for terminal sialic acid or terminal galactose, normalized to the amount of total anti-lgE detection antibody is bound. In some embodiments, the measurement for the amount of lgE-bound labelled lectin is by, but not limited to, fluorescence or a colorimetric enzymatic reaction. Also provided is an in vitro assay method to determine the pathogenicity to a specific allergen of circulating lgEs in humans; the method can include measuring the levels of sialic acid sugar residues or galactose residues on said lgEs, isolated from the sera of human patients and which bind to a specific allergen, wherein higher levels of sialylation on allergen-bound lgEs predict susceptibility to a pathogenic reaction (e.g. anaphylaxis) to said allergen. In some embodiments, the in vitro assay is an ELISA in which allergen-specific lgE is captured, and sialylation levels are quantified by the ratio of the amount of lgE-bound labelled lectin, specific for terminal sialic acid or terminal galactose, normalized to the amount of total anti-lgE detection antibody is bound. In some embodiments, the measurement for the amount of lgE-bound labelled lectin is by, but not limited to, fluorescence or a colorimetric enzymatic reaction.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
The following materials and methods were used in this Example.
All human samples were collected under IRB approved protocols. Serum samples from MGH IRB approved and consented peanut allergic were collected prior to treatment. Peanut allergy was confirmed by clinical history, allergen-specific IgE screening, and double-blind placebo-controlled oral challenge (PNOIT2, NCT01750879, Table 1). IRB-approved non-atopic adults were recruited (Research Blood Components, Boston, Mass.) on the basis of self-identification as non-allergic donors. Non-atopy was confirmed by clinical history, and allergen-specific IgE screening (Table 1). Total IgE, Ara h 2-specific IgE, Fel d 1-specific IgE, Der p 1-specific IgE, and Bet v 1-specific IgE were determined by ImmunoCap Assay (Phalleon, Thermo Scientific) according to manufacturer's protocols. Primary IgE was enriched from serum samples by serially depleting IgG by protein G agarose (GE Healthcare) followed by anti-IgE conjugated NHS-beads (GE Healthcare). IgE purity was confirmed by protein electrophoresis and coomassie gel staining. Recombinant OVA-specific IgE was generated as described23. Briefly, cDNA sequences for generating OVA-specific heavy ε and light κ chain of mouse and human IgE23 were cloned into pcDNA3.4 using restriction enzyme sites Xbal and AgeI. To generate recombinant OVA-specific mouse or human IgE, plasmids containing OVA-specific heavy and light chain were transiently co-transfected at 1:1 ratio using Expi293 Expression System Kit (Life Technologies) according to the manufacturer's protocol. The cells expressing IgE were selected by addition of 400 μg/mL G418 in the culture media for two weeks and maintained before expanding to a larger scale production. OVA-specific IgE was purified from cell culture supernatant by OVA-coupled agarose beads23.
Sandwich ELISA for quantifying mIgE and OVA-specific binding were conducted as previously described23. Briefly, 96-well Nunc plates were coated with goat polyclonal anti-mouse IgE (Bethyl Laboratories) or OVA and blocked with BSA in PBS (1% BSA for mIgE and 2% for OVA) prior to sample incubation. Samples were probed with goat polyclonal anti-mouse IgE-HRP (2 ng/ml; Bethyl Laboratories) and the reactions were detected by 3,3,5,5-tetramethylbenzidine (TMB; Thermo Fisher Scientific) and stopped by 2 M sulfuric acid, and the absorbance was measured at 450 nm.
Site specific glycosylation was quantified for IgE isolated from non-allergic donors and from peanut allergic donors using nano LC-MS/MS following enzymatic digestion of the proteins as described previously, with minor modifications22-24 (Tables 2, 3).
The isolated polyclonal primary hIgE and myeloma hIgE (Sigma Aldrich AG30P) was prepared for proteolysis by denaturing the protein in 6M guanidine HCl followed by reduction with dithiothreitol and alkylation with iodoacetamide followed by dialysis into 25 mM ammonium bicarbonate pH 7.8. Proteolysis was done with either trypsin to quantify N218, N371 and N394 or chymotrypsin to quantify N140, N168 and N265. For the tryptic digest IgE was incubated with trypsin (Trypsin Gold Promega) at a 1:50 enzyme to substrate ratio overnight at 37 C. For the chymotryptic digest IgE was incubated with chymotrypsin (Sequencing Grade Promega) at a 1:100 enzyme to substrate ratio for 4 hours at 25 C. Both enzymes were quenched with formic acid added to 2% w/w. The separation was performed on a Thermo EasySpray C18 nLC column 0.75 um×50 cm using water and acetonitrile with 0.1% formic acid for mobile phase A and mobile phase B respectively. A linear gradient from 1% to 35% mobile phase B 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. Glycopeptides were quantified based on the extracted ion area of the Y1 ion (
Five- to six-week-old female BALB/c mice were purchased from the Jackson Laboratory and used in these studies. All mice were housed in specific pathogen-free conditions according to the National Institutes of Health (NTH), and all animal experiments were conducted under protocols approved by the MGH IACUC. For all experiments, age- and sex-matched mice were randomized allocating to experimental group, with 4-5 mice per group, and repeated at least three independent times.
Passive Cutaneous Anaphylaxis (PCA) was conducted as previously described23. In brief, monoclonal SiamIgE or AsmIgE specifically for OVA or dinitrophenyl (DNP, clone SPE-7; Sigma-Aldrich) was injected intradermally in the mice ears. For experiments where OVA-specific AsmIgE was added to OVA-specific SiamIgE, a mIgE isotype control (clone MEA-36, Biolegend) was included. The next day mice were intravenously challenged with PBS containing 125 μg OVA (Sigma-Aldrich) or DNP-Human Serum Albumin (DNP-HSA; Sigma-Aldrich) and 2% Evans blue dye in PBS. 45 min after challenge, the ears were excised and minced before incubation in N,N-dimethyl-formamide (EMD Millipore) at 55° C. for 3 h. The degree of blue dye in the ears was quantitated by the absorbance at 595 nm.
Passive Systemic Anaphylaxis (PSA) was elicited as previously described with minor modifications42,43. Briefly, mice were injected intravenously with monoclonal mIgE specific for OVA or DNP (clone SPE-7; Sigma-Aldrich) in PBS and challenged the next day intravenously with PBS containing 1 mg OVA (Sigma-Aldrich) or DNP-HSA (Sigma-Aldrich). For examining the therapeutic potential of AsmIgE, mice that had been injected intravenously with 10 μg DNP-specific mIgE (clone SPE-7; Sigma-Aldrich) the first day were injected intravenously with PBS, 20 μg OVA-specific SiamIgE or 20 μg OVA-specific AsmIgE the next day and challenged with 1 mg DNP-HSA or OVA (Sigma-Aldrich) the third day. For testing the therapeutic potential of NEUFcε, mice injected intravenously with 10 μg OVA-specific mIgE the first day were further injected intravenously with PBS, 100 μg NEUFcε or 100 μg mIgE isotype control (clone MEA-36, Biolegend) the next day and challenged with 1 mg OVA (Sigma-Aldrich) the third day. Core temperature was recorded at the baseline and every 10 min after the allergen challenge by a rectal microprobe thermometer (Physitemp). Histamine in the blood was quantified by histamine enzyme immunoassay kit (SPI-Bio) according to the manufacturer's protocol. Briefly, histamine in the blood was derivatized and incubated with plate precoated with monoclonal anti-histamine antibodies and histamine-AchE tracer at 4° C. for 24 h. The plate was then washed and developed with Ellman's reagent and the absorbance measured at 405 nm.
Passive Food Anaphylaxis (PFA) was elicited by adapting PSA described above. Briefly, mice injected intravenously with 20 μg monoclonal mIgE specific for TNP (clone MEA-36; Biolegend) in PBS the first day were administered with 20 mg TNP-OVA in PBS (Biosearch Technologies) by oral gavage the next day. Core temperature was recorded at the baseline and every 10 min after the challenge by a rectal microprobe thermometer (Physitemp).
To determine in vivo half-lives of SiamIgE or AsmIgE, mice were injected intraperitoneally with 30 μg DNP-specific SiamIgE or AsmIgE and the blood collected at the indicated times after injection into a Microtainer blood collection tube with clot actiator/SST gel (BD Diagonistics). The level of mIgE was quantified by mIgE ELISA described below.
Human LAD2 mast cell line was a generous gift of Dr. Metcalfe (MAID, NIH) and was maintained as previously described2344. Briefly, LAD2 cells were cultured in StemPro-34 SFM medium (Life Technologies) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 100 ng/ml recombinant human stem cell factor (PeproTech). The cells were hemi-depleted each week with fresh medium and maintained at 2-5×105 cells/ml at 37° C. and 5% CO2.
Degranulation assays were performed as previously described (Shade, 2015), LAD2 cells were sensitized overnight with 1 μg/mL OVA-specific hIgE at 1 μg/mL or 50 ng/mL peanut-allergic hIgE. The following day, the cells were pelleted by centrifugation, resuspended in HEPES buffer, plated in 96-well plates, and stimulated with allergen OVA or crude peanut extract at defined concentrations. Upon allergen challenge, mast cell degranulation was determined by the amount of substrate p-nitrophenyl N-acetyl-β-D-glucosamide digested by β-hexosaminidase release from the mast cell granules at the absorbance of 405 nm. To assess the effect of sialic acid removal on IgE-bound mast cells, IgE-sensitized LAD2 cells were treated with NEUFcε, heat-inactivated NEUFcε, mIgE isotype control (clone MEA-36, Biolegend) for 20 min before allergen challenge. To inactivate NEUFcε, the enzyme was heated at 95° C. for 10 min. To determine whether addition of a surrogate asialylated glycoprotein could recapitulate the phenotype of sialic acid removal from IgE, LAD2 cells sensitized with OVA-specific SiahIgE were incubate with sialylated fetuin (SiaFetuin) or asialylated fetuin (AsFetuin) at defined amount for 20 min before allergen challenge.
Unsalted dry-roasted peanuts (Blanched Jumbo Runner cultivar; Planters) were ground to a smooth paste, followed by washing with 20 volumes of cold acetone, filtered using Whatman paper, and dried as previously described23. Protein was extracted by agitating the peanut flour overnight with PBS containing protease inhibitor cocktail without EDTA (Roche). The peanut protein extracts were collected as the supernatant after centrifugation at 24,000×g for 30 min.
To remove sialic acids on IgE, IgE was digested with Glyko Sialidase A (Prozyme) at 37° C. for 72 h according to the manufacturer's instructions. To re-sialylate AsmIgE by in vitro sialylation reaction, AsmIgE was incubated with 5 mM Cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac2; Nacalai USA) in the sialylation buffer (150 mM NaCl, 20 mM HEPES, pH7.4) overnight at room temperature. Following reactions, OVA-specific SiaIgE or AsIgE were purified by OVA-coupled beads to remove glycosylation modifying enzymes as described23. All digestion or sialylation reactions were verified by lectin blotting or HPLC.
Equal amounts of SiaIgE or AsIgE were resolved on 3-8% Tris-Acetate protein gels (Life Technologies) in SDS-PAGE under nonreducing conditions. For protein stain, gels were incubated in AcquaStain Protein Gel Stain (Bulldog Bio) for 1 h at room temperature and destained in distilled water. For lectin blotting, the protocol was conducted as described23. Briefly, after resolved proteins on the gel were transferred to Immobilon-PSQ polyvinylidene difluoride membranes (Millipore Sigma), the membranes were blocked with 0.2% BSA in TBS for 1 hour at room temp, washed in TBS, followed by incubation with biotinylated Sambucus nigra lectin (SNA; 0.4 μg/ml; Vector Laboratories) in TBS with 0.1 M Ca2+ and 0.1 M Mg2+ for 1 hour at room temp to determine the level of terminal α2,6 sialic acids on N-linked glycans of proteins. The membrane was then washed in TBS and incubated with alkaline phosphatase conjugated goat anti-biotin (1:5000 dilution; Vector Laboratories) in TBS for 1 hour at room temp. Sialylated proteins on membranes were visualized by incubation with 1-Step NBT/BCIP plus Suppressor Substrate Solution.
Basophil activation was performed as previously described45. Buffy coats of human blood from healthy, de-identified, consenting donors were obtained from the MGH Blood Transfusion Service. Peripheral blood mononuclear cells (PBMCs) were separated from buffy coats by a density gradient centrifugation using Ficoll Paque Plus (GE Healthcare) and resuspended in 0.5% BSA in RPMI 1640 Media (GE Healthcare). PBMCs were incubated for 2 min with ice-cold lactic acid buffer (13.4 mM lactate, 140 mM NaCl, 5 mM KCl, pH 3.9) to remove endogenous human IgE on the cell surface prior to neutralization by 12% Tris (pH 8). Cells were then washed and incubated 1 hour at 37° C. with 1 μg OVA-specific SiahIgE or AshIgE per 1×106 cells in basophil activation buffer (0.5% BSA, 2 mM CaCl2 and 2 mM MgCl2 in RPMI 1640 Media). Sensitized cells were washed and resuspended in basophil activation buffer supplemented with 10 ng/mL human IL3 (Peprotech) prior to 30 min OVA activation. Activation was stopped by addition of ice-cold 0.2 M EDTA in FACS buffer. Activated cells were washed and resuspended in FACS buffer to proceed antibody staining for basophil activation markers.
Antibodies used for surface allergen staining are listed in Table A. For staining for mouse cells, suspension cells were incubated with anti-mouse CD16/CD32 (clone 2.4G2, BD Biosciences) prior to antibody staining. Cells were incubated in FACS buffer with desired staining antibodies for 20 minutes at 4° C. Cells were then washed in FACS buffer before being acquired by an LSRII flow cytometer (BD Biosciences) or CytoFLEX (Beckman Coulter). Data were analyzed using FlowJo software version 10.4 software (Tree Star). To quantify hIgE loading following sensitization, PBS or 1 μg/mL OVA-specific SiahIgE or AshIgE were incubated with 2.5×105 cells/mL LAD2 mast cells overnight before wash with FACS buffer and stained with OVA-A647. To quantify dermal mast cell IgE loading, single cell suspensions were generated from mouse ears as described23. Ears were intradermally injection of 40 ng OVA-specific SiamIgE or AsmIgE. The following day, ears were removed, separated into dorsal and ventral halves, and minced before incubation in DMEM containing 2% FCS, 1% HEPES, 500 u/mL collagenase type 4 (Worthington), 0.5 mg/mL hyaluronidase (Sigma) and Dnase I (Roche) at 37° C. for 1 h at 180 RPM. The digested sample was then subjected to disruption by Gentle MACS and filtered through a 70 μm cell strainer followed by a 40 μm cell strainer in FACS buffer (2 mM EDTA and 0.5% Bovine Serum Albumins (BSA) in PBS).
Binding kinetics and affinity of protein interaction studies were performed by the Octet K2 system (Molecular Devices) using Octet buffer (PBS with 0.025% Tween and 1% BSA). For measuring hFcεRIα interaction, ligand 0.25 ng/mL His-tagged hFcεRIα (Acro Biosystems) was loaded onto Anti-Penta-HIS (HIS1K) Biosensors (Molecular Devices). For OVA interaction, ligand 100 ng/mL OVA was immobilized onto Amine Reactive Second-Generation (AR2G) Biosensors in 10 mM sodium acetate, pH 5 using EDC/Sulfo-NHS based chemistry. Association of analyte OVA-specific SiahIgE or AshIgE was performed in 3-fold serial dilution from 90 to 1 nM or NEUFcε in 3-fold serial dilution from 24 to 0.3 nM in Octet buffer. Analyte dissociation was measured in Octet buffer. Analysis of binding kinetic parameters were performed by Octet data analysis software 10.0 using interaction of ligand-loaded biosensor with no analyte during association phase as the reference sensor.
1.5×106 LAD2 cells were sensitized with PBS or 1 μg/mL OVA-specific SiahIgE or AshIgE. Sensitized cells were washed and resuspended in HEPES buffer the next day followed by OVA stimulation at 10 ng/mL OVA at 37° C. for indicated times. Cells were immediately centrifuged after OVA stimulation and the cell pellets lysed in ice-cold lysis buffer for 30 min on ice (RIPA buffer (Boston BioProducts), 1× Halt Protease Inhibitor Cocktail (Thermo Scientific), 1× Halt™ Phosphatase Inhibitor Cocktail (Thermo Scientific) and 2.5 mM EDTA). After incubation on ice, lysed pellets were passed rapidly through a 27 G needle on ice and centrifuged at maximal speed at 4° C. for 15 min to clear the membrane and nuclei. The protein concentration was quantified using Pierce BCA Protein Assay kit (Thermo Scientific) and 20 μg of protein lysate was loaded per well on 4-12% Bis-Tris protein gels (Life Technologies) in SDS-PAGE under denaturing and reducing conditions. Briefly, after protein transferred to PVDF membranes described as above, the membranes were blocked with 5% milk in TBS with 0.1% Tween (TBST) for 1 hour at room temp, washed in TBST, followed by incubation with 1:2000 Rabbit anti-Phospho-Syk (Tyr352) Antibody (Cell Signaling Techology) in 5% BSA in TBST overnight at 4° C. The membrane was then washed in TBST before incubating with anti-rabbit-HRP for 1 hour at room temp and washed in TBST again followed by chemiluminescent detection using Immobilon Western Chemiluminescent HRP Substrate (Millipore Sigma). To detect total Syk on the membrane, after chemiluminescent detection using autoradiography film, the membrane was stripped by incubating in stripping buffer (2% SDS and 0.1 M β-mecaptoethanol in Tris buffer) at 50° C. for 30 min. The stripped membranes were then blocked, washed as above and then incubated with 1:2000 Rabbit anti-Syk Antibody (Cell Signaling Techology) for 2 h in 5% BSA in TBST at room temp before incubating with 1:30,000 anti-rabbit-HRP for 1 hour at room temp. To probe for β-Actin, the stripped membranes were incubated with 1:150,000 anti-β-Actin HRP (Santa Cruz Biotechnology) for 1 hour at room temp, washed and signal determined by chemiluminescent detection.
5×105 LAD2 cells were sensitized overnight with PBS or 500 ng/mL OVA-specific SiahIgE or AshIgE. Next day, sensitized cells were washed before loading with 2 μM Fluo-4-AM (Invitrogen) at 37° C. in HEPES buffer for 20 minutes. After loading, the cells were washed and resuspended in HEPES buffer. Fluorescence was filtered through the 530/30 band pass filter and collected in FL-1/FITC. Baseline Ca2+ fluorescence levels were recorded for 1 minute on the Accuri C6 (BD Biosciences) before the addition of indicated allergen or buffer to each sample. At the end of allergen stimulation, cells were added 2 μM Ca2+ ionophore A23187 (Sigma) as a positive control.
The neuraminidase fusion protein was designed by fusing a kappa light chain secretion signal sequence and the sialidase gene from Arthrobacter urefaciens (EC 3.2.1.18, gene AU104)46. Stop codon of the AU104 was omitted, instead, a short flexible linker peptide (GGGGGG), mouse IgE Cε2, Cε3, Cε4, and His6-tag was inserted to the C-terminus of the sialidase. The gene was codon optimized for human and synthesized by GenScript. The protein of 288 kDa was then produced by WuXi biologics. Sialidase activity of NEUFcε was determined by the level of p-nitrophenol released from 250 μM 2-O-(p-Nitrophenyl)-α-D-N-acetylneuraminic acid (Sigma) in 100 mM sodium phosphate (pH 5.5) for 10 min at 37° C. The reaction was terminated by adding 0.5 M sodium carbonate and the absorbance quantified at 405 nm.
All statistical analyses were performed using Prism 8 (GraphPad Software), and results are shown as means with SEM. Un-paired and paired Student's t test were used for parametric comparisons of two unmatched and matched groups, respectively. For comparisons of two sample groups of multiple non-parametric conditions, a two-way ANOVA with Sidak's multiple comparison test was used. For parametric comparison between three or more groups, one-way or two-way ANOVA with Tukey's multiple comparison test was used. Accuracy of individual IgE glycan moieties capacity to distinguish allergic IgE was analyzed by receiver operating characteristic (ROC) curves. Area under each ROC curve (AUC) was calculated for each glycan moiety. AUC was interpreted as follows, where a maximum AUC of 1 indicates the specific glycan moiety is able to distinguish allergic IgE from non-allergic IgE. An AUC of 0.5 indicates the differentiation capacity of a specific glycan moiety is poor.
The present study examined whether allergic disease-specific glycosylation patterns existed for IgE, and if so, whether those patterns influenced IgE biological activity. Non-atopic adults reported no history of atopy, had low total IgE titers, and had little IgE reactivity to peanut allergen (Ara h 2), birch tree pollen allergen (Bet v 1), house dust mite allergen (Der p 1), or cat allergen (Fel d 1) (
Next, the N-glycan residues on total IgE enriched from non-atopic and allergic serum was analyzed by mass spectrometry22,24,27. This revealed similar numbers of mannose moieties between total non-atopic and allergic IgE (
To determine whether the differences in glycan residues on total IgE were predictive of allergic disease, we assessed the variable glycan content on non-atopic and allergic IgE using Receiver Operating Characteristics (ROC) curves (
These findings raised the possibility that sialic acid content modifies IgE effector functions. Indeed, sialylation has been implicated in regulating most other antibody classes, including IgG1 anti-inflammatory activity, IgA neuropathy and influenza neutralization, and IgM-induced inhibitory signaling on B and T cells28-32. However, the role for sialylation in modulating IgE function has not been described. Sialic acid was attached in α2,6 linkages on hIgE and mouse IgE (mIgE) as determined by neuraminidase (NEU) digestion assays and lectin blotting (
Next, we systemically sensitized mice with SiamIgE, AsmIgE, or PBS and challenged with allergen the following day in a model of passive systemic anaphylaxis (PSA). SiamIgE-sensitized mice elicited a robust anaphylactic response underscored by 3° C. loss in temperature 20 minutes after allergen challenge (
We next asked whether sialylation similarly regulated hIgE. We sensitized human LAD2 mast cells with PBS, sialylated or asialylated human IgE (SiahIgE and AshIgE, respectively,
Because sialylation does not alter IgE interactions to allergen and receptor, we tested whether signaling downstream of FcεRI was affected by IgE sialylation. LAD2 mast cells sensitized with SiahIgE or AshIgE were stimulated with allergen and cellular lysates collected at defined intervals. Western blotting of mast cell lysates for Syk revealed reduced phosphorylation at 5 and 30 minutes after stimulation (
The observation that an asialylated glycoprotein could inhibit mast cell degranulation in vitro raised the possibly that AsIgE could actively inhibit anaphylaxis in vivo. We therefore sensitized mice intradermally in the ears with PBS, OVA-specific SiamIgE, a combination of OVA-specific SiamIgE and ten-fold more OVA-specific AsmIgE, or a combination of OVA-specific SiamIgE and ten-fold more TNP-specific SiamIgE isotype control. The next day mice were challenged with OVA and blue coloration of the ears quantified. Extensive vascular leakage occurred in ears sensitized with OVA-specific SiamIgE alone (
As sialic acid removal attenuated IgE effector functions, we explored whether targeting sialic acid on IgE-bearing cells is a viable strategy for attenuating allergic inflammation. Thus, we genetically fused a neuraminidase to the N-terminus of IgE Fc CE2-4 domains (NeuFcε,
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.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/808,449, filed on Feb. 21, 2019, and 62/808,456, filed on Feb. 21, 2019. The entire contents of the foregoing are hereby incorporated by reference.
This invention was made with Government support under Grant Nos. AR068272 and AI139669 awarded by the National Institutes of Health (NIH). The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/018380 | 2/14/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62808456 | Feb 2019 | US | |
| 62808449 | Feb 2019 | US |
