Compositions and methods for neutralizing antigens

Abstract

Compositions and methods for neutralizing allergens or treating environmental allergies are provided. These compositions and methods can reduce, minimize, or prevent allergic reactions to environmental allergens.

Description

[Technical Field]

[0001] cross reference This application claims the benefits of U.S. Provisional Patent Application No. 63 / 496,508, filed on 17 April 2023, which is incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in an XML file and incorporated herein by reference. This XML file, created on 2 April 2024, is named 65491-701_601_SL.xml and has a size of 70,130 bytes. [Background technology]

[0003] Many people suffer from allergies to animals, plants, or dust mites, particularly other allergies or asthma. Despite efforts to develop approaches to reduce, minimize, or prevent allergic responses to animal allergens, the need to overcome the inherent limitations of conventional methods at a reasonable cost remains unmet. This disclosure addresses these needs and provides relevant advantages. [Overview of the Initiative]

[0004] This specification provides allergen-binding proteins that bind to or neutralize allergens, and which include nanobodies, monobodies, DARPins, or small peptides having a molecular weight of less than about 25 kilodaltons (kDa) or a length of less than about 300 amino acids.

[0005] Furthermore, this specification also provides allergen-binding proteins that bind to or neutralize allergens, comprising nanobodies or small peptides having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1-57 and 67-73.

[0006] In some embodiments, allergen-binding proteins are formulated for food coating or for spraying, misting, or brushing onto animal or household surfaces.

[0007] In some embodiments, the food is pet food.

[0008] In some embodiments, the allergen is a pet allergen.

[0009] In some embodiments, the allergen-binding protein is formulated to be applied as a topping to food.

[0010] In some embodiments, the allergen-binding protein is formulated to be mixed with food.

[0011] In some embodiments, the allergen-binding protein is present in solution or after suspension at a concentration of approximately 0.01 milligrams / milliliter (mg / ml) to approximately 500 mg / ml.

[0012] In some embodiments, the allergen induces an allergic reaction in humans.

[0013] In some embodiments, the allergen includes environmental allergens.

[0014] In some embodiments, the allergen includes animal allergens.

[0015] In some embodiments, the allergen includes pet allergens.

[0016] In some embodiments, the allergen includes allergens of cats, dogs, rabbits, mice, or cockroaches.

[0017] In some embodiments, the allergen is selected from the group consisting of Fel d 1, Fel d 2, Fel d 3, Fel d 4, Can f 1, Can f 2, Can f 4, Can f 7, Ory C 1, Mus M 1, and Bla G 2.

[0018] In some embodiments, the allergen is Fel d 1.

[0019] In some embodiments, the allergen-binding protein includes a nanobody having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 49 - 57.

[0020] In some embodiments, the allergen-binding protein includes a nanobody having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 49.

[0021] In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 49.

[0022] In some embodiments, the allergen-binding protein includes a nanobody having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 57.

[0023] In some embodiments, the allergen-binding protein comprises a nanobody having the sequence of SEQ ID NO: 57.

[0024] In some embodiments, the allergen is Can f 1.

[0025] In some embodiments, the allergen-binding protein comprises a nanobody comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 67-73.

[0026] In some embodiments, the allergen-binding protein comprises a nanobody comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 67.

[0027] In some embodiments, the allergen-binding protein comprises a nanobody having the sequence consisting of SEQ ID NO: 67.

[0028] In some embodiments, the allergen is Can f 2.

[0029] In some embodiments, the allergen-binding protein comprises a nanobody comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 9-23.

[0030] In some embodiments, the allergen-binding protein comprises a nanobody comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 16.

[0031] In some embodiments, the allergen-binding protein includes a nanobody containing the amino acid sequence of SEQ ID NO: 16.

[0032] In some embodiments, the allergen includes dust allergens.

[0033] In some embodiments, the dust allergen includes Der p1 or Der p2.

[0034] In some embodiments, the dust allergen is Der p1.

[0035] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 24-34.

[0036] In some embodiments, the allergen-binding protein comprises a peptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 35 to 48.

[0037] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with the sequence described in SEQ ID NO: 27.

[0038] In some embodiments, the allergen-binding protein includes a nanobody having the sequence SEQ ID NO: 27.

[0039] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO: 28.

[0040] In some embodiments, the allergen-binding protein includes a nanobody having the sequence SEQ ID NO: 28.

[0041] In some embodiments, the dust allergen is Der p2.

[0042] In some embodiments, the allergen-binding protein includes nanobodies having at least 80%, at least 85%, at least 90%, or 100% sequence identity with respect to a sequence selected from the group consisting of SEQ ID NOs: 1 to 8.

[0043] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with the sequence described in SEQ ID NO: 3.

[0044] In some embodiments, the allergen-binding protein includes a nanobody having the sequence corresponding to SEQ ID NO: 3.

[0045] In some embodiments, the allergen includes plant or plant pollen allergens.

[0046] In some embodiments, the plant or pollen allergen is selected from the group consisting of Bet v1, Phl p 5, Phl p 1, Poa p 1, Cyn d 1, Bet v 2, Ol e 1, Amb a 1, Amb a 11, and Art v 1.

[0047] In some embodiments, the allergen includes mold allergens.

[0048] In some embodiments, the mold allergen is selected from the group consisting of Alt a 1, Asp f 1, Asp f 2, Cla h 8, Pen ch 13, and Pen ch 18.

[0049] In some embodiments, the allergen is a food allergen.

[0050] In some embodiments, the food allergen is selected from the group consisting of Pen a 1, Ara h 1, and Ara h 3.

[0051] In some embodiments, the allergen-binding protein includes at least one modified amino acid.

[0052] In some embodiments, the allergen-binding protein contains about 1, about 5, about 10, about 15, about 20, about 25, or about 30 modified amino acids.

[0053] In some embodiments, the modified amino acids include non-standard amino acids.

[0054] In some embodiments, the non-standard amino acid is selected from the group consisting of para-benzoylphenylalanine, 3,4-dihydroxyphenylalanine, tetrazine, chlorooctene, homopropagylglycine, para-propagyloxyphenylalanine, para-azidophenylalanine, para-isothiocyanatephenylalanine, para-benzoylphenylalanine, para-cyanophenylalanine, para-nitrophenylalanine, m-halogenated tyrosine analogs, halogenated proline analogs, halogenated tryptophan analogs, and halogenated leucine analogs.

[0055] In some embodiments, the solubility of the allergen-binding protein increases by approximately 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, or more compared to the unmodified allergen-binding protein.

[0056] In some embodiments, the binding affinity of the allergen-binding protein is increased by approximately 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or more, compared to the unmodified allergen-binding protein.

[0057] In some embodiments, the thermal stability of the allergen-binding protein is increased by approximately 10%, 25%, 50%, 75%, 100%, 200%, 300%, or more compared to the unmodified allergen-binding protein.

[0058] In some embodiments, the allergen-binding protein is a polyvalent allergen-binding protein.

[0059] In some embodiments, the polyvalent allergen-binding protein is a bivalent allergen-binding protein.

[0060] In some embodiments, the binding affinity of the polyvalent allergen-binding protein is increased by approximately 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or more compared to the monovalent allergen-binding protein.

[0061] In some embodiments, the allergen-binding protein further comprises a carrier.

[0062] In some embodiments, the carrier includes a solvent, diluent, dispersion medium, or coating.

[0063] In some embodiments, the carrier includes silica.

[0064] In some embodiments, the allergen-binding protein is in a dry form.

[0065] In some embodiments, the carrier contains water.

[0066] In some embodiments, the allergen-binding protein contains a liquid.

[0067] In some embodiments, the allergen-binding protein further comprises a preservative.

[0068] In some embodiments, the preservative includes potassium sorbate, EDTA, benzoic acid, phenoxyethanol, or maltol.

[0069] In some embodiments, the allergen-binding protein further comprises a stabilizer or a thickener.

[0070] In some embodiments, the stabilizer or thickener includes dextrin, maltodextrin, glycerol, glucose, sucrose, or trehalose.

[0071] In some embodiments, the allergen-binding protein further comprises an isotonic agent.

[0072] In some embodiments, the allergen-binding protein is suspended or resuspended in a solvent.

[0073] In some embodiments, the solvent includes water.

[0074] The Specified also provides compositions comprising an allergen-binding protein that binds to or neutralizes an allergen, wherein the allergen-binding protein comprises a nanobody, monobody, DARPin, or small peptide having a molecular weight of less than about 25 kilodaltons or a length of less than about 300 amino acids.

[0075] The Specified also provides compositions comprising an allergen-binding protein that binds to or neutralizes an allergen, wherein the allergen-binding protein comprises a nanobody or small peptide with a molecular weight of less than about 25 kilodaltons or a length of less than about 300 amino acids, and the allergen-binding protein comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1-57 and 67-73.

[0076] In some embodiments, the composition is formulated for coating food, or for spraying, misting, or brushing onto animal or household surfaces.

[0077] In some embodiments, the food is pet food.

[0078] In some embodiments, the allergen is a pet allergen.

[0079] In some embodiments, the composition is formulated to be applied as a topping to food.

[0080] In some embodiments, the composition is formulated to be mixed with food.

[0081] In some embodiments, the allergen-binding protein is present in solution or after suspension at a concentration of approximately 0.01 milligrams / milliliter (mg / ml) to approximately 500 mg / ml.

[0082] In some embodiments, the allergen induces an allergic reaction in humans.

[0083] In some embodiments, the allergen includes environmental allergens.

[0084] In some embodiments, the allergen includes animal allergens.

[0085] In some embodiments, the allergen includes pet allergens.

[0086] In some embodiments, the allergens include cat, dog, rabbit, mouse, or cockroach allergens.

[0087] In some embodiments, the allergen is selected from the group consisting of Fel d 1, Fel d 2, Fel d 3, Fel d 4, Can f 1, Can f 2, Can f 4, Can f 7, Ory C 1, Mus M 1, and Bla G 2.

[0088] In some embodiments, the allergen is Fel d 1.

[0089] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 49 to 57.

[0090] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 49.

[0091] In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 49.

[0092] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 57.

[0093] In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 57.

[0094] In some embodiments, the allergen is Can f 1.

[0095] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 67-73.

[0096] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO: 67.

[0097] In some embodiments, the allergen-binding protein includes a nanobody having the sequence SEQ ID NO: 67.

[0098] In some embodiments, the allergen is Can f 2.

[0099] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 9 to 23.

[0100] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with the sequence described in SEQ ID NO: 16.

[0101] In some embodiments, the allergen-binding protein includes a nanobody containing the amino acid sequence of SEQ ID NO: 16.

[0102] In some embodiments, the allergen includes dust allergens.

[0103] In some embodiments, the dust allergen includes Der p1 or Der p2.

[0104] In some embodiments, the dust allergen is Der p1.

[0105] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 24-34.

[0106] In some embodiments, the allergen-binding protein comprises a peptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 35 to 48.

[0107] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with the sequence described in SEQ ID NO: 27.

[0108] In some embodiments, the allergen-binding protein includes a nanobody having the sequence SEQ ID NO: 27.

[0109] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO: 28.

[0110] In some embodiments, the allergen-binding protein includes a nanobody having the sequence SEQ ID NO: 28.

[0111] In some embodiments, the dust allergen is Der p2.

[0112] In some embodiments, the allergen-binding protein includes nanobodies having at least 80%, at least 85%, at least 90%, or 100% sequence identity with respect to a sequence selected from the group consisting of SEQ ID NOs: 1 to 8.

[0113] In some embodiments, the allergen-binding protein includes a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with the sequence described in SEQ ID NO: 3.

[0114] In some embodiments, the allergen-binding protein includes a nanobody having the sequence corresponding to SEQ ID NO: 3.

[0115] In some embodiments, the allergen includes plant or plant pollen allergens.

[0116] In some embodiments, the plant or pollen allergen is selected from the group consisting of Bet v1, Phl p 5, Phl p 1, Poa p 1, Cyn d 1, Bet v 2, Ol e 1, Amb a 1, Amb a 11, and Art v 1.

[0117] In some embodiments, the allergen includes mold allergens.

[0118] In some embodiments, the mold allergen is selected from the group consisting of Alt a 1, Asp f 1, Asp f 2, Cla h 8, Pen ch 13, and Pen ch 18.

[0119] In some embodiments, the allergen is a food allergen.

[0120] In some embodiments, the food allergen is selected from the group consisting of Pen a 1, Ara h 1, and Ara h 3.

[0121] In some embodiments, the allergen-binding protein includes at least one amino acid modification.

[0122] In some embodiments, the allergen-binding protein includes about 1, about 5, about 10, about 15, about 20, about 25, or about 30 amino acid modifications.

[0123] In some embodiments, amino acid modification includes the introduction of non-standard amino acids.

[0124] In some embodiments, the non-standard amino acid is selected from the group consisting of para-benzoylphenylalanine, 3,4-dihydroxyphenylalanine, tetrazine, chlorooctene, homopropagylglycine, para-propagyloxyphenylalanine, para-azidophenylalanine, para-isothiocyanatephenylalanine, para-benzoylphenylalanine, para-cyanophenylalanine, para-nitrophenylalanine, m-halogenated tyrosine analogs, halogenated proline analogs, halogenated tryptophan analogs, and halogenated leucine analogs.

[0125] In some embodiments, the solubility of the composition increases by approximately 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, or more, compared to unmodified allergen-binding proteins.

[0126] In some embodiments, the binding affinity of the allergen-binding protein is increased by approximately 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or more, compared to the unmodified allergen-binding protein.

[0127] In some embodiments, the thermal stability of the allergen-binding protein is increased by approximately 10%, 25%, 50%, 75%, 100%, 200%, 300%, or more compared to the unmodified allergen-binding protein.

[0128] In some embodiments, the allergen-binding protein is a polyvalent allergen-binding protein.

[0129] In some embodiments, the polyvalent allergen-binding protein is a bivalent allergen-binding protein.

[0130] In some embodiments, the binding affinity of the polyvalent allergen-binding protein composition is increased by approximately 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or more compared to the monovalent allergen-binding protein.

[0131] In some embodiments, the composition further comprises a carrier.

[0132] In some embodiments, the carrier includes a solvent, diluent, dispersion medium, or coating.

[0133] In some embodiments, the carrier includes silica.

[0134] In some embodiments, the composition is in a dry form.

[0135] In some embodiments, the carrier contains water.

[0136] In some embodiments, the composition includes a liquid.

[0137] In some embodiments, the composition further includes a preservative.

[0138] In some embodiments, the preservative includes potassium sorbate, EDTA, benzoic acid, phenoxyethanol, or maltol.

[0139] In some embodiments, the composition further comprises a stabilizer or a thickener.

[0140] In some embodiments, the stabilizer or thickener includes dextrin, maltodextrin, glycerol, glucose, sucrose, or trehalose.

[0141] In some embodiments, the composition further comprises an isotonic agent.

[0142] In some embodiments, the composition is suspended or resuspended in a solvent.

[0143] In some embodiments, the solvent includes water.

[0144] Furthermore, this specification also provides a method comprising the steps of spraying or atomizing an allergen-binding protein or a composition described in any one of the embodiments described above, or bringing a surface into contact with a composition described in any one of the embodiments described above.

[0145] In some embodiments, the contact step includes coating or brushing.

[0146] In some embodiments, the surface includes the surface of an air filter or humidifier.

[0147] In some embodiments, the surface contains allergens.

[0148] In some embodiments, the composition neutralizes allergens on a surface.

[0149] In some embodiments, the surface contains food.

[0150] In some embodiments, the food is ingested by an animal that is the source of the antigen.

[0151] In some embodiments, the surface includes pet accessories (e.g., collars or brushes) or areas / products where animals can sit or walk (e.g., dog beds).

[0152] In some embodiments, spraying is performed by a humidifier or a spray bottle.

[0153] In some embodiments, spraying approximately 0.01 milliliters (ml) to 20 ml of the composition per area of ​​approximately 1 square meter neutralizes the allergenicity of the antigen.

[0154] Furthermore, this specification also provides a method for preparing an allergen-binding protein or a composition described in any one of the embodiments described above, the method comprising the step of collecting the allergen-binding protein from an engineered microorganism or from a secretion by a microorganism, wherein the microorganism comprises a heterologous nucleic acid encoding the allergen-binding protein.

[0155] In some embodiments, the method further includes the step of incorporating a heterologous nucleic acid encoding an allergen-binding protein into a cell-free protein expression system.

[0156] In some embodiments, the microorganisms include yeast or bacteria.

[0157] In some embodiments, the microorganisms are bacteria, including Escherichia coli (E. coli).

[0158] In some embodiments, the microorganism is a yeast, including Pichia pastoris.

[0159] In some embodiments, allergen-binding proteins are purified or concentrated from microbial secretions.

[0160] In some embodiments, purification or concentration includes a filtration step.

[0161] In some embodiments, the filtration step includes passing the secretion through a filter of approximately 0.01 nm, 0.1 nm, 1 nm, 5 nm, or larger.

[0162] In some embodiments, this method does not include a centrifugal separation step.

[0163] Furthermore, this specification also provides a method for treating an allergy to a subject requiring allergy treatment, the method comprising the step of administering an allergen-binding protein or a composition described in any one of the embodiments described above to the subject.

[0164] Furthermore, this specification also provides a method for treating an allergy in a subject requiring allergy treatment, comprising the step of administering to the subject a therapeutically effective amount of a microorganism manipulated to produce an allergen-binding protein or a composition described in any one of the embodiments described above.

[0165] In some embodiments, the microorganism is yeast or bacteria.

[0166] In some embodiments, the microorganisms are bacteria, including Escherichia coli (E. coli).

[0167] In some embodiments, the microorganism is a yeast, including Pichia pastoris.

[0168] In some embodiments, the microorganisms are administered orally to the subject.

[0169] In some embodiments, microorganisms produce allergen-binding proteins or compositions when ingested.

[0170] Furthermore, this specification also provides a method for neutralizing an allergen, comprising the steps of (i) aerosolizing an allergen-binding protein or a composition described in any one of the embodiments described above into a mist, and (ii) contacting the mist with a surface containing the allergen.

[0171] In some embodiments, the aerosolization process is carried out by an aerosolizing machine.

[0172] In some embodiments, the aerosolizing machine is worn by the subject.

[0173] In some embodiments, the surface includes pet accessories or areas / products on which animals can sit or walk.

[0174] Furthermore, this specification also provides a method for neutralizing an allergen, comprising the steps of (i) aerosolizing an allergen-binding protein or a composition described in any one of the embodiments described above into a mist, and (ii) contacting a food with the mist.

[0175] In some embodiments, the food is pet food.

[0176] In some embodiments, the allergen is a pet allergen.

[0177] In some embodiments, the mist is applied to food as a topping.

[0178] In some embodiments, the mist is mixed into the food.

[0179] Furthermore, this specification also provides a method for treating an allergy to a subject requiring allergy treatment, comprising the step of aerosolizing an allergen-binding protein or a composition described in any one of the embodiments described above into a mist.

[0180] In some embodiments, the aerosolization process is carried out by an aerosolizing machine.

[0181] In some embodiments, the aerosolizing machine is worn.

[0182] In some embodiments, the aerosolizing machine is worn by the subject.

[0183] In some embodiments, the method further includes the inhalation of mist by the subject.

[0184] In some embodiments, the subject is human.

[0185] In some embodiments, the subject is a non-human animal.

[0186] Furthermore, this specification also provides pharmaceutical compositions comprising (i) an allergen-binding protein described in any one of the embodiments described above or a composition described in any one of the embodiments described above, and (ii) a pharmaceutically acceptable excipient.

[0187] Furthermore, this specification also provides allergen-binding proteins containing any one of the sequences 1-57 or 67-73.

[0188] Furthermore, this specification also provides allergen-binding proteins containing the sequence of Sequence ID No. 3.

[0189] This specification also provides allergen-binding proteins containing the sequence of SEQ ID NO: 16.

[0190] This specification also provides an allergen-binding protein containing the sequence of SEQ ID NO: 27.

[0191] This specification also provides an allergen-binding protein containing the sequence of Sequence ID No. 28.

[0192] This specification also provides an allergen-binding protein containing the sequence of SEQ ID NO: 49.

[0193] This specification also provides allergen-binding proteins containing the sequence of SEQ ID NO: 57. This specification also provides allergen-binding proteins containing the sequence of SEQ ID NO: 67.

[0194] Additional aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the present disclosure. As will be understood, other embodiments and different embodiments are possible of the present disclosure, and some of its details can be modified in various obvious ways without departing from the present disclosure. Accordingly, the drawings and description are illustrative in nature and should not be considered limiting.

[0195] Reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as when each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. [Brief explanation of the drawing]

[0196] Novel features of the present invention are specifically stated in the appended claims. The features and advantages of the present invention will be better understood by referring to the following detailed description, which specifies exemplary embodiments in which the principles of the present invention are utilized, and to the appended drawings (also referred to herein as "Figure" and "FIG.").

[0197] [Figure 1]This is a schematic diagram of a functional assay according to one or more embodiments of the present disclosure. [Figure 2] This figure shows SDS-PAGE Western blot images of recombinant His-tagged Der p2 protein according to one or more embodiments of the present disclosure. [Figure 3A] This figure shows results from a Der p2 yeast display VHH library showing sequence alignments of 50 randomly selected clones according to one or more embodiments of the present disclosure. [Figure 3B] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 3C] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 3D] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 3E] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 3F] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 3G] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 3H] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 3I]This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 4] This figure shows SDS-PAGE Western blot images of recombinant His-tagged Can f1 protein according to one or more embodiments of the present disclosure. [Figure 5A] This figure shows results from a Can f1 yeast display VHH library, illustrating the sequence alignment of 50 randomly selected clones according to one or more embodiments of the present disclosure. [Figure 5B] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f1 VHH clones according to one or more embodiments of the present disclosure. [Figure 5C] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f1 VHH clones according to one or more embodiments of the present disclosure. [Figure 5D] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f1 VHH clones according to one or more embodiments of the present disclosure. [Figure 5E] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f1 VHH clones according to one or more embodiments of the present disclosure. [Figure 5F] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f1 VHH clones according to one or more embodiments of the present disclosure. [Figure 5G] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f1 VHH clones according to one or more embodiments of the present disclosure. [Figure 5H] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f1 VHH clones according to one or more embodiments of the present disclosure. [Figure 6]This figure shows SDS-PAGE Western blot images of recombinant His-tagged Can f2 protein according to one or more embodiments of the present disclosure. [Figure 7A] This figure shows results from a Can f2 yeast display VHH library, illustrating the sequence alignment of 50 randomly selected clones according to one or more embodiments of the present disclosure. [Figure 7B] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7C] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7D] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7E] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7F] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7G] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7H] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7I] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7J]This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7K] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7L] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7M] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7N] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7O] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 7P] This figure shows binding curves obtained from ELISA assays performed using identified anti-Can f2 VHH clones according to one or more embodiments of the present disclosure. [Figure 8] This figure shows SDS-PAGE Western blot images of recombinant His-tagged Der p1 protein according to one or more embodiments of the present disclosure. [Figure 9A] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 9B] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 9C]This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 9D] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 9E] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 9F] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 9G] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 9H] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 9I] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 9J] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 9K] This figure shows binding curves obtained from ELISA assays performed using identified anti-Der p2 VHH clones according to one or more embodiments of the present disclosure. [Figure 10] This figure shows the results of a Der p2 activity assay when Der p2 is incubated with a designated anti-Der p2 VHH clone, according to one or more embodiments of the present disclosure. [Figure 11A]This figure shows the results of a Der p2 activity assay when various concentrations of Der p2 were incubated with a specified anti-Der p2 VHH clone, according to one or more embodiments of the present disclosure. [Figure 11B] This figure shows the results of a Der p2 activity assay when various concentrations of Der p2 were incubated with a specified anti-Der p2 VHH clone, according to one or more embodiments of the present disclosure. [Figure 11C] This figure shows the results of a Der p2 activity assay when various concentrations of Der p2 were incubated with a specified anti-Der p2 VHH clone, according to one or more embodiments of the present disclosure. [Figure 11D] This figure shows the results of a Der p2 activity assay when various concentrations of Der p2 were incubated with a specified anti-Der p2 VHH clone, according to one or more embodiments of the present disclosure. [Figure 11E] This figure shows the results of a Der p2 activity assay when various concentrations of Der p2 were incubated with a specified anti-Der p2 VHH clone, according to one or more embodiments of the present disclosure. [Figure 11F] This figure shows the results of a Der p2 activity assay when various concentrations of Der p2 were incubated with a specified anti-Der p2 VHH clone, according to one or more embodiments of the present disclosure. [Figure 11G] This figure shows the results of a Der p2 activity assay when various concentrations of Der p2 were incubated with a specified anti-Der p2 VHH clone, according to one or more embodiments of the present disclosure. [Figure 11H] This figure shows the results of a Der p2 activity assay when various concentrations of Der p2 were incubated with a specified anti-Der p2 VHH clone, according to one or more embodiments of the present disclosure. [Figure 12]This figure shows the results from a Der p1 phage display 7-amino acid peptide library, illustrating the sequence alignment of the obtained peptides according to one or more embodiments of the present disclosure. [Figure 13] This figure shows binding curves obtained from an ELISA assay performed using the identified peptide clone 2-D3 according to one or more embodiments of the present disclosure. [Figure 14] This figure shows SDS-PAGE Western blot images of recombinant His-tagged Fel d1 protein according to one or more embodiments of the present disclosure. [Figure 15A] This figure shows results from a Fel d1 yeast display VHH library, illustrating the sequence alignment of 50 randomly selected clones, according to one or more embodiments of the present disclosure. [Figure 15B] This figure shows binding curves obtained from ELISA assays performed using identified anti-Fel d1 VHH clones specific to the Fel d1 epitope ENARILKNCVDAKM (SEQ ID NO: 61) according to one or more embodiments of the present disclosure. [Figure 15C] This figure shows binding curves obtained from ELISA assays performed using identified anti-Fel d1 VHH clones specific to the Fel d1 epitope ENARILKNCVDAKM (SEQ ID NO: 61) according to one or more embodiments of the present disclosure. [Figure 15D] This figure shows binding curves obtained from ELISA assays performed using identified anti-Fel d1 VHH clones specific to the Fel d1 epitope ENARILKNCVDAKM (SEQ ID NO: 61) according to one or more embodiments of the present disclosure. [Figure 16] This figure shows binding curves obtained from ELISA assays performed using identified anti-Fel d1 VHH clones specific to the Fel d1 epitope FAVANGNELLLDLS (SEQ ID NO: 59) according to one or more embodiments of the present disclosure. [Figure 17A]This figure shows binding curves obtained from ELISA assays performed using identified anti-Fel d1 VHH clones specific to the Fel d1 epitope AKMTEEDKENALS (SEQ ID NO: 60) according to one or more embodiments of the present disclosure. [Figure 17B] This figure shows binding curves obtained from ELISA assays performed using identified anti-Fel d1 VHH clones specific to the Fel d1 epitope AKMTEEDKENALS (SEQ ID NO: 60) according to one or more embodiments of the present disclosure. [Figure 17C] This figure shows binding curves obtained from ELISA assays performed using identified anti-Fel d1 VHH clones specific to the Fel d1 epitope AKMTEEDKENALS (SEQ ID NO: 60) according to one or more embodiments of the present disclosure. [Figure 17D] This figure shows binding curves obtained from ELISA assays performed using identified anti-Fel d1 VHH clones specific to the Fel d1 epitope AKMTEEDKENALS (SEQ ID NO: 60) according to one or more embodiments of the present disclosure. [Figure 17E] This figure shows binding curves obtained from ELISA assays performed using identified anti-Fel d1 VHH clones specific to the Fel d1 epitope AKMTEEDKENALS (SEQ ID NO: 60) according to one or more embodiments of the present disclosure. [Figure 18] This figure shows the binding curves obtained from an ELISA assay performed using an identified anti-Fel d1 VHH clone specific to the Fel d1 epitope VAQYKALPVVLENA (SEQ ID NO: 58). [Figure 19A] This figure shows the results obtained from a thermal stability assay performed using an identified anti-Fel d1 VHH clone according to one or more embodiments of the present disclosure. [Figure 19B] This figure shows the results obtained from a thermal stability assay performed using an identified anti-Fel d1 VHH clone according to one or more embodiments of the present disclosure. [Figure 19C]This figure shows the results obtained from a thermal stability assay performed using an identified anti-Fel d1 VHH clone according to one or more embodiments of the present disclosure. [Figure 19D] This figure shows the results obtained from a thermal stability assay performed using an identified anti-Fel d1 VHH clone according to one or more embodiments of the present disclosure. [Figure 19E] This figure shows the results obtained from a thermal stability assay performed using an identified anti-Fel d1 VHH clone according to one or more embodiments of the present disclosure. [Figure 19F] This figure shows the results obtained from a thermal stability assay performed using an identified anti-Fel d1 VHH clone according to one or more embodiments of the present disclosure. [Figure 19G] This figure shows the results obtained from a thermal stability assay performed using an identified anti-Fel d1 VHH clone according to one or more embodiments of the present disclosure. [Figure 19H] This figure shows the results obtained from a thermal stability assay performed using an identified anti-Fel d1 VHH clone according to one or more embodiments of the present disclosure. [Figure 19I] This figure shows the results obtained from a thermal stability assay performed using an identified anti-Fel d1 VHH clone according to one or more embodiments of the present disclosure. [Figure 20A] This figure shows a size exclusion chromatography (SEC) graph obtained by testing the neutralization ability of anti-Fel d1 VHH clone C3 according to one or more embodiments of the present disclosure. [Figure 20B] This figure shows a size exclusion chromatography (SEC) graph obtained by testing the neutralization ability of anti-Fel d1 VHH clone C3 according to one or more embodiments of the present disclosure. [Figure 20C] This figure shows a size exclusion chromatography (SEC) graph obtained by testing the neutralization ability of anti-Fel d1 VHH clone C3 according to one or more embodiments of the present disclosure. [Modes for carrying out the invention]

[0198] Allergies can involve an abnormal response from the immune system to exposure to allergens. The human body's innate immune system can produce antibodies. Antibodies can include Y-shaped proteins that bind to various foreign proteins or chemicals that interact with them. Allergic reactions can occur when the immune system reacts abnormally to a foreign substance (allergen), entering an over-defense state (such as the immune cell TH2 response). This defense can sometimes be triggered by immunoglobulin E antibodies (IgE) that mast cells use to sense foreign substances, in this case allergens. When people inhale or come into contact with allergens, the immune system can react, causing an inflammatory response in the nasal passages or lungs. Long-term exposure to allergens can also lead to persistent inflammation associated with asthma.

[0199] Approximately 7 out of 10 households in the United States own pets, and about 15-30% of the population is allergic to pets, particularly dogs or cats. Cat and canine allergens can be found in the animals' shed skin cells (scalculi), saliva, urine, sweat, and fur. Scalculi are very small and can remain airborne, which can cause problems. Cats can be one of the main sources of indoor inhaled allergens. The global incidence of cat allergies is rapidly increasing and is considered a major public health issue. Some examples of cat allergens may include Fel d 1 to Fel d 8. More than 95% of cat allergies can be caused by a secretoglobin called Fel d 1. Cats secrete the Fel d 1 protein from their salivary and sebaceous glands and can spread it throughout their fur during regular grooming (tongue baths). Fel d 1 is easily airborne and can remain in the indoor environment. Two allergens present in dog hair or scale extracts may be Canis familiaris allergen 1 (Can f1) and Canis familiaris allergen 1 (Can f2). Canine allergens can also be present in scales, saliva, urine, and blood. Allergen levels vary between breeds, and allergies can be triggered in all breeds, including hairless dogs. House dust mite allergies involve group I (e.g., Der p I and Der f I) and group II (e.g., Der p II and Der f II) protein allergens.

[0200] Several other allergens may also induce allergic reactions. Examples of pet allergens include, but are not limited to, Fel d 1, Fel d 2, Fel d 3, Fel d 4, Can f 1, Can f 2, Can f 4, Can f 7, Ory C 1, Mus M 1, or Bla G 2. Examples of plant or pollen allergens include, but are not limited to, Bet v 1, Phl p 5, Phl p 1, Poa p 1, Cyn d 1, Bet v 2, Olee 1, Amb a 1, Amb a 11, and Art v 1. Examples of mold allergens include, but are not limited to, Alt a 1, Asp f 1, Asp f 2, Cla h 8, Pen ch 13, and Pen ch 18. Examples of food allergens include, but are not limited to, Pen a 1, Ara h 1, and Ara h 3.

[0201] The human body can produce antibodies that bind to allergens and neutralize allergic reactions. Antibodies can be produced by cells outside the human body and used to neutralize allergens. However, antibody production can be expensive because it is done using human, mammalian, or avian cells. For example, a conventional approach might involve immunizing eggs / chickens with Fel d 1 and then feeding these eggs to cats to reduce production costs. This approach may temporarily neutralize Fel d 1 in the mouths of cats, but the results are not complete.

[0202] Alpacas, llamas, and camels can produce similar immune proteins. Scientists can isolate small portions of immune proteins called single-domain antibodies (sdAbs) or nanobodies. The usefulness and advantages of single-domain antibodies or nanobodies may include, but are not limited to, their small size, a large number of accessible epitopes, relatively low production costs due to production in bacteria or yeast, and improved robustness compared to their full-length antibodies. They can also be produced from bacteria or yeast at very low cost. Peptides with short amino acid chains can also be designed to bind to allergens. Other small protein conjugates exist that can be designed to bind to allergens and neutralize them, such as monobodies and engineered ankyrin repeat proteins (DARPin).

[0203] Allergen-binding proteins In certain embodiments, the disclosure provides allergen-binding proteins. In some embodiments, the allergen-binding protein may bind to or neutralize an allergen. In some embodiments, the allergen-binding protein comprises an antibody fragment. The antibody fragment may be monoclonal. The antibody fragment may be polyclonal. In some embodiments, the allergen-binding protein comprises a nanobody, a monobody, a DARPin, or a small peptide. In some embodiments, the allergen-binding protein may be synthetic. In some embodiments, the allergen-binding protein may be manipulated. In some embodiments, the allergen-binding protein may be recombinant.

[0204] In some embodiments, the disclosure provides allergen-binding proteins that bind to or neutralize allergens. In some embodiments, the allergen-binding proteins include nanobodies, monobodies, DARPin, or small peptides with a molecular weight of less than about 25 kilodaltons (kDa) or a length of less than about 300 amino acids. In some embodiments, the allergen-binding proteins may be formulated for coating food or for spraying, misting, or brushing onto animal or household surfaces. In some embodiments, the food is pet food. In some embodiments, the allergen is a pet allergen. In some embodiments, the allergen-binding proteins are formulated to be applied as a topper on food. In some embodiments, the allergen-binding proteins are formulated to be mixed with food.

[0205] In some embodiments, the allergen-binding protein may be a nanobody. In some embodiments, the allergen-binding protein may be a monobody. In some embodiments, the allergen-binding protein may be DARPin. In some embodiments, DARPin is a small, single-domain protein of about 14 kDa that can be selected to bind to any given target protein with high affinity and specificity. In some embodiments, the allergen-binding protein may be a small peptide.

[0206] In some embodiments, the allergen-binding proteins described herein may be nanobodies. In some embodiments, the allergen-binding proteins may be single-domain antibodies. The utility and advantages of single-domain antibodies (sdAbs) may include, but are not limited to, smaller size, a large number of accessible epitopes, relatively lower production costs, or improved robustness compared to their full-length antibodies.

[0207] In some embodiments, the allergen-binding protein includes nanobodies or peptides having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 1-57 and 67-73. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO. 1. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO. 2. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 3. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 4. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 5. In some embodiments, the allergen-binding protein comprises nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 6.In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 7. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 8. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 9. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 10. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 11. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 12. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 13.In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 14. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 15. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 16. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 17. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 18. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 19. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 20.In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 21. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 22. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 23. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 24. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 25. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 26. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 27.In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 28. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 29. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 30. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 31. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 32. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 33. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 34.In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 49. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 50. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 97% sequence identity with respect to the sequence of SEQ ID NO: 51. The allergen-binding protein includes nanobodies having 9% or 100% sequence identity. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 52. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 53. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 54. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 55. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 56. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 57. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 67.In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 68. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 69. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 70. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 71. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 72. In some embodiments, the allergen-binding protein includes nanobodies having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO: 73.

[0208] In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 1. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 2. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 3. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 4. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 5. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 6. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 7. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 8. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 9. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 10. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 11. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 12. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 13. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 14. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 15. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 16. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 17. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 18. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 19. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 20. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 21.In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 22. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 23. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 24. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 25. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 26. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 27. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 28. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 29. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 30. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 31. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 32. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 33. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 34. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 49. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 50. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 51. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 52. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 53. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 54. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 55. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 56.In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 57. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 67. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 68. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 69. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 70. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 71. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 72. In some embodiments, the allergen-binding protein includes a nanobody having the sequence of SEQ ID NO: 73.

[0209] In some embodiments, the allergen-binding protein contains a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 35. In some embodiments, the allergen-binding protein contains a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 36. In some embodiments, the allergen-binding protein contains a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 37. In some embodiments, the allergen-binding protein contains a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 38. In some embodiments, the allergen-binding protein contains a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 39. In some embodiments, the allergen-binding protein contains a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 40. In some embodiments, the allergen-binding protein comprises a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 41.In some embodiments, the allergen-binding protein contains a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 42. In some embodiments, the allergen-binding protein contains a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 43. In some embodiments, the allergen-binding protein contains a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 44. In some embodiments, the allergen-binding protein contains a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 45. In some embodiments, the allergen-binding protein contains a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 46. In some embodiments, the allergen-binding protein contains a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 47. In some embodiments, the allergen-binding protein comprises a peptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 48.

[0210] In some embodiments, the allergen-binding protein includes a peptide having the sequence of SEQ ID NO: 35. In some embodiments, the allergen-binding protein includes a peptide having the sequence of SEQ ID NO: 36. In some embodiments, the allergen-binding protein includes a peptide having the sequence of SEQ ID NO: 37. In some embodiments, the allergen-binding protein includes a peptide having the sequence of SEQ ID NO: 38. In some embodiments, the allergen-binding protein includes a peptide having the sequence of SEQ ID NO: 39. In some embodiments, the allergen-binding protein includes a peptide having the sequence of SEQ ID NO: 40. In some embodiments, the allergen-binding protein includes a peptide having the sequence of SEQ ID NO: 41. In some embodiments, the allergen-binding protein includes a peptide having the sequence of SEQ ID NO: 42. In some embodiments, the allergen-binding protein includes a peptide having the sequence of SEQ ID NO: 43. In some embodiments, the allergen-binding protein includes a peptide having the sequence of SEQ ID NO: 44. In some embodiments, the allergen-binding protein includes a peptide having the sequence of SEQ ID NO: 45. In some embodiments, the allergen-binding protein includes a peptide having the sequence of SEQ ID NO: 46. In some embodiments, the allergen-binding protein comprises a peptide having the sequence of SEQ ID NO: 47. In some embodiments, the allergen-binding protein comprises a peptide having the sequence of SEQ ID NO: 48.

[0211] In some embodiments, the allergen-binding proteins described herein may be humanized antibody fragments, nanobodies, variants, or derivatives thereof, which can be formulated, for example, for administration to humans. In some embodiments, the humanized antibody may be a chimeric humanized antibody or a fully human antibody, and may include, for example, an amino acid sequence derived from or similar to a human antibody amino acid sequence, and a non-human amino acid sequence. For example, portions of the heavy and / or light chain of a chimeric humanized antibody may be identical or similar to the corresponding sequence in a human antibody, while the remainder of the chain may be identical or similar to the corresponding sequence in a non-human antibody, for example, derived from another species or belonging to another antibody class or subclass. The non-human sequence may be humanized to reduce the potential for immunogenicity while preserving target specificity, for example, by incorporating human DNA into the gene sequence of a gene that produces an antibody in a non-human animal. The humanized antibody may be a fully human antibody containing, for example, a human antibody amino acid sequence.

[0212] In some embodiments, the allergen-binding proteins described herein include signal peptidases or peptide-protein fusions. Signal peptides can lead to higher levels of protein expression and / or secretion by cells. Signal peptidases can, for example, cleave the signal peptide from an antibody or its antigen-binding fragment during the secretion process to produce a mature antibody that does not contain the signal peptide sequence.

[0213] The constant regions of the allergen-binding proteins described herein can mediate various effector functions while having minimal involvement in antigen binding.

[0214] The allergen-binding proteins described herein include a constant region that is selected or modified to provide suitable protein features, for example, features suitable for treating the diseases or illnesses disclosed herein.

[0215] The variable (V) region mediates antigen binding and can determine the specificity of a particular antibody against an antigen.

[0216] Within the hypervariable region, there are amino acid residues that primarily determine the binding specificity of antibodies. Sequences containing these residues are known as complementarity-determining regions (CDRs). A single antigen-binding site on an allergen-binding protein may consist of several variable loops that confer specificity to the allergen-binding protein.

[0217] In some embodiments, the allergen-binding proteins described herein include variants or derivatives thereof. For example, non-human animals, bacteria, yeasts, or plants may be genetically modified to produce protein variants or derivatives. In some embodiments, the allergen-binding protein may be a single-domain antibody (sdAb), such as a heavy-chain-only antibody (HCAb)VHH, nanobody, monobody, DARPin, small peptide, or scFV.

[0218] In other embodiments, the allergen-binding protein described herein may be its binding fragment. The allergen-binding protein described herein may be a humanized antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a multispecific antibody or its binding fragment, a bispecific antibody or its binding fragment, or a single-domain antibody thereof (e.g., nanobody®). The allergen-binding protein described herein may be a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or its binding fragment, or a chemically modified derivative thereof.

[0219] In some embodiments, the allergen-binding proteins described herein may be multispecific antibodies. In some cases, the multispecific protein comprises two or more target-binding moieties, each specifically binding to a different antigen. In some cases, the multispecific antibody comprises target-binding moieties specifically binding to three or more different antigens, four or more different antigens, or five or more different antigens. In some embodiments, the antibody may be a bispecific antibody. Depending on the case, the bispecific antibodies or conjugate fragments may include Knobs-into-Holes (KiH), Asymmetric Re-engineering Technology-immunoglobulin (ART-Ig), Triomab quadroma, bispecific monoclonal antibodies (BiMAb, BsmAb, BsAb, bsMab, BS-Mab, or Bi-MAb), FcΔAdp, XmAb, Azymetric, Bispecific Engagement by Antibodies based on the T-cell receptor (BEAT), Bispecific T-cell Engagers (BiTE), Biclonics, Fab-scFv-Fc, Two-in-one / Dual Action Fab (DAF), FinomAb, scFv-Fc-(Fab)-fusion, Dock-aNd-Lock (DNL), Adaptir (formerly SCORPION), Tandem This includes, but is not limited to, diAbody (TandAb), Dual-affinity-ReTargeting (DART), or nanobody. In some embodiments, the bispecific antibody may be a triplicate antibody or a bispecific mini-antibody. In some cases, the bispecific antibody may be a triplicate antibody. The triplicate antibody may be a full-length monoclonal antibody containing binding sites for two different antigens.

[0220] In some embodiments, the allergen-binding proteins described herein include one or more mutations for stabilizing the protein and / or increasing its half-life.

[0221] In some embodiments, the allergen-binding protein described herein comprises a humanized antibody or its binding fragment, or a chimeric antibody or its binding fragment. In some embodiments, the composition comprises a multispecific antibody or its binding fragment. In some embodiments, the antibody comprises a bispecific antibody or its binding fragment. In some embodiments, the antibody may be IgG-scFv, nanobody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, triplebody, mini-antibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, or intrabody. Depending on the case, the antibody may be monovalent Fab', bivalent Fab2, F(ab)'3 fragment, single-chain variable fragment (scFv), bis-scFv, (scFv)2, diabody, minibody, nanobody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), single-domain antibody (e.g., nanobody), Ig NAR, camelid antibody, or its conjugated fragment, or a chemically modified derivative thereof.

[0222] In some embodiments, the allergen-binding proteins described herein (e.g., single-domain antibodies) may bind to epitopes expressed by target cells associated with the disease or illness described herein. In some embodiments, the antibodies or their antigen-binding fragments may bind to epitopes associated with the microenvironment described herein.

[0223] In some embodiments, exogenous proteins may function as agonists or antagonists, and when they bind to any one of the epitopes described herein, the binding of an antibody or single-domain antibody induces an agonist or antagonist effect.

[0224] In some embodiments, the antigen-binding protein may have a length of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nm. In some embodiments, the antigen-binding protein may have a length of up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 nm. In some embodiments, the antigen-binding protein may have a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nm. In some embodiments, the antigen-binding protein may have a length between the two values ​​above, for example, about 1 to about 10, about 2 to about 9, about 3 to about 8, about 4 to about 7, or about 5 to about 6 nm. In some embodiments, the antigen-binding protein may have a length between the two values ​​above, for example, 1 to 10, 2 to 9, 3 to 8, 4 to 7, or 5 to 6 nm. In some embodiments, the antigen-binding protein may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nm.

[0225] In some embodiments, the antigen-binding protein may have a molecular weight of about 9, about 11, about 13, about 15, about 17, about 19, about 21, about 25, or about 30 kDa. In some embodiments, the antigen-binding protein may have a molecular weight of up to 9, up to 11, up to 13, up to 15, up to 17, up to 19, up to 21, up to 25, or up to 30 kDa. In some embodiments, the antigen-binding protein may have a molecular weight of at least 9, at least 11, at least 13, at least 15, at least 17, at least 19, at least 21, at least 25, or at least 30 kDa. In some embodiments, the antigen-binding protein may have a molecular weight between the two values ​​above, for example, about 9 to about 30, about 11 to about 25, about 13 to about 21, about 15 to about 19, or about 17 to about 30 kDa. In some embodiments, the antigen-binding protein may have a molecular weight between the two values ​​above, for example, 9–30, 11–25, 13–21, 15–19, or 17–30 kDa.

[0226] In some embodiments, the antigen-binding protein contains about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 200, or about 250 amino acids. In some embodiments, the antigen-binding protein contains up to 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 110, up to 120, up to 130, up to 140, up to 150, up to 200, or up to 250 amino acids. In some embodiments, the antigen-binding protein contains at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 200, or at least 250 amino acids. In some embodiments, the antigen-binding protein contains a number of amino acids between the above two values, for example, about 5 to about 250, about 10 to about 200, about 20 to about 150, about 30 to about 140, about 50 to about 130, about 60 to about 120, about 70 to about 110, about 80 to about 100, or about 90 to about 250 amino acids. In some embodiments, the antigen-binding protein contains a number of amino acids between the two values ​​above, for example, 5-250, 10-200, 20-150, 30-140, 50-130, 60-120, 70-110, 80-100, or 90-250 amino acids. In some embodiments, the antigen-binding protein contains 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, or 250 amino acids.

[0227] In some embodiments, the nanobody may have a length of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nm. In some embodiments, the nanobody may have a length of up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 nm. In some embodiments, the nanobody may have a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nm. In some embodiments, the nanobody may have a length between the two values ​​above, for example, about 1 to about 10, about 2 to about 9, about 3 to about 8, about 4 to about 7, or about 5 to about 6 nm. In some embodiments, the nanobody may have a length between the two values ​​above, for example, 1 to 10, 2 to 9, 3 to 8, 4 to 7, or 5 to 6 nm. In some embodiments, the nanobody may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nm.

[0228] In some embodiments, the nanobody may have a molecular weight of about 9, about 11, about 13, about 15, about 17, about 19, or about 21 kDa. In some embodiments, the nanobody may have a molecular weight of up to 9, up to 11, up to 13, up to 15, up to 17, up to 19, or up to 21 kDa. In some embodiments, the nanobody may have a molecular weight of at least 9, at least 11, at least 13, at least 15, at least 17, at least 19, or at least 21 kDa. In some embodiments, the nanobody may have a molecular weight between the two values ​​above, for example, about 9 to about 21, about 11 to about 19, about 13 to about 17, about 15 to about 21, or about 12 to about 15 kDa. In some embodiments, the nanobody may have a molecular weight between the two values ​​above, for example, 9 to 21, 11 to 19, 13 to 17, 15 to 21, or 12 to 15 kDa. In some embodiments, the nanobody may have a molecular weight of 9, 11, 13, 15, 17, 19, or 21 kDa.

[0229] In some embodiments, the nanobody contains about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, or about 150 amino acids. In some embodiments, the nanobody contains up to 70, up to 80, up to 90, up to 100, up to 110, up to 120, up to 130, up to 140, or up to 150 amino acids. In some embodiments, the nanobody contains at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, or at least 150 amino acids. In some embodiments, the nanobody contains a number of amino acids between the two values ​​above, for example, about 70 to about 150, about 80 to about 140, about 90 to about 130, about 100 to about 120, or about 110 to about 150 amino acids. In some embodiments, the nanobody contains a number of amino acids between the two values ​​above, for example, 70-150, 80-140, 90-130, 100-120, or 110-150 amino acids.

[0230] In some embodiments, the antigen-binding protein includes a multispecific protein. In some embodiments, the antigen-binding protein may be conjugated with a protein or peptide.

[0231] In some embodiments, the small peptide or protein binder includes modified amino acids or non-natural amino acids, or modified non-natural amino acids, or combinations thereof. In some embodiments, the modified amino acids or modified non-natural amino acids include post-translational modifications. In some embodiments, the small peptide includes modifications including, but not limited to, acetylation, acrylication, ADP-ribosylation, amidation, covalent bonding of flavin, covalent bonding of heme moiety, covalent bonding of nucleotide or nucleotide derivative, covalent bonding of lipid or lipid derivative, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gammacarboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolysis, phosphorylation, prenylation, racemization, selenoylation, sulfated, arginylation, and other RNA-mediated addition of amino acids to proteins, as well as ubiquitination. Modifications can occur anywhere on small peptides, including the peptide backbone, amino acid side chains, and terminals.

[0232] In some embodiments, the allergen-binding protein comprises at least one modified amino acid. In some embodiments, the allergen-binding protein comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 65, or about 70 modified amino acids. In some embodiments, the allergen-binding protein comprises a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, or 70 modified amino acids. In some embodiments, the allergen-binding protein comprises a minimum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, or 70 modified amino acids. In some embodiments, the allergen-binding protein comprises about 1 to about 70, about 2 to about 65, about 3 to about 60, about 4 to about 50, about 5 to about 45, about 6 to about 40, about 7 to about 35, about 8 to about 30, about 9 to about 25, about 10 to about 20, or about 11 to about 15 modified amino acids. In some embodiments, the allergen-binding protein comprises 1 to 70, 2 to 65, 3 to 60, 4 to 50, 5 to 45, 6 to 40, about 7 to 35, 8 to 30, 9 to 25, 10 to 20, or 11 to 15 modified amino acids. In some embodiments, the allergen-binding protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, or 70 modified amino acids.

[0233] In some embodiments, the modified amino acid includes a non-standard amino acid. In some embodiments, the non-standard amino acid is selected from the group consisting of para-benzoylphenylalanine, 3,4-dihydroxyphenylalanine, tetrazine, chlorooctene, homopropagylglycine, para-propagyloxyphenylalanine, para-azidophenylalanine, para-isothiocyanatephenylalanine, para-benzoylphenylalanine, para-cyanophenylalanine, para-nitrophenylalanine, m-halogenated tyrosine analogs, halogenated proline analogs, halogenated tryptophan analogs, and halogenated leucine analogs. In some embodiments, the non-standard amino acid is para-benzoylphenylalanine. In some embodiments, the non-standard amino acid is 3,4-dihydroxyphenylalanine. In some embodiments, the non-standard amino acid is tetrazine. In some embodiments, the non-standard amino acid is chlorooctene. In some embodiments, the non-standard amino acid is homopropagylglycine. In some embodiments, the non-standard amino acid is para-propagyloxyphenylalanine. In some embodiments, the non-standard amino acid is para-azidophenylalanine. In some embodiments, the non-standard amino acid is para-isothiocyanatephenylalanine. In some embodiments, the non-standard amino acid is para-benzoylphenylalanine. In some embodiments, the non-standard amino acid is para-cyanophenylalanine. In some embodiments, the non-standard amino acid is para-nitrophenylalanine. In some embodiments, the non-standard amino acid is an m-halogenated tyrosine analog. In some embodiments, the non-standard amino acid is a halogenated proline analog. In some embodiments, the non-standard amino acid is a halogenated tryptophan analog. In some embodiments, the non-standard amino acid is a halogenated leucine analog.

[0234] In some embodiments, the solubility of the allergen-binding protein increases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, or more, compared to the unmodified allergen-binding protein. In some embodiments, the solubility of the allergen-binding protein increases by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 90%, up to 100%, up to 150%, up to 200%, up to 250%, up to 300%, up to 350%, up to 400%, up to 450%, up to 500%, up to 600%, up to 700%, up to 800%, up to 900%, up to 1000%, or more, compared to the unmodified allergen-binding protein. In some embodiments, the solubility of the allergen-binding protein increases by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or more, compared to the unmodified allergen-binding protein. In some embodiments, the solubility of the allergen-binding protein increases by approximately 5% to 1000%, 10% to 900%, 15% to 800%, 20% to 700%, 25% to 600%, 30% to 500%, 35% to 400%, 40% to 300%, 45% to 200%, 50% to 100%, 55% to 90%, or 60% to 80% compared to the unmodified allergen-binding protein.In some embodiments, the solubility of the allergen-binding protein is increased by 5% to 1000%, 10% to 900%, 15% to 800%, 20% to 700%, 25% to 600%, 30% to 500%, 35% to 400%, 40% to 300%, 45% to 200%, 50% to 100%, 55% to 90%, or 60% to 80% compared to the unmodified allergen-binding protein. In some embodiments, the solubility of the allergen-binding protein increases by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, compared to the unmodified allergen-binding protein.

[0235] In some embodiments, the binding affinity of the allergen-binding protein increases by about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, about 1000%, about 1500%, about 2000%, about 3000%, or more, compared to the unmodified allergen-binding protein. In some embodiments, the binding affinity of the allergen-binding protein increases by up to 10%, up to 15%, up to 20%, 25%, up to 50%, up to 75%, up to 100%, up to 150%, up to 200%, up to 250%, up to 300%, up to 350%, up to 400%, up to 450%, up to 500%, up to 550%, up to 600%, up to 650%, up to 700%, up to 750%, up to 800%, up to 850%, up to 900%, up to 950%, up to 1000%, up to 1500%, up to 2000%, up to 3000%, or more, compared to the unmodified allergen-binding protein. In some embodiments, the binding affinity of the allergen-binding protein increases by at least 10%, at least 15%, at least 20%, 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 1500%, at least 2000%, at least 3000%, or more, compared to the unmodified allergen-binding protein. In some embodiments, the binding affinity of the allergen-binding protein is increased by approximately 10% to 3000%, 15% to 2000%, 20% to 1500%, 25% to 1000%, 50% to 950%, 75% to 900%, 100% to 850%, 150% to 800%, 200% to 750%, 250% to 700%, 300% to 650%, 350% to 600%, 400% to 550%, or 450% to 500% compared to the unmodified allergen-binding protein.In some embodiments, the binding affinity of the allergen-binding protein is increased by 10% to 3000%, 15% to 2000%, 20% to 1500%, 25% to 1000%, 50% to 950%, 75% to 900%, 100% to 850%, 150% to 800%, 200% to 750%, 250% to 700%, 300% to 650%, 350% to 600%, 400% to 550%, or 450% to 500% as compared to the unmodified allergen-binding protein. In some embodiments, the binding affinity of the allergen-binding protein is increased by 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 3000%, or more as compared to the unmodified allergen-binding protein.

[0236] In some embodiments, the thermal stability of the allergen-binding protein is increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, or more, compared to the unmodified allergen-binding protein. In some embodiments, the thermal stability of the allergen-binding protein is increased by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 90%, up to 100%, up to 150%, up to 200%, up to 250%, up to 300%, up to 350%, up to 400%, up to 450%, up to 500%, up to 600%, up to 700%, up to 800%, up to 900%, up to 1000%, or more, compared to the unmodified allergen-binding protein. In some embodiments, the thermal stability of the allergen-binding protein is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or more, compared to the unmodified allergen-binding protein. In some embodiments, the thermal stability of the allergen-binding protein is increased by approximately 5% to 1000%, 10% to 900%, 15% to 800%, 20% to 700%, 25% to 600%, 30% to 500%, 35% to 400%, 40% to 300%, 45% to 200%, 50% to 100%, 55% to 90%, or 60% to 80% compared to the unmodified allergen-binding protein.In some embodiments, the thermal stability of the allergen-binding protein is increased by 5%-1000%, 10%-900%, 15%-800%, 20%-700%, 25%-600%, 30%-500%, 35%-400%, 40%-300%, 45%-200%, 50%-100%, 55%-90%, or 60%-80% compared to the unmodified allergen-binding protein. In some embodiments, the thermal stability of the allergen-binding protein is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000%, or more compared to the unmodified allergen-binding protein.

[0237] In some embodiments, the allergen-binding protein is polyvalent. In some embodiments, the allergen-binding protein is divalent. In some embodiments, the allergen-binding protein is trivalent. In some embodiments, the allergen-binding protein is tetravalent. In some embodiments, the solubility of the allergen-binding protein increases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, or more, compared to a monovalent allergen-binding protein. In some embodiments, the solubility of the allergen-binding protein increases by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 90%, up to 100%, up to 150%, up to 200%, up to 250%, up to 300%, up to 350%, up to 400%, up to 450%, up to 500%, up to 600%, up to 700%, up to 800%, up to 900%, up to 1000%, or more, compared to the monovalent allergen-binding protein. In some embodiments, the solubility of the allergen-binding protein increases by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or more, compared to the monovalent allergen-binding protein.In some embodiments, the solubility of the allergen-binding protein increases by approximately 5% to 1000%, 10% to 900%, 15% to 800%, 20% to 700%, 25% to 600%, 30% to 500%, 35% to 400%, 40% to 300%, 45% to 200%, 50% to 100%, 55% to 90%, or 60% to 80% compared to the monovalent allergen-binding protein. In some embodiments, the solubility of the allergen-binding protein is increased by 5% to 1000%, 10% to 900%, 15% to 800%, 20% to 700%, 25% to 600%, 30% to 500%, 35% to 400%, 40% to 300%, 45% to 200%, 50% to 100%, 55% to 90%, or 60% to 80% compared to the monovalent allergen-binding protein. In some embodiments, the solubility of the allergen-binding protein increases by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, compared to the monovalent allergen-binding protein.

[0238] In some embodiments, the small peptide may have a molecular weight of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 28, about 30, about 32, or about 35 kDa. In some embodiments, the small peptide may have a molecular weight of up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25, up to 26, up to 28, up to 30, up to 32, or up to 35 kDa. In some embodiments, the small peptide may have a molecular weight of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 28, at least 30, at least 32, or at least 35 kDa. In some embodiments, the small peptide may have a molecular weight between the two values ​​above, for example, about 1 to about 35, about 2 to about 34, about 3 to about 33, about 4 to about 32, about 5 to about 31, about 6 to about 30, about 7 to about 29, about 8 to about 28, about 9 to about 27, about 10 to about 26, about 11 to about 25, about 12 to about 24, about 13 to about 23, about 14 to about 21, about 15 to about 20, about 16 to about 19, or about 17 to about 18 kDa. In some embodiments, the small peptide may have a molecular weight of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 30, 32, or 35 kDa.

[0239] In some embodiments, the small peptides contain amino acids with lengths of approximately 5, 10, 20, 60, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400. In some embodiments, the small peptides contain amino acids with lengths of up to 5, 10, 20, 60, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400. In some embodiments, the small peptide contains amino acids with lengths of at least 5, at least 10, at least 20, at least 60, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, at least 280, at least 300, at least 320, at least 340, at least 360, at least 380, or at least 400. In some embodiments, the small peptide contains a number of amino acids between the above two values, for example, amino acids with lengths of about 5 to about 400, about 10 to about 380, about 20 to about 360, about 60 to about 340, about 120 to about 320, about 140 to about 300, about 160 to about 280, about 180 to about 260, or about 200 to about 240. In some embodiments, the small peptide contains a number of amino acids with lengths between 5–400, 10–380, 20–360, 60–340, 120–320, 140–300, 160–280, 180–260, or 200–240. In some embodiments, the small peptide contains amino acids with lengths of 5, 10, 20, 60, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400.

[0240] The allergen-binding proteins disclosed herein can be formulated in any preferred physical form. Preferred forms may include, but are not limited to, aerosols, liquids, gels, semi-solids, solids, or powders. In some embodiments, the allergen-binding proteins can be formulated as toners, creams, emulsions, lotions, ointments, pastes, gels, suspensions, serums, oils, sprays, shampoos, foams, cleansers, mousses, aerosols, or powders that are resuspended in a solvent such as water.

[0241] In some embodiments, the allergen-binding protein may be formulated for coating food. In some embodiments, the allergen-binding protein may be mixed with any type of food for animals. In some embodiments, the allergen-binding protein may be provided as part of a normal food intake or added to it. In some embodiments, the allergen-binding protein may be provided as an additive to liquid intakes for animals, including drinking water. In some embodiments, the allergen-binding protein may be formulated as a food.

[0242] In some embodiments, the allergen-binding protein may be formulated for spraying, misting, or brushing onto animals or household surfaces. In some embodiments, the allergen-binding protein may be formulated for use in humidifiers and air filters. In some embodiments, the allergen-binding protein may be applied to environmental surfaces, for example, by spraying, misting, depositing, wiping, or other preferred application methods. In some embodiments, the surface may be the surface of an animal that is a source of allergens.

[0243] In some embodiments, the allergen-binding proteins disclosed herein can reduce, minimize, or prevent at least one symptom of an allergic response to an environmental or animal allergen. In some embodiments, the allergen-binding proteins disclosed herein can reduce at least one symptom of an allergic response to an environmental or animal allergen. In some embodiments, the allergen-binding proteins disclosed herein can minimize at least one symptom of an allergic response to an environmental or animal allergen. In some embodiments, the allergen-binding proteins disclosed herein can prevent at least one symptom of an allergic response to an environmental or animal allergen. In some embodiments, the allergen-binding proteins can come into contact with environmental allergens, bind to allergens, and prevent allergic reactions from being induced by the allergen in a subject suspected of or suffering from an allergy caused by the allergen. In some embodiments, the allergen-binding proteins can come into contact with environmental allergens. In some embodiments, the allergen-binding proteins can bind to allergens.

[0244] In some embodiments, the allergen-binding protein may be resuspended at concentrations ranging from about 0.01 milligrams / milliliter (mg / ml) to about 500 mg / ml, or may be present in the solution. In some embodiments, the allergen-binding protein may be present at a concentration of approximately 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 mg / ml. In some embodiments, the allergen-binding protein may be at concentrations of up to 0.001, up to 0.005, up to 0.01, up to 0.05, up to 0.1, up to 0.5, up to 1, up to 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 120, up to 140, up to 160, up to 180, up to 200, up to 220, up to 240, up to 260, up to 280, up to 300, up to 320, up to 340, up to 360, up to 380, up to 400, up to 420, up to 440, up to 460, up to 480, up to 500, up to 520, up to 540, up to 560, up to 580, or up to 600 mg / ml. In some embodiments, the allergen-binding protein may be present at a concentration of at least 0.001, at least 0.005, at least 0.01, at least 0.05, at least 0.1, at least 0.5, at least 1, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, at least 280, at least 300, at least 320, at least 340, at least 360, at least 380, at least 400, at least 420, at least 440, at least 460, at least 480, at least 500, at least 520, at least 540, at least 560, at least 580, or at least 600 mg / ml.In some embodiments, the allergen-binding protein may be present at concentrations of approximately 0.001 to 600, 0.005 to 580, 0.01 to 560, 0.05 to 540, 0.1 to 520, 0.5 to 500, 1 to 480, 5 to 460, 10 to 440, 20 to 420, 30 to 400, 40 to 380, 50 to 360, 60 to 340, 70 to 320, 80 to 300, 90 to 280, 100 to 260, 120 to 240, 140 to 220, 160 to 200, or 180 to 500 mg / ml. In some embodiments, the allergen-binding protein may be present in concentrations of 0.001-600, 0.005-580, 0.01-560, 0.05-540, 0.1-520, 0.5-500, 1-480, 5-460, 10-440, 20-420, 30-400, 40-380, 50-360, 60-340, 70-320, 80-300, 90-280, 100-260, 120-240, 140-220, 160-200, or 180-500 mg / ml. In some embodiments, the allergen-binding protein may be present at concentrations of 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 mg / ml.

[0245] In some embodiments, the allergen may induce an allergic reaction in humans. In some embodiments, the allergen may include environmental allergens. In some embodiments, the allergen may include animal allergens. In some embodiments, the allergen may include pet allergens. In some embodiments, the allergen may include cat, dog, rabbit, mouse, or cockroach allergens. In some embodiments, the allergen may include cat allergens. In some embodiments, the allergen may include dog allergens. In some embodiments, the allergen may include rabbit allergens. In some embodiments, the allergen may include mouse allergens. In some embodiments, the allergen may include cockroach allergens.

[0246] In some embodiments, the allergen may include dust allergens.

[0247] In some embodiments, the allergen may include plant or plant pollen allergens. In some embodiments, the plant may include trees, grasses, or weeds. In some embodiments, the plant or pollen allergen may be selected from the group consisting of Bet v1, Phl p 5, Phl p 1, Poa p 1, Cyn d 1, Bet v 2, Ole e 1, Amb a 1, Amb a 11, and Art v 1. In some embodiments, the plant or pollen allergen may be Bet v1. In some embodiments, the plant or pollen allergen may be Phl p 5. In some embodiments, the plant or pollen allergen may be Phl p 1. In some embodiments, the plant or pollen allergen may be Poa p 1. In some embodiments, the plant or pollen allergen may be Cyn d 1. In some embodiments, the plant or pollen allergen may be Bet v 2. In some embodiments, the plant or pollen allergen may be Ole e 1. In some embodiments, the plant or pollen allergen may be Amb a 1. In some embodiments, the plant or pollen allergen may be Amb a 11. In some embodiments, the plant or pollen allergen may be Art v 1.

[0248] In some embodiments, the allergen includes a mold allergen. In some embodiments, the mold allergen may be selected from the group consisting of Alt a 1, Asp f 1, Asp f 2, Cla h 8, Pen ch 13, and Pen ch 18. In some embodiments, the mold allergen may be Alt a 1. In some embodiments, the mold allergen may be Asp f 1. In some embodiments, the mold allergen may be Asp f 2. In some embodiments, the mold allergen may be Cla h 8. In some embodiments, the mold allergen may be Pen ch 13. In some embodiments, the mold allergen may be Pen ch 18.

[0249] In some embodiments, the allergen includes a food allergen. In some embodiments, the food allergen may be selected from the group consisting of Pen a 1, Ara h 1, and Ara h 3. In some embodiments, the food allergen may be Pen a 1. In some embodiments, the food allergen may be Ara h 1. In some embodiments, the food allergen may be Ara h 1. In some embodiments, the food allergen may be Ara h 3.

[0250] In some embodiments, the allergen may include Fel d 1, Fel d 2, Fel d 3, Fel d 4, Can f1, Can f2, Can f4, Can f7, Der P1, Der P2, Ory C1, Mus M1, Bla G2, Bet v 1, Phl p 5, or a combination thereof. In some embodiments, the allergen may include Fel d 1. In some embodiments, the allergen may include Fel d 2. In some embodiments, the allergen may include Fel d 3. In some embodiments, the allergen may include Fel d 4. In some embodiments, the allergen may include Can f1. In some embodiments, the allergen may include Can f2. In some embodiments, the allergen may include Can f4. In some embodiments, the allergen may include Can f7. In some embodiments, the allergen may include Der P1. In some embodiments, the allergen may include Der P2. In some embodiments, the allergen may include Ory C1. In some embodiments, the allergen may include Mus M1. In some embodiments, the allergen may include Bla G2. In some embodiments, the allergen may include Bet v 1. In some embodiments, the allergen may include Phl p 5.

[0251] composition In certain embodiments, this disclosure provides compositions comprising allergen-binding proteins disclosed herein. In some embodiments, the compositions may be pharmaceutical compositions. In some embodiments, the compositions may further comprise a carrier. In some embodiments, the carrier may be a pharmaceutically acceptable carrier. The carrier may include, but is not limited to, a solvent, stabilizer, diluent, dispersion medium, and coating. In some embodiments, the carrier may comprise silica. In some embodiments, the compositions may be in a dry form.

[0252] In some embodiments, the carrier may contain water. In some embodiments, the composition may contain a liquid. In some embodiments, the composition may contain a buffer, including a phosphate buffer or a saline buffer. In some embodiments, the composition may contain phosphate-buffered saline (PBS), citrate buffer, bicarbonate buffer, sodium hydrogen phosphate-citrate buffer, citrate-sodium citrate buffer, sodium hydrogen phosphate-sodium dihydrogen phosphate buffer, or acetate-sodium acetate buffer. In some embodiments, the pH of the composition may be about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or about 11. In some embodiments, the pH of the composition may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11. In some embodiments, the pH of the composition may be at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, or at most 11. In some embodiments, the pH of the composition may be about 2 to about 11, about 3 to about 10, or about 4 to about 9. In some embodiments, the pH of the composition may be 2 to 11, 3 to 10, or 4 to 9. In some embodiments, the pH of the composition may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0253] Non-limiting examples of carriers may include water, dimethyl sulfoxide (DMSO), α-toluene, α-pinene, camphene, sabinene, β-pinene, α-terpinene, limonene, peltay2-carene, trans-sabinene hydrate, terpinolene, 3-cyclohexen-1-ol, terpinen-4-ol, 1,2-benzenediol, linalyl acetate, borneol, bornyl acetate, α-thujone, terpinyl acetate, isolongrifolene, epit-bicyclosesquiphellandrene, α-humulene, guadiol, elemol, cedrol, β-eudesmol, rosifoliol, limien, hexadecanoic acid, sembren, verticerol, totarol, totara-1,9-octadecenamide, tatarol, 2-(hexylthiol)decanal, and combinations thereof.

[0254] In some embodiments, the composition may further contain a preservative. In some embodiments, the preservative may contain potassium sorbate. In some embodiments, the composition may contain one or more preservatives. The preservatives include benzyl alcohol, methylparaben, ethylparaben, propylparaben, butylparaben, isobutylparaben, glycerin, ethylhexylglycerin, phenoxyethanol, sodium benzoate, ethylenediaminetetraacetic acid (EDTA), benzoic acid, phenoxyethanol, maltol, potassium sorbate, imidazolidinyl urea, diazolidinyl urea, sorbic acid, methylisothiazolinone, chlorhexidine digluconate, polyaminopropyl biguanide, and dehydro Examples of ingredients include, but are not limited to, sodium acetate, grapefruit seed extract, salicylic acid, DMDM ​​hydantoin, formaldehyde, chlorphenism, triclosan, dehydroacetic acid, quaternium-15, stearalkonium chloride, zinc pyrithione, sodium metabisulfite, 2-bromo-2-nitropropane, benzalkonium chloride, sodium sulfite, sodium salicylate, citric acid, neem oil, essential oils, lactic acid, vitamin E (tocopherol), and combinations thereof.

[0255] The compositions disclosed herein may optionally further comprise at least one excipient. The at least one excipient may be a pharmaceutically acceptable excipient. In some embodiments, the at least one excipient may be selected from the group consisting of animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, zinc oxide, lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, polyamide powder, and combinations thereof. In some embodiments, the at least one excipient may be naturally occurring. In another embodiment, the at least one excipient may not be naturally occurring.

[0256] The compositions disclosed herein may optionally further comprise at least one additive. Non-limiting examples of the at least one additive may include fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blockers, humectants, essential oils, dyes, pigments, hydrophilic or hydrophobic activators, lipid vesicles, insect repellents, stabilizers, hydrating agents, emulsification promoters or salts and / or buffers for osmotic pressure control, and combinations thereof. In some embodiments, the at least one additive may be naturally occurring. In another embodiment, the at least one additive may not be naturally occurring. In some embodiments, the at least one additive may include other useful substances. In further embodiments, the at least one additive may further comprise absorption promoters, penetration enhancers, thickeners, viscosity enhancers, agents for adjusting and / or maintaining pH, agents for adjusting osmotic pressure, preservatives, surfactants, buffers, salts, suspending agents, dispersants, solubilizers, stabilizers, and / or isotonic agents.

[0257] In some embodiments, the composition may further contain a stabilizer or thickener. In some embodiments, the stabilizer or thickener may include dextrin, maltodextrin, glycerol, glucose, sucrose, or trehalose. In some embodiments, the composition may further contain an isotonic agent. In some embodiments, the stabilizer may be glucose, sucrose, glycerol, or trehalose.

[0258] In some embodiments, the carrier may include maltodextrin, dextrin, potassium sorbate, silica, water, or a combination thereof.

[0259] In other embodiments, the compositions of the present invention may include pharmaceutically acceptable carriers. Non-limiting examples of pharmaceutically acceptable carriers may include solvents, dispersions, coatings, adjuvants, stabilizers, diluents, preservatives, antimicrobial and antifungal agents, isotonic agents, or combinations thereof. In some embodiments, diluents may include water, saline, dextrose, ethanol, glycerol, or combinations thereof. In some embodiments, isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, lactose, or combinations thereof.

[0260] In some embodiments, the carrier may be at a concentration of up to about 80 wt / v (grams per milliliter of solvent) of the allergen-binding protein. In some embodiments, the carrier may be at a concentration of about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, or about 95 wt / v% of the composition. In some embodiments, the carrier may be at a concentration of up to 50, up to 55, up to 60, up to 65, up to 70, up to 75, up to 80, up to 85, up to 90, or up to 95 wt / v% of the composition. In some embodiments, the carrier may be at a concentration of at least 0.01, at least 0.05, at least 0.1, at least 0.5, at least 1, at least 2, at least 5, at least 10, or at least 20 wt / v% of the composition. In some embodiments, the carrier may be at a concentration of about 0.01 to about 95, about 0.1 to about 90, about 0.5 to about 85, about 1 to about 80, about 5 to about 75, about 10 to about 70, or about 20 to about 65 wt / v% of the composition. In some embodiments, the carrier may be at a concentration of 0.01 to about 95, 0.1 to about 90, 0.5 to about 85, 1 to about 80, 5 to 75, 10 to 70, or 20 to 65 wt / v% of the composition. In some embodiments, the carrier may be at a concentration of 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 wt / v% of the composition.

[0261] In some embodiments, the composition contains one or more allergen-binding proteins disclosed herein. In some embodiments, the composition contains two allergen-binding proteins disclosed herein. In some embodiments, the composition contains three or more allergen-binding proteins disclosed herein.

[0262] This composition can be formulated in any suitable physical form. Suitable forms may include, but are not limited to, aerosols, liquids, gels, semi-solids, solids, or powders. In some embodiments, this composition can be formulated as a toner, cream, emulsion, lotion, ointment, paste, gel, suspension, serum, oil, spray, shampoo, foam, cleanser, mousse, aerosol, or powder that can be resuspended in a solvent such as water.

[0263] In some embodiments, the composition may be formulated to coat food. In some embodiments, the composition may be edible. In some embodiments, the composition may be mixed with any type of food for animals. In some embodiments, the composition may be provided as a normal food intake or added to it. In some embodiments, the composition may be provided as an additive to liquid intakes for animals, including drinking water. In some embodiments, the composition may be formulated as a food. In some embodiments, the food is pet food. In some embodiments, the allergen is a pet allergen. In some embodiments, the allergen-binding protein is formulated to be applied as a topper on food. In some embodiments, the allergen-binding protein is formulated to be mixed with food.

[0264] In some embodiments, the composition may be formulated for spraying, misting, or brushing onto animals or household surfaces. In some embodiments, the composition may be formulated for use in humidifiers and air filters. In some embodiments, the composition may be applied to environmental surfaces by, for example, spraying, misting, depositing, wiping, or other preferred application methods. In some embodiments, the surface may be the surface of an animal that is a source of allergens.

[0265] In some embodiments, the compositions disclosed herein can reduce, minimize, or prevent at least one symptom of an allergic response to an environmental or animal allergen. In some embodiments, the compositions disclosed herein can reduce at least one symptom of an allergic response to an environmental or animal allergen. In some embodiments, the compositions disclosed herein can minimize at least one symptom of an allergic response to an environmental or animal allergen. In some embodiments, the compositions disclosed herein can prevent at least one symptom of an allergic response to an environmental or animal allergen. In some embodiments, the compositions can come into contact with environmental allergens, bind to allergens, and prevent allergic reactions from being induced by the allergens in subjects suspected of or suffering from allergies caused by the allergens. In some embodiments, the compositions can come into contact with environmental allergens. In some embodiments, allergen-binding proteins can bind to allergens.

[0266] method In certain embodiments, the Disclosure provides a method for reducing, minimizing, or preventing at least one symptom of an allergic response to an environmental allergen. In some embodiments, the Disclosure provides a method for reducing at least one symptom of an allergic response to an environmental allergen. In some embodiments, the Disclosure provides a method for minimizing at least one symptom of an allergic response to an environmental allergen. In some embodiments, the Disclosure provides a method for preventing at least one symptom of an allergic response to an environmental allergen. The method may include neutralizing the allergen. Non-limiting examples of allergic reaction symptoms include congestion, nasal or pharyngeal itching, sneezing, runny nose and itching, watery eyes, pain or tenderness around the cheeks, eyes, or forehead, cough, wheezing or shortness of breath, itchy or raised skin rash (hives), feeling or being sick, swelling of the eyes, lips, mouth, or throat, poor and rapid pulse, nausea, vomiting or diarrhea, dizziness or fainting, or a combination thereof. In some embodiments, a composition containing an allergen-binding protein may come into contact with an environmental allergen, bind to the allergen, and prevent an allergic reaction from being induced by the allergen in a subject suspected of or suffering from an allergy caused by the allergen. In some embodiments, the method includes the step of administering the allergen-binding protein disclosed herein to a subject. In some embodiments, the method includes the step of administering a composition containing the allergen-binding protein disclosed herein to a subject.

[0267] In some embodiments, the method may include the steps of spraying or atomizing a composition disclosed herein, or resuspending an allergen-binding protein or composition in a solvent disclosed herein and then contacting a surface with any one of the allergen-binding proteins or compositions disclosed herein. In some embodiments, the contact step may include coating or brushing. In some embodiments, the surface may contain an allergen. In some embodiments, the surface may include the surface of an air filter or humidifier. In some embodiments, the surface may contain food. In some embodiments, the food may be ingested by an animal that is a source of the antigen.

[0268] In some embodiments, the composition can neutralize allergens on a surface.

[0269] In some embodiments, the surface may include pet accessories (e.g., collars or brushes), air filters, or areas / products where animals can sit or walk (e.g., dog beds).

[0270] In some embodiments, spraying may be performed by a humidifier or a spray bottle.

[0271] In some embodiments, the allergenicity of an antigen can be neutralized by spraying about 0.01 milliliters (ml) to about 20 ml of the composition per area of ​​about 1 square meter. In some embodiments, the allergenicity of an antigen can be neutralized by spraying about 0.001, about 0.005, about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 ml of the composition per area of ​​about 1 square meter. In some embodiments, the allergenicity of the antigen may be neutralized by spraying up to 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ml of the composition per area of ​​about 1 square meter. In some embodiments, the allergenicity of the antigen may be neutralized by spraying a minimum of 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ml of the composition per area of ​​about 1 square meter. In some embodiments, the allergenic neutralization of the antigen may be performed by spraying about 0.001 to about 30, about 0.005 to about 29, about 0.01 to about 28, about 0.05 to about 27, about 0.1 to about 26, about 0.5 to about 25, about 1 to about 24, about 2 to about 23, about 3 to about 22, about 4 to about 21, about 5 to about 20, about 6 to about 19, about 7 to about 18, about 8 to about 17, about 9 to about 16, about 10 to about 15, about 11 to about 14, or about 12 to about 13 ml of the composition per area of ​​about 1 square meter.In some embodiments, the allergenic neutralization of the antigen may be performed by spraying 0.001-30, 0.005-29, 0.01-28, 0.05-27, 0.1-26, 0.5-25, 1-24, 2-23, 3-22, 4-21, 5-20, 6-19, 7-18, 8-17, 9-16, 10-15, 11-14, or 12-13 ml of the composition per area of ​​about 1 square meter. In some embodiments, the allergenic neutralization of the antigen may be performed by spraying 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ml of the composition per area of ​​about 1 square meter.

[0272] In certain embodiments, this disclosure provides a method for preparing allergen-binding proteins disclosed herein. In some embodiments, the method may include the step of collecting allergen-binding proteins from an engineered microorganism or from a secretion by a microorganism. In some embodiments, the microorganism may include heterologous nucleic acids encoding allergen-binding proteins. In some embodiments, the microorganism may include yeast or bacteria. In some embodiments, the microorganism may include bacteria. In some embodiments, the microorganism may include bacteria, including Escherichia coli. In some embodiments, the microorganism may include yeast, including Pichia pastoris. In some embodiments, the allergen-binding protein may be purified or concentrated from a secretion by a microorganism. In some embodiments, the allergen-binding protein may be secreted by a microorganism via a secretion tag and further purified. In some embodiments, the allergen-binding protein may be purified by affinity purification. In some embodiments, the allergen-binding protein may be purified by microfiltration. In some embodiments, the purification or concentration includes a filtration step. In some embodiments, the filtration step includes passing the secretion through a filter of about 0.01 nm, 0.1 nm, 1 nm, 5 nm, or larger. In some embodiments, the filter is about 0.0001 nm, about 0.0005 nm, about 0.001 nm, about 0.005 nm, about 0.01 nm, about 0.05 nm, about 0.1 nm, nm, about 0.5 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, or larger. In some embodiments, the filters are up to 0.0001nm, up to 0.0005nm, up to 0.001nm, up to 0.005nm, up to 0.01nm, up to 0.05nm, up to 0.1nm, nm, up to 0.5nm, up to 1nm, up to 2nm, up to 3nm, up to 4nm, up to 5nm, up to 6nm, up to 7nm, up to 8nm, up to 9nm, up to 10nm, or larger.In some embodiments, the filter size is at least 0.0001nm, at least 0.0005nm, at least 0.001nm, at least 0.005nm, at least 0.01nm, at least 0.05nm, at least 0.1nm, at least 0.5nm, at least 1nm, at least 2nm, at least 3nm, at least 4nm, at least 5nm, at least 6nm, at least 7nm, at least 8nm, at least 9nm, at least 10nm, or larger. In some embodiments, the filter size may be between the two values ​​mentioned above, for example, about 0.0001nm to about 10nm, about 0.0005nm to about 9nm, about 0.001nm to about 8nm, about 0.005nm to about 7nm, about 0.01nm to about 6nm, about 0.05nm to about 5nm, about 0.1nm to about 4nm, about 0.5nm to about 3nm, or about 1nm to about 2nm. In some embodiments, the filter size may be 0.0001 nm, 0.0005 nm, 0.001 nm, 0.005 nm, 0.01 nm, 0.05 nm, 0.1 nm, nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or 10 nm. In some embodiments, the method does not include a centrifugation step.

[0273] In some embodiments, the method may further include the step of incorporating a heterologous nucleic acid encoding an allergen-binding protein into a cell-free protein expression system. In some embodiments, the cell-free protein expression system includes ribosomes.

[0274] This specification also provides methods for treating allergies in subjects requiring allergy treatment. In some embodiments, the method includes administering a therapeutically effective dose to a subject of microorganisms engineered to produce one of the allergen-binding proteins disclosed herein. In some embodiments, the method includes administering a therapeutically effective dose to a subject of microorganisms engineered to produce one of the compositions comprising allergen-binding proteins disclosed herein. In some embodiments, the microorganisms may be yeast. In some embodiments, the microorganisms may be bacteria. In some embodiments, the microorganisms may include bacteria. In some embodiments, the microorganisms may include bacteria, including Escherichia coli. In some embodiments, the microorganisms may include yeast, including Pichia pastoris. In some embodiments, the microorganisms may be administered orally to the subject. In some embodiments, the microorganisms may be ingested. In some embodiments, the microorganisms, upon ingestion, produce allergen-binding proteins or compositions.

[0275] This specification also provides methods for neutralizing allergens. In some embodiments, the method comprises the steps of aerosolizing one of the allergen-binding proteins described herein into a mist, and contacting the mist with a surface containing the allergen. In some embodiments, the method comprises the steps of aerosolizing one of the compositions containing the allergen-binding proteins described herein into a mist, and contacting the mist with a surface containing the allergen. In some embodiments, the aerosolizing step may be performed by an aerosolizing machine. In some embodiments, the aerosolizing machine may be worn by the subject. In some embodiments, the surface includes pet accessories. In some embodiments, the surface includes areas / products on which an animal can sit or walk.

[0276] This specification also provides methods for neutralizing allergens. In some embodiments, the method includes the steps of aerosolizing one of the allergen-binding proteins described herein into a mist and contacting food with the mist. In some embodiments, the method includes the steps of aerosolizing one of the compositions containing the allergen-binding proteins described herein into a mist and contacting food with the mist. In some embodiments, the food is pet food. In some embodiments, the allergen is a pet allergen. In some embodiments, the mist is applied to the food as a topper. In some embodiments, the mist is mixed into the food.

[0277] This specification also provides methods for neutralizing allergens. In some embodiments, the method includes the step of contacting food with one of the allergen-binding proteins described herein. In some embodiments, the method includes the step of contacting food with one of the compositions containing the allergen-binding proteins described herein. In some embodiments, the food is pet food. In some embodiments, the allergen is a pet allergen. In some embodiments, the allergen-binding protein is applied to food as a topper. In some embodiments, the allergen-binding protein is mixed into a mist. In some embodiments, the composition is applied to food as a topper. In some embodiments, the composition is mixed into food.

[0278] This specification also provides methods for treating allergies in subjects requiring allergy treatment. In some embodiments, the method includes a step of aerosolizing one of the allergen-binding proteins described herein into a mist. In some embodiments, the method includes a step of aerosolizing one of the compositions containing the allergen-binding proteins described herein into a mist. In some embodiments, the aerosolizing step may be performed by an aerosolizing machine. In some embodiments, the aerosolizing machine may be worn by the subject. In some embodiments, the method further includes inhalation of the mist by the subject. In some embodiments, the subject may be a human. In some embodiments, the subject may be a non-human animal.

[0279] This specification also provides methods for identifying allergen-binding proteins. In some embodiments, the method includes the step of transfecting target cells with human Fc epsilon receptor I (FceR1). In some embodiments, the method includes the step of contacting target cells with IgE isolated from a subject with a pet allergy. In some embodiments, the method includes the step of contacting target cells with a candidate allergen-binding protein and a pet allergen. In some embodiments, the subject with a pet allergy is a subject with a cat allergy. In some embodiments, the pet allergen is a cat allergen. In some embodiments, the cat allergen is FelD1. In some embodiments, the method includes the step of measuring histamine release by target cells. In some embodiments, the method includes the step of measuring β-hexosaminidase release by target cells.

[0280] kit In some embodiments of this specification, kits for using the compositions described herein are disclosed. In some embodiments, the kits disclosed herein may be used to reduce, minimize, or prevent at least one symptom of an allergic response to an environmental allergen. In some embodiments, the kit may include an assembly of materials or components other than the compositions. In some embodiments, the kit may require additional external materials, including water.

[0281] Instructions for use may be included in the kit. In some embodiments, the kit may include instructions for administering the composition to a subject requiring administration. In some embodiments, the kit may include instructions for measuring the survival rate of the recovered composition to ensure its effectiveness for its intended purpose (e.g., therapeutic effectiveness when used to treat a subject).

[0282] Optionally, this kit may also include other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, or measuring tools, or other useful accessories. The materials or components assembled in this kit may be provided to healthcare professionals in any convenient and suitable form that maintains their operability and usefulness. For example, components may be in dissolved, dehydrated, or lyophilized form, and may be provided at room temperature, refrigerated, or freezing temperature. Components may typically be contained in suitable packaging materials.

[0283] definition Unless otherwise specified, all technical terms, notations, and other technical and scientific terms used herein are intended to have the same meaning as those generally understood by those skilled in the art in the field to which the claimed subject matter pertains. Where applicable, terms with a generally understood meaning are defined herein for clarity and / or easy reference, but the inclusion of such definitions herein should not necessarily be construed as representing something substantially different from the generally understood meaning in the art.

[0284] Throughout this application, various embodiments may be presented in scope form. It should be understood that scope form is merely for convenience and brevity and should not be interpreted as an inflexible limitation to the scope of this disclosure. Therefore, a scope description should be considered to encompass all possible sub-scopes specifically disclosed, as well as the individual numerical values ​​within those scopes. For example, a scope description such as 1–6 should be interpreted to encompass not only the specifically disclosed sub-scopes such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, as well as the individual numbers within those scopes, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the scope.

[0285] As used herein and in the claims, the singular forms "a," "an," and "the" include multiple subjects unless otherwise explicitly indicated by the context. For example, the term "sample" includes multiple samples, including mixtures thereof.

[0286] Whenever the terms "at least," "greater than," or "greater than or equal to" precede the first number in a set of two or more numbers, the terms "at least," "greater than," or "greater than or equal to" apply to each number in the set. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0287] Whenever the terms "no more than," "less than," or "less than or equal to" precede the first number in a set of two or more numbers, the terms "no more than," "less than," or "less than or equal to" apply to each number in the set. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0288] The expressions "at least one of A and B" and "at least one of A or B" can be interpreted as meaning at least A, at least B, or at least A and B (i.e., a set including A and B, which may also include one or more additional elements). The term "A and / or B" can be interpreted as meaning A only, B only, or both A and B.

[0289] The phrases "at least about A, B, and C" and "at least about A, B, or C" can be interpreted as meaning at least about A, at least about B, or at least about C. The phrases "at most about A, B, and C" and "at most about A, B, or C" can be interpreted as meaning at most about A, at most about B, or at most about C.

[0290] The expression "approximately between A and B, between C and D, and between E and F" can be interpreted as meaning approximately between A and B, approximately between C and D, and approximately between E and F. The expression "approximately between A and B, C and D, or E and F" can be interpreted as meaning approximately between A and B, approximately between C and D, or approximately between E and F.

[0291] The expression "approximately A to B and C to D" can be interpreted as meaning the range between approximately A and B, and the range between approximately C and D. The expression "approximately A to B or C to D" can be interpreted as meaning the range between approximately A and B, or the range between approximately C and D.

[0292] As used herein, the term “exemplary” means “serving as an example, case, or illustration.” No embodiment described herein as “exemplary” should be construed as preferable or advantageous to any other embodiment.

[0293] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. These terms include determining whether or not an element is present (e.g., detection). These terms may include quantitative, qualitative, or both quantitative and qualitative determinations. Assessments may be relative or absolute. “Detecting the presence” may, depending on the context, include determining the quantity of something that is present, in addition to determining whether or not it is present.

[0294] The terms “subject,” “individual,” and “patient” are often used interchangeably herein. “Subject” may be a biological entity containing expressed genetic material. A biological entity may be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject may be a tissue, cell, or offspring of a biological entity obtained in vivo or cultured in vitro. A subject may be a mammal. A mammal may be a human. A subject may be diagnosed or suspected of being at high risk of disease. In some cases, a subject may not necessarily be diagnosed or suspected of being at high risk of disease.

[0295] The term "pharmaceutically acceptable" means that it is approved or eligible for approval by a federal or state regulatory authority, or is listed in the United States Pharmacopeia (USP) or other generally accepted pharmacopoeia for use in animals, including humans. A "pharmaceutically acceptable excipient" is an excipient that can be administered to a target along with the active ingredient (i.e., the allergen-binding protein), does not destroy its pharmacological activity, and is non-toxic when administered in a dose sufficient to deliver a therapeutic portion. The term "pharmaceutically acceptable carrier" refers to any non-toxic substance that can be safely administered to a patient and does not interfere with the efficacy of the biological activity of the active ingredient. The term "pharmaceutically acceptable carrier" may also refer to a "biologically compatible carrier," which is a substance that does not interfere with the ability of the allergen-binding protein to bind and is non-toxic for use in humans or animals. Carriers can be solid, liquid, or gaseous and help deliver the active ingredient in a pharmaceutical to a target area of ​​the body. pharmaceutically acceptable carriers may be selected based on their compatibility with the active ingredient and their compatibility with the route of administration. The carrier may include substances that perform functions to stabilize, solubilize, emulsify, suspend, or otherwise promote the functional dispersion of the active ingredient in the pharmaceutical composition. Examples include, but are not limited to, binders, fillers, diluents, solvents, buffers, preservatives, and surfactants commonly used in the field of pharmaceutical formulations.

[0296] The term "therapeutic dose" refers to the amount of compound, composition, or drug that, when administered to a subject or patient, produces a beneficial therapeutic response over time. This response may include, but is not limited to, relief of one or more symptoms, modification or cessation of disease progression, or complete elimination of the disease state. The therapeutic dose can vary based on various factors, including the specific disease being treated, the specific compound, composition, or drug used, the severity of the disease, the patient's age and weight, and the route of administration. Determining the therapeutic dose is within the realm of what a person skilled in the art can conceive, and often involves the use of time-adjusted dosage regimens.

[0297] The term "in vivo" is used to describe events that occur within the body of a subject.

[0298] The term "ex vivo" is used to describe events that occur outside the body of a subject. Ex vivo assays are not performed on the subject itself; rather, they are performed on samples isolated from the subject. An example of an ex vivo assay performed on a sample is an "in vitro" assay.

[0299] The term "in vitro" is used to describe events that occur in a container used to hold laboratory reagents so that they are separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays that utilize living or dead cells. In vitro assays can also encompass cell-free assays that do not utilize intact cells.

[0300] As used herein, the term "about" refers to a number that is plus or minus 10% of that number. The term "about" refers to a range that is 10% of the lowest value plus 10% of the highest value.

[0301] As used herein, the terms “treatment” or “treating” are used in reference to pharmaceutical or other intervention regimens for obtaining beneficial or desired outcomes for a recipient. Beneficial or desired outcomes include, but are not limited to, therapeutic benefits and / or preventive benefits. A therapeutic benefit may refer to the eradication or improvement of the treated symptom or underlying disease. A therapeutic benefit may also be achieved by the eradication or improvement of one or more physiological symptoms associated with an underlying disorder, such that improvement is observed in the subject, even though the subject may still suffer from the underlying disorder. Preventive effects include delaying, preventing, or eliminating the onset of a disease or illness; delaying or eliminating the onset of symptoms of a disease or illness; slowing, stopping, or reversing the progression of a disease or illness; or any combination thereof. In the case of a preventive benefit, a subject at risk of developing a particular disease, or a subject reporting one or more physiological symptoms of a disease, may receive treatment even if a diagnosis of the disease has not been made.

[0302] The term "antibody" can include fully constructed antibodies, antibody fragments capable of binding to antigens, such as Fab, F(ab')2, Fv, single-chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, and single VHH domains derived from camelids (also known as nanobodies).

[0303] The term "complementarity-determining region" or "CDR" refers to a portion of the variable region in an antibody or allergen-binding protein that is structurally complementary to the epitope to which the antibody binds and is more variable than the rest of the variable region. Therefore, CDRs are sometimes referred to as hypervariable regions. A variable region contains three CDRs. CDR peptides can be obtained by constructing a gene encoding the CDR of the antibody of interest. Such genes are prepared, for example, by synthesizing the variable region from RNA of antibody-producing cells using polymerase chain reaction. For example, see Larrick et al., Methods: A Companion to Methods in Enzymology 2:106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pp. 166-179 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), pp. 137-185 (Wiley-Liss, Inc. 1995).

[0304] The term "Fab" refers to a protein containing a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments in that they have several residues added to the carboxyl terminus of the heavy chain CH1 domain containing one or more cysteines derived from the antibody hinge region. Fab'-SH is the herein designation for Fab' fragments in which the cysteine ​​residue of the constant domain has a free thiol group. Fab' fragments are produced by reducing the heavy chain disulfide crosslink of the F(ab')2 fragment. Other chemical couplings of antibody fragments are also known.

[0305] A "single-chain variable fragment (scFv)" is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an antibody, linked to a short linker peptide of 10 to approximately 25 amino acids. The linker is typically rich in glycine for flexibility and serine or threonine for solubility, and can be linked from the N-terminus of VH to the C-terminus of VL, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991), pp. 46-96. Furthermore, the antibody fragment contains a single-chain polypeptide that possesses the characteristics of a VH domain, i.e., can be constructed with a VL domain, or possesses the characteristics of a VL domain, i.e., can be constructed with a VH domain to provide a functional antigen-binding site, thereby conferring the antigen-binding properties of a full-length antibody.

[0306] A "nanobody" is typically a single-domain antibody, or a portion of a single-domain antibody, usually derived from alpacas, llamas, or other camelid animals. Sometimes, a nanobody refers to a single variable domain (VHH domain) in the heavy chain of an antibody. Nanobodies can be modified to increase solubility, stability, efficacy, or any other important characteristics.

[0307] A "monobody" is a synthetic binding protein derived from mutations in the fibronectin type III domain (FN3). The binding interface of the FN3 domain undergoes mutations within the monobody in order to develop protein binders that bind to the target protein.

[0308] "DARPin" is a genetically engineered antibody-mimicking protein that represents a designed ankyrin repeat protein.

[0309] As used herein, the terms “polypeptide,” “peptide,” and “protein” may be used interchangeably with respect to polymers of amino acid residues. A protein may refer to a full-length polypeptide translated from a coding open reading frame or processed into its mature form, while a polypeptide or peptide may also refer to a protein degradation or processing fragment that is still uniquely or identifiablely mapped to a particular protein. A polypeptide may be a single linear polymer chain of amino acids linked to one another by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Polypeptides may be modified, for example, by carbohydrate addition or phosphorylation.

[0310] As used herein, the terms “fragment” or “part,” or equivalent terms, may also refer to a portion of a protein that is less than its full length and, optionally, retains the function of the protein.

[0311] The term "allergen" refers to any naturally occurring protein or mixture of proteins that has been reported to induce an allergic reaction, or IgE-mediated reaction, in individuals upon repeated exposure.

[0312] The section headings used herein are for organizational purposes only and should not be construed as limiting the scope of the subject matter. [Examples]

[0313] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0314] Example 1: ELISA binding assay of anti-Fel D1 nanobodies A 2 microgram / milliliter (mg / mL) target antigen is coated onto a 96-well plate, and an ELISA assay is performed after incubation with candidate VHH. The binding EC50 of each clone is calculated using Prism GraphPad. A positive control antibody is included as an internal control.

[0315] Example 2: Functional blocking assay of Fel D1 Human FcεR1 (FcεRI) is stably transfected into RBL-2H3 cells. The resulting stable cells, along with human IgE from a cat allergy population, cat saliva, a positive control antibody, and candidate VHH cells, are used to evaluate the function of the candidate VHH cells. The EC50 of each clone is calculated using Prism GraphPad. A schematic representation of the functional assay is shown in Figure 1.

[0316] Example 3: Use of a composition to alleviate allergy symptoms To an individual allergic to cat or dog scales, provide the composition disclosed herein, which contains nanobodies that neutralize pet allergens. The composition is mixed with water in a bottle fitted with a sprayer. The water may be tap water. The composition is thoroughly suspended in the water by shaking the bottle. The suspended composition is sprayed into the air, around the house, and on various household surfaces, including pet bedding, personal bedding, air filters, couches, pillows, walls, or floors. In the view of those skilled in the art, the composition reduces allergic symptoms.

[0317] Example 4: Identification of anti-Der p2 VHH To obtain a single variable domain on the heavy chain specific to the house dust mite allergen Der p2, i.e., VHH, highly purified recombinant His-tagged Der p2 was prepared, as shown by SDS-Page Western blotting in Figure 2. To confirm this, ELISA was performed. Briefly, Der p2-HIS was coated onto an ELISA plate, and a known Der p2 antibody (clone 6OY4) was added. Next, a secondary detection antibody that could bind to the Der p2 antibody was added. The secondary detection antibody was conjugated with horseradish peroxidase, which was used to induce a colorimetric reaction. The optical density was then measured. As summarized in Table 1, recombinant Der p2-His was effectively conjugated by the antibody.

[0318] [Table 1]

[0319] Next, recombinant Der p2 was administered to two alpacas four times. The alpacas received 250 μg of recombinant Der p2 on days 0, 14, 28, and 49. ELISA was performed using titrated serum derived from blood to detect the level of anti-Der p2 VHH. The results are summarized in Table 2. Each measurement was performed in pairs. Negative serum was collected from untreated alpacas and used as a negative control.

[0320] [Table 2]

[0321] After the third immunization cycle, peripheral blood mononuclear cells (PBMCs) were obtained from immunized alpacas. RNA was extracted from the PBMCs and reverse transcribed into cDNA. Subsequently, the VHH sequence was amplified from the cDNA using a combination of single-domain antibody cloning primers, subcloned into the yeast display vector pYDisplay, and electrotransformed to produce EYB100 competent cells, thereby constructing a single-domain antibody yeast display library.

[0322] The sequences of 50 randomly selected clones are shown in Figure 3A, demonstrating minimal overlap and library diversity. A single clone was subjected to a flow cytometry assay, where biotinylated recombinant Der p2 was incubated with the VHH clone. After a washing step, the clone was identified as a VHH candidate by detecting the bound Der p2. The VHH candidate was then constructed into a prokaryotic expression vector.

[0323] The isolated VHH clones were further validated by performing the ELISA assay described in Example 1. (Melting temperature (T)) m ), starting temperature (T onset ), and condensation temperature (T agg Measures of thermal stability, including ), were also tested by increasing the temperature from 20°C to 95°C at a rate of 1°C / min. These measurements were performed using a Nanotemper DSF. Where applicable, T m 1 refers to the first observed melting point, T m 2 refers to the second observed melting point. Sequence information and binding assay results for the VHH clones are summarized in Table 3. Individual binding curves are shown in Figures 3B to 3I for each candidate. For each clone, low EC 50 The strong coupling indicated by the values ​​was demonstrated.

[0324] [Table 3-1]

[0325] [Table 3-2]

[0326] Example 5: Identification of anti-Can f1 VHH To obtain a single variable domain (VHH) on the heavy chain specific to the canine allergen Can f1, recombinant His-tagged Can f1 was prepared to high purity, as shown by SDS-Page Western blotting in Figure 4. To confirm this, ELISA was performed to measure the optical density when recombinant Can f1-His was incubated with a biotinylated anti-His antibody. As summarized in Table 4, recombinant Can f1-His was effectively conjugated by the antibody.

[0327] [Table 4]

[0328] Next, recombinant Can f1 was administered to two alpacas four times. The alpacas received 250 μg of recombinant Can f1 on days 0, 14, 28, and 49. ELISA was performed using titrated serum derived from blood to detect the level of anti-Can f1 VHH. The results are summarized in Table 5. Each measurement was performed in pairs. Negative serum was collected from untreated alpacas and used as a negative control.

[0329] [Table 5]

[0330] After the third immunization cycle, peripheral blood mononuclear cells (PBMCs) were obtained from immunized alpacas. RNA was extracted from the PBMCs and reverse transcribed into cDNA. Subsequently, the VHH sequence was amplified from the cDNA using a combination of single-domain antibody cloning primers, subcloned into the yeast display vector pYDisplay, and electrotransformed to produce EYB100 competent cells, thereby constructing a single-domain antibody yeast display library.

[0331] The sequences of 50 randomly selected clones are shown in Figure 5A, demonstrating that there is little overlap and the library is diverse. Single clones were subjected to an assay using flow cytometry, where biotinylated recombinant Can f1 was incubated with the VHH clones, and after a washing step, the clones were identified as VHH candidates by detection of the bound Can f1. The VHH candidates were then constructed into prokaryotic expression vectors.

[0332] The isolated VHH clones were further verified by performing the ELISA assay described in Example 1. The thermal stability metrics, including the melting temperature (T m ), onset temperature (T onset ), and aggregation temperature (T agg ), were also tested by raising the temperature from 20 °C to 95 °C at a rate of 1 °C / min. The sequence information and binding assay results of the VHH clones are summarized in Table 6. The individual binding curves are shown in Figures 5B - 5H for each candidate. Strong binding, indicated by low EC 50 values, was demonstrated for each clone.

[0333]

Table 6-1

[0334]

Table 6-2

[0335] Example 6: Identification of anti-Can f2 VHH To obtain a single variable domain (VHH) on the heavy chain specific to the canine allergen Can f2, recombinant His-tagged Can f2 was prepared to high purity, as shown by SDS-Page Western blotting in Figure 6. To confirm this, ELISA was performed to measure the optical density when recombinant Can f2-His was incubated with a biotinylated anti-His antibody. Recombinant Can f2-His was effectively conjugated by the antibody, as summarized in Table 7.

[0336] [Table 7]

[0337] Next, recombinant Can f2 was administered to two alpacas four times. The alpacas received 250 μg of recombinant Can f2 on days 0, 14, 28, and 49. ELISA was performed using titrated serum derived from blood to detect the level of anti-Can f2 VHH. The results are summarized in Table 8. This was further repeated on alpacas immunized with SUMO-modified Can f2-His, and the results are shown in Table 9. The effectiveness of the Suomo tag in increasing solubility was tested. Each measurement was performed in a double-chain configuration. Negative serum was collected from untreated alpacas and used as a negative control.

[0338] [Table 8]

[0339] [Table 9]

[0340] After the third immunization cycle, peripheral blood mononuclear cells (PBMCs) were obtained from immunized alpacas. RNA was extracted from the PBMCs and reverse transcribed into cDNA. Subsequently, the VHH sequence was amplified from the cDNA using a combination of single-domain antibody cloning primers, subcloned into the yeast display vector pYDisplay, and electrotransformed to produce EYB100 competent cells, thereby constructing a single-domain antibody yeast display library.

[0341] The sequences of 50 randomly selected clones are shown in Figure 7A, demonstrating minimal overlap and library diversity. A single clone was subjected to a flow cytometry assay, where biotinylated recombinant Can f2 was incubated with the VHH clone. After a washing step, the clone was identified as a VHH candidate by detecting the bound Can f2. The VHH candidate was then constructed into a prokaryotic expression vector.

[0342] The isolated VHH clones were further validated by performing the ELISA assay described in Example 1. (Melting temperature (T)) m ), starting temperature (T onset ), and condensation temperature (T agg Measures of thermal stability, including ), were also tested by increasing the temperature at a rate of 1°C / min in the range of 20°C to 95°C. Sequence information and binding assay results for VHH clones are summarized in Table 10. Individual binding curves are shown in Figures 7B to 7P for each candidate. For each clone, low EC 50 The strong coupling indicated by the values ​​was demonstrated.

[0343] [Table 10-1]

[0344] [Table 10-2]

[0345] [Table 10-3]

[0346] Example 7: Identification of anti-Der p1 VHH To obtain a single variable domain (VHH) on the heavy chain specific to the house dust mite allergen Der p1, recombinant His-tagged Der p1 was prepared to high purity, as shown by SDS-Page Western blotting in Figure 8. To confirm this, ELISA was performed to measure the optical density when recombinant Der p1-His was incubated with a known Der p1 antibody (clone 10B9). As summarized in Table 11, recombinant Der p1-His was effectively conjugated by the antibody.

[0347] [Table 11]

[0348] Next, recombinant Der p1 was administered to two alpacas four times. The alpacas received 250 μg of recombinant Der p1 on days 0, 14, 28, and 49. On day 56, the fifth immunization was completed in one alpaca. ELISA was performed using titrated serum derived from blood to detect the level of anti-Der p1 VHH. The results are summarized in Table 12. Each measurement was performed in pairs. Negative serum was collected from untreated alpacas and used as a negative control.

[0349] [Table 12]

[0350] After the fourth and fifth immunization cycles, peripheral blood mononuclear cells (PBMCs) were obtained from each of the two immunized alpacas. RNA was then extracted from the PBMCs and reverse transcribed into cDNA. The VHH sequence was then amplified from the cDNA using a combination of single-domain antibody cloning primers, subcloned into the yeast display vector pYDisplay, and electrotransformed to produce EYB100 competent cells, thereby constructing a single-domain antibody yeast display library.

[0351] The sequences of 50 randomly selected clones demonstrated low overlap, indicating library diversity. A single clone was subjected to a flow cytometry assay, where biotinylated recombinant Der p1 was incubated with the VHH clone. After a washing step, the clone was identified as a VHH candidate by detecting the bound Der p1. The VHH candidate was then constructed into a prokaryotic expression vector.

[0352] The isolated VHH clones were further validated by performing the ELISA assay described in Example 1. The sequence information and binding assay results of the VHH clones are summarized in Table 13. Individual binding curves for each candidate are shown in Figures 9A to 9K. For each clone, a low EC was observed. 50 The strong coupling indicated by the values ​​was demonstrated.

[0353] [Table 13-1]

[0354] [Table 13-2]

[0355] The cysteine ​​protease activity of Der p1 is a major contributor to allergenicity by enhancing total IgE and Der p1-specific IgE synthesis. To screen for VHH candidates capable of inhibiting this enzymatic activity and thus induced IgE synthesis, VHH candidates were incubated with Der p1 and cysteine ​​solution, and the cysteine ​​protease activity of Der p1 was measured by a continuous-rate assay using a fluorescence-generating substrate. Der p1 was pre-activated with 5 mM cysteine ​​(Sigma Chemical Co.) to regenerate its thiol groups, which were then oxidized during purification. The catalytic activity of Derp1 was measured in a continuous-rate (kinetic) assay using the fluorescent peptide substrate N-tert-butoxy-carbonyl (Boc)-Gln-Ala-Arg-7-amino-4-methyl-coumarin (AMC). This assay was performed at 37°C in 1 ml total volume of 50 mM sodium phosphate buffer at pH 7.0 containing 2.5 mM EDTA and 2.5 mM dithiothreitol (DTT). Hydrolysis of the AMC substrate was monitored using Hitachi F-2000 fluorescence at λex=380 nm and λem=460 nm. As summarized in Figure 10, many VHHs demonstrated a blocking effect on Der p1 enzyme activity. These candidates were further tested by titration experiments to confirm their activity. Each VHH was 3-fold diluted up to 5 times in sodium phosphate buffer, starting at 0.6 mg / ml as shown in Table 14. The activity of these substances in blocking Der p1 cysteine ​​protease activity was measured. The titration results for each VHH are shown in Figures 11A to 11H and summarized in Table 15.

[0356] [Table 14]

[0357] [Table 15]

[0358] Example 8: Identification of Der p1-binding peptide A phage display library was prepared using the recombinant Der p1 obtained in Example 7, and 7-amino acid peptides capable of binding to Der p1 were screened. This was carried out in the same manner as the display library described in the previous example, using nucleic acids encoding peptides expressed in the phage. As shown in Figure 12, a large number of peptides were identified, demonstrating a high degree of diversity. The sequences of these peptides are summarized in Table 16.

[0359] [Table 16]

[0360] The identified peptides were validated using ELISA. Der p1 was bound to the bottom of the plate. Various concentrations of selected (biotinylated) peptides were added and then washed. Next, HRP-streptavidin (which binds to biotin) was added, and the OD was measured at 450 nm. The results are summarized in Table 17. Duplicate measurements are separated by commas. An exemplary binding curve for peptide 2-D3 is shown in Figure 13.

[0361] [Table 17]

[0362] Example 9: Identification of anti-Fel D1 VHH To obtain a single variable domain (VHH) on the heavy chain specific to the cat allergen Fel D1, recombinant His-tagged Fel D1 was prepared to high purity, as shown by SDS-Page Western blotting in Figure 14. To confirm this, ELISA was performed to measure the optical density when recombinant Fel d1-His was incubated with two known Fel D1 antibodies summarized in Table 18. As summarized in Table 19, recombinant Fel d1-His was effectively conjugated by both antibodies.

[0363] [Table 18]

[0364] [Table 19]

[0365] Four distinct epitopes of Fel D1 were selected for further identification. These epitopes are summarized in Table 20. Peptides were synthesized for the four epitopes for VHH identification.

[0366] [Table 20]

[0367] Next, recombinant Fel D1 was administered to two alpacas four times. The alpacas received 250 μg of recombinant Fel D1 on days 0, 14, 28, and 49. On day 56, the fifth immunization was completed in one alpaca. ELISA was performed using titrated serum derived from blood to detect the level of anti-Fel D1 VHH. The results are summarized in Table 21. Each measurement was performed in pairs. Negative serum was collected from untreated alpacas and used as a negative control.

[0368] [Table 21]

[0369] After the fourth and fifth immunization cycles, peripheral blood mononuclear cells (PBMCs) were obtained from each of the two immunized alpacas. RNA was then extracted from the PBMCs and reverse transcribed into cDNA. The VHH sequence was then amplified from the cDNA using a combination of single-domain antibody cloning primers, subcloned into the yeast display vector pYDisplay, and electrotransformed to produce EYB100 competent cells, thereby constructing a single-domain antibody yeast display library.

[0370] The sequences of 50 randomly selected clones demonstrated low overlap, as shown in Figure 15A, indicating library diversity. Single clones were subjected to a flow cytometry assay, where biotinylated recombinant Fel D1 was incubated with the VHH clone. After a washing step, the clone was identified as a VHH candidate by detecting the bound Fel D1. The VHH candidate was then constructed into a prokaryotic expression vector.

[0371] The isolated VHH clones were further validated by performing the ELISA assay described in Example 1. The clones were incubated with each of the epitopes listed in Table 20. The binding results for the epitope of SEQ ID NO: 61 are shown in Figures 15B-15D. The binding results for the epitope of SEQ ID NO: 59 are shown in Figure 16. The binding results for the epitope of SEQ ID NO: 60 are shown in Figures 17A-17E. The binding results for the epitope of SEQ ID NO: 58 are shown in Figure 18. The sequence information and binding assay results for the VHH clones are summarized in Table 22. Each clone exhibited low EC values. 50 The strong coupling indicated by the values ​​was demonstrated.

[0372] [Table 22-1]

[0373] [Table 22-2]

[0374] Detailed thermal stability graphs used to determine Tm, Tonset, and Tag are shown in Figures 19A–19I, and the raw data is summarized in Table 23. All subsequent melting points (Tm) observed are shown below. m 1. T m 2, T m Separate terms like 3 with commas.

[0375] [Table 23-1]

[0376] [Table 23-2]

[0377] Functional validation was also performed to test the ability of an exemplary clone to neutralize Fel D1. Fel D1 is a weaker tetramer that typically causes allergic reactions by causing cross-linking of adjacent IgE on immune cells (such as mast cells). Size exclusion chromatography (SEC) is performed, and when the protein is passed through a mesh, larger proteins elute more quickly because they do not flow through the mesh as easily. In SEC, two distinct peaks are present for pure Fel D1, the earlier peak representing the tetramer and the later (laker) peak representing the monomer (Figure 20A). The conditions for this SEC graph are as follows: Column: Zenix-C SEC-80, 7.8 × 300 mm, 3 μm, 80 Å; Sample: Fel D1; Sample volume: 30 μL (25 μg); Mobile phase: 150 mM phosphate buffer, pH 7.0; Flow rate: 0.7 mL / min; Detection: UV 280 nm; System: Thermo Vanquish Flex.

[0378] Assaying clone C3 revealed that it was smaller than the Fel D1 monomer and had a major peak at approximately 14 minutes (Figure 20B). The conditions for this SEC graph were as follows: Column: Zenix-C SEC-80, 7.8 × 300 mm, 3 μm, 80 Å; Sample: 1-C3; Sample volume: 30 μL; Mobile phase: 150 mM phosphate buffer, pH 7.0; Flow rate: 0.7 mL / min; Detection: UV 280 nm; System: Thermo Vanquish Flex.

[0379] When clones C3 and Fel D1 were incubated together at 37°C for 4 hours and then assayed, the two Fel D1 peaks were no longer detectable, and only the C3-Fel D1 complex was detected. In particular, since this complex appeared later than the Fel D1 tetramer, it is demonstrated that the C3 nanobody prevents the formation of the Fel D1 tetramer (Figure 20C). The conditions for this SEC graph were as follows: Column: Zenix-C SEC-80, 7.8 × 300 mm, 3 μm, 80 Å; Sample: 30 μg Fel D1 protein and 30 μg 1-C3 pre-incubated in a 60 μl incubation system at 37°C for 4 hours; Sample volume: 30 μL (25 μg); Mobile phase: 150 mM phosphate buffer, pH 7.0; Flow rate: 0.7 mL / min; Detection: UV 280 nm; System: Thermo Vanquish Flex.

[0380] Preferred embodiments of the present invention have been shown and described herein, but it will be obvious to those skilled in the art that such embodiments are provided merely as examples. Numerous variations, modifications, and substitutions will be conceivable to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be adopted in the practice of the present invention. The following claims define the scope of the present invention, and the methods and structures within these claims, as well as their equivalents, are intended to be encompassed by the claims.

Claims

1. An allergen-binding protein that binds to or neutralizes an allergen, comprising a nanobody, monobody, DARPin, or small peptide with a molecular weight of less than approximately 25 kilodaltons or a length of less than approximately 300 amino acids.

2. An allergen-binding protein that binds to or neutralizes an allergen, comprising a nanobody or small peptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1-57 and 67-73.

3. The allergen-binding protein according to claim 1 or 2, wherein the allergen-binding protein is formulated for coating food or for spraying, misting, or brushing animal or household surfaces.

4. The allergen-binding protein according to claim 3, wherein the food is pet food.

5. The allergen-binding protein according to claim 4, wherein the allergen is a pet allergen.

6. The allergen-binding protein according to any one of claims 3 to 5, wherein the allergen-binding protein is formulated to be applied as a topping on food.

7. The allergen-binding protein according to any one of claims 3 to 5, wherein the allergen-binding protein is formulated to be mixed with food.

8. The allergen-binding protein according to any one of claims 1 to 7, wherein the allergen-binding protein has a concentration of about 0.01 milligrams / milliliter (mg / ml) to about 500 mg / ml in solution or after suspension.

9. The allergen-binding protein according to any one of claims 1 to 8, wherein the allergen induces an allergic reaction in humans.

10. The allergen-binding protein according to any one of claims 1 to 9, wherein the allergen includes an environmental allergen.

11. The allergen-binding protein according to any one of claims 1 to 10, wherein the allergen includes an animal allergen.

12. The allergen-binding protein according to any one of claims 1 to 11, wherein the allergen includes a pet allergen.

13. The allergen-binding protein according to any one of claims 1 to 12, wherein the allergen includes allergens of cats, dogs, rabbits, mice, or cockroaches.

14. The allergen-binding protein according to claim 13, wherein the allergen is selected from the group consisting of Fel d 1, Fel d 2, Fel d 3, Fel d 4, Can f 1, Can f 2, Can f 4, Can f 7, Ory C 1, Mus M 1, and Bla G 2.

15. The allergen-binding protein according to claim 14, wherein the allergen is Fel d 1.

16. The allergen-binding protein according to claim 15, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 49 to 57.

17. The allergen-binding protein according to claim 16, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of SEQ ID NO:

49.

18. The allergen-binding protein according to claim 17, wherein the allergen-binding protein comprises a nanobody having the sequence of SEQ ID NO:

49.

19. The allergen-binding protein according to claim 16, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of Sequence ID No.

57.

20. The allergen-binding protein according to claim 19, wherein the allergen-binding protein comprises a nanobody having the sequence of SEQ ID NO:

57.

21. The allergen-binding protein according to claim 14, wherein the allergen is Can f 1.

22. The allergen-binding protein according to claim 21, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 67 to 73.

23. The allergen-binding protein according to claim 22, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO:

67.

24. The allergen-binding protein according to claim 23, wherein the allergen-binding protein includes a nanobody having the sequence corresponding to sequence number 67.

25. The allergen-binding protein according to claim 14, wherein the allergen is Can f 2.

26. The allergen-binding protein according to claim 25, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 9 to 23.

27. The allergen-binding protein according to claim 26, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO:

16.

28. The allergen-binding protein according to claim 27, wherein the allergen-binding protein includes a nanobody containing the amino acid sequence of SEQ ID NO:

16.

29. The allergen-binding protein according to any one of claims 1 to 10, wherein the allergen includes a dust allergen.

30. The allergen-binding protein according to claim 29, wherein the dust allergen comprises Der p1 or Der p2.

31. The allergen-binding protein according to claim 30, wherein the dust allergen is Der p1.

32. The allergen-binding protein according to claim 31, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 24 to 34.

33. The allergen-binding protein according to claim 32, wherein the allergen-binding protein comprises a peptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to a sequence selected from the group consisting of SEQ ID NOs. 35 to 48.

34. The allergen-binding protein according to claim 33, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO:

27.

35. The allergen-binding protein according to claim 34, wherein the allergen-binding protein includes a nanobody having the sequence of sequence number 27.

36. The allergen-binding protein according to claim 33, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO:

28.

37. The allergen-binding protein according to claim 36, wherein the allergen-binding protein includes a nanobody having the sequence of sequence number 28.

38. The allergen-binding protein according to claim 30, wherein the dust allergen is Der p2.

39. The allergen-binding protein according to claim 38, wherein the allergen-binding protein comprises nanobodies having at least 80%, at least 85%, at least 90%, or 100% sequence identity with respect to a sequence selected from the group consisting of SEQ ID NOs: 1 to 8.

40. The allergen-binding protein according to claim 39, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO:

3.

41. The allergen-binding protein according to claim 40, wherein the allergen-binding protein includes a nanobody having the sequence of sequence number 3.

42. The allergen-binding protein according to any one of claims 1 to 10, wherein the allergen includes a plant or plant pollen allergen.

43. The allergen-binding protein according to claim 42, wherein the allergen of the plant or plant pollen is selected from the group consisting of Bet v1, Phl p 5, Phl p 1, Poa p 1, Cyn d 1, Bet v 2, Ole e 1, Amb a 1, Amb a 11, and Art v 1.

44. The allergen-binding protein according to any one of claims 1 to 10, wherein the allergen includes a mold allergen.

45. The allergen-binding protein according to claim 44, wherein the mold allergen is selected from the group consisting of Alt a 1, Asp f 1, Asp f 2, Cl h 8, Pen ch 13, and Pen ch 18.

46. The allergen-binding protein according to any one of claims 1 to 10, wherein the allergen is a food allergen.

47. The allergen-binding protein according to claim 46, wherein the food allergen is selected from the group consisting of Pen a 1, Ara h 1, and Ara h 3.

48. The allergen-binding protein according to any one of claims 1 to 47, wherein the allergen-binding protein comprises at least one modified amino acid.

49. The allergen-binding protein according to any one of claims 1 to 48, wherein the allergen-binding protein comprises about 1, about 5, about 10, about 15, about 20, about 25, or about 30 modified amino acids.

50. The allergen-binding protein according to claim 48 or 49, wherein the modified amino acid is a non-standard amino acid.

51. The allergen-binding protein according to claim 50, wherein the non-standard amino acid is selected from the group consisting of para-benzoylphenylalanine, 3,4-dihydroxyphenylalanine, tetrazine, chlorooctene, homopropagylglycine, para-propagyloxyphenylalanine, para-azidophenylalanine, para-isothiocyanatephenylalanine, para-benzoylphenylalanine, para-cyanophenylalanine, para-nitrophenylalanine, m-halogenated tyrosine analog, halogenated proline analog, halogenated tryptophan analog, and halogenated leucine analog.

52. The allergen-binding protein according to any one of claims 48 to 51, wherein the solubility of the allergen-binding protein is increased by about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, or more compared to an unmodified allergen-binding protein.

53. The allergen-binding protein according to any one of claims 48 to 51, wherein the binding affinity of the allergen-binding protein is increased by about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or more, compared to the unmodified allergen-binding protein.

54. The allergen-binding protein according to any one of claims 48 to 51, wherein the thermal stability of the allergen-binding protein is increased by about 10%, 25%, 50%, 75%, 100%, 200%, 300%, or more compared to an unmodified allergen-binding protein.

55. The allergen-binding protein according to any one of claims 1 to 54, wherein the allergen-binding protein is a polyvalent allergen-binding protein.

56. The allergen-binding protein according to claim 55, wherein the polyvalent allergen-binding protein is a bivalent allergen-binding protein.

57. The allergen-binding protein according to claim 55 or 56, wherein the binding affinity of the polyvalent allergen-binding protein is increased by about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or more, compared to the monovalent allergen-binding protein.

58. The allergen-binding protein according to any one of claims 1 to 57, further comprising a carrier.

59. The allergen-binding protein according to claim 58, wherein the carrier comprises a solvent, a diluent, a dispersion medium, or a coating.

60. The allergen-binding protein according to claim 59, wherein the carrier contains silica.

61. The allergen-binding protein according to any one of claims 1 to 60, wherein the allergen-binding protein is in a dried form.

62. The allergen-binding protein according to claim 58, wherein the carrier contains water.

63. The allergen-binding protein according to any one of claims 1 to 62, wherein the allergen-binding protein comprises a liquid.

64. The allergen-binding protein according to any one of claims 1 to 63, wherein the allergen-binding protein further comprises a preservative.

65. The allergen-binding protein according to claim 64, wherein the preservative comprises potassium sorbate, EDTA, benzoic acid, phenoxyethanol, or maltol.

66. The allergen-binding protein according to any one of claims 1 to 65, wherein the allergen-binding protein further comprises a stabilizer or a thickener.

67. The allergen-binding protein according to claim 66, wherein the stabilizer or thickener comprises dextrin, maltodextrin, glycerol, glucose, sucrose, or trehalose.

68. The allergen-binding protein according to any one of claims 1 to 67, wherein the allergen-binding protein further comprises an isotonic agent.

69. The allergen-binding protein according to claim 1 or 2, wherein the allergen-binding protein is suspended or resuspended in a solvent.

70. The allergen-binding protein according to claim 69, wherein the solvent contains water.

71. A composition comprising an allergen-binding protein that binds to or neutralizes an allergen, wherein the allergen-binding protein comprises a nanobody, monobody, DARPin, or small peptide having a molecular weight of less than about 25 kilodaltons or a length of less than about 300 amino acids.

72. A composition comprising an allergen-binding protein that binds to or neutralizes an allergen, wherein the allergen-binding protein comprises a nanobody or small peptide with a molecular weight of less than about 25 kilodaltons or a length of less than about 300 amino acids, and the allergen-binding protein comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1 to 57 and 67 to 73.

73. The composition according to claim 71 or 72, wherein the composition is formulated for coating food or for spraying, misting, or brushing onto animal or household surfaces.

74. The composition according to claim 73, wherein the food is pet food.

75. The composition according to claim 74, wherein the allergen is a pet allergen.

76. The composition according to any one of claims 73 to 75, wherein the allergen-binding protein is formulated to be applied as a topping on food.

77. The composition according to any one of claims 73 to 75, wherein the allergen-binding protein is formulated to be mixed with food.

78. The composition according to any one of claims 71 to 73, wherein the allergen-binding protein is concentrated in solution or after suspension at a concentration of about 0.01 milligrams / milliliter (mg / ml) to about 500 mg / ml.

79. The composition according to any one of claims 71 to 74, wherein the allergen induces an allergic reaction in humans.

80. The composition according to any one of claims 71 to 79, wherein the allergen includes an environmental allergen.

81. The composition according to any one of claims 71 to 80, wherein the allergen includes an animal allergen.

82. The composition according to any one of claims 71 to 81, wherein the allergen includes a pet allergen.

83. The composition according to any one of claims 71 to 82, wherein the allergen includes an allergen of a cat, dog, rabbit, mouse, or cockroach.

84. The composition according to claim 83, wherein the allergen is selected from the group consisting of Fel d 1, Fel d 2, Fel d 3, Fel d 4, Can f 1, Can f 2, Can f 4, Can f 7, Ory C 1, Mus M 1, and Bla G 2.

85. The composition according to claim 84, wherein the allergen is Fel d 1.

86. The composition according to claim 85, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 49 to 57.

87. The composition according to claim 86, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of Sequence ID No.

49.

88. The composition according to claim 86, wherein the allergen-binding protein comprises a nanobody having the sequence of Sequence ID No.

49.

89. The composition according to claim 86, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence of Sequence ID No.

57.

90. The composition according to claim 86, wherein the allergen-binding protein comprises a nanobody having the sequence of Sequence ID No.

57.

91. The composition according to claim 84, wherein the allergen is Can f 1.

92. The composition according to claim 91, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 67 to 73.

93. The composition according to claim 92, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO:

67.

94. The composition according to claim 92, wherein the allergen-binding protein comprises a nanobody having the sequence corresponding to sequence number 67.

95. The composition according to claim 84, wherein the allergen is Can f 2.

96. The composition according to claim 95, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 9 to 23.

97. The composition according to claim 95, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO:

16.

98. The composition according to claim 97, wherein the allergen-binding protein comprises a nanobody containing the amino acid sequence of SEQ ID NO:

16.

99. The composition according to any one of claims 71 to 80, wherein the allergen includes a dust allergen.

100. The composition according to claim 99, wherein the dust allergen comprises Der p1 or Der p2.

101. The composition according to claim 100, wherein the dust allergen is Der p1.

102. The composition according to claim 101, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 24 to 34.

103. The composition according to claim 101, wherein the allergen-binding protein comprises a peptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to a sequence selected from the group consisting of SEQ ID NOs. 35 to 48.

104. The composition according to claim 101, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO:

27.

105. The composition according to claim 104, wherein the allergen-binding protein comprises a nanobody having the sequence of sequence number 27.

106. The composition according to claim 101, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO:

28.

107. The composition according to claim 104, wherein the allergen-binding protein comprises a nanobody having the sequence of sequence number 28.

108. The composition according to claim 100, wherein the dust allergen is Der p2.

109. The composition according to claim 106, wherein the allergen-binding protein comprises nanobodies having at least 80%, at least 85%, at least 90%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1 to 8.

110. The composition according to claim 107, wherein the allergen-binding protein comprises a nanobody containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to the sequence described in SEQ ID NO:

3.

111. The composition according to claim 108, wherein the allergen-binding protein comprises a nanobody having the sequence corresponding to Sequence ID No.

3.

112. The composition according to any one of claims 71 to 80, wherein the allergen includes a plant or plant pollen allergen.

113. The composition according to claim 112, wherein the allergen of the plant or plant pollen is selected from the group consisting of Bet v1, Phl p 5, Phl p 1, Poa p 1, Cyn d 1, Bet v 2, Ole e 1, Amb a 1, Amb a 11, and Art v 1.

114. The composition according to any one of claims 71 to 80, wherein the allergen includes a mold allergen.

115. The composition according to claim 114, wherein the mold allergen is selected from the group consisting of Alt a 1, Asp f 1, Asp f 2, Cl h 8, Pen ch 13, and Pen ch 18.

116. The composition according to any one of claims 72 to 80, wherein the allergen is a food allergen.

117. The composition according to claim 116, wherein the food allergen is selected from the group consisting of Pen a 1, Ara h 1, and Ara h 3.

118. The composition according to any one of claims 71 to 117, wherein the allergen-binding protein comprises at least one amino acid modification.

119. The composition according to claim 118, wherein the allergen-binding protein comprises about 1, about 5, about 10, about 15, about 20, about 25, or about 30 amino acid modifications.

120. The composition according to claim 118 or 119, wherein the amino acid modification includes the introduction of a non-standard amino acid.

121. The composition according to claim 120, wherein the non-standard amino acid is selected from the group consisting of para-benzoylphenylalanine, 3,4-dihydroxyphenylalanine, tetrazine, chlorooctene, homopropagylglycine, para-propagyloxyphenylalanine, para-azidophenylalanine, para-isothiocyanatephenylalanine, para-benzoylphenylalanine, para-cyanophenylalanine, para-nitrophenylalanine, m-halogenated tyrosine analog, halogenated proline analog, halogenated tryptophan analog, and halogenated leucine analog.

122. The composition according to any one of claims 118 to 121, wherein the solubility of the composition increases by about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, or more, compared to an unmodified allergen-binding protein.

123. The composition according to any one of claims 118 to 121, wherein the binding affinity of the allergen-binding protein is increased by about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or more, compared to the unmodified allergen-binding protein.

124. The composition according to any one of claims 118 to 121, wherein the thermal stability of the allergen-binding protein is increased by about 10%, 25%, 50%, 75%, 100%, 200%, 300%, or more, compared to an unmodified allergen-binding protein.

125. The composition according to any one of claims 71 to 124, wherein the allergen-binding protein is a polyvalent allergen-binding protein.

126. The composition according to claim 125, wherein the polyvalent allergen-binding protein is a bivalent allergen-binding protein.

127. The composition according to claim 124 or 125, wherein the binding affinity of the polyvalent allergen-binding protein composition is increased by about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or more, compared to a monovalent allergen-binding protein.

128. The composition according to any one of claims 71 to 127, further comprising a carrier.

129. The composition according to any one of claims 71 to 128, wherein the carrier comprises a solvent, a diluent, a dispersion medium, or a coating.

130. The composition according to claim 128, wherein the carrier contains silica.

131. The composition according to any one of claims 71 to 130, wherein the composition is in a dry state.

132. The composition according to claim 128, wherein the carrier contains water.

133. The composition according to any one of claims 71 to 132, wherein the composition comprises a liquid.

134. The composition according to any one of claims 71 to 133, further comprising a preservative.

135. The composition according to claim 134, wherein the preservative comprises potassium sorbate, EDTA, benzoic acid, phenoxyethanol, or maltol.

136. The composition according to any one of claims 71 to 135, wherein the composition further comprises a stabilizer or a thickener.

137. The composition according to claim 136, wherein the stabilizer or thickener comprises dextrin, maltodextrin, glycerol, glucose, sucrose, or trehalose.

138. The composition according to any one of claims 71 to 137, further comprising an isotonic agent.

139. The composition according to claim 71 or 72, wherein the composition is suspended or resuspended in a solvent.

140. The composition according to claim 139, wherein the solvent comprises water.

141. A method comprising the steps of spraying or atomizing an allergen-binding protein according to any one of claims 3 to 70 or a composition according to any one of claims 73 to 140, or bringing a surface into contact with a composition according to any one of claims 73 to 140.

142. The method according to claim 141, wherein the contact step includes coating or brushing.

143. The method according to claim 141 or 142, wherein the surface includes the surface of an air filter or a humidifier.

144. The method according to any one of claims 141 to 143, wherein the surface contains an allergen.

145. The method according to any one of claims 141 to 144, wherein the composition neutralizes the allergen on the surface.

146. The method according to any one of claims 141 to 145, wherein the surface contains food.

147. The method according to claim 146, wherein the food is consumed by an animal that is a source of antigen.

148. The method according to any one of claims 141 to 145, wherein the surface includes a pet accessory (e.g., a collar or brush) or an area / product on which an animal can sit or walk (e.g., a dog bed).

149. The method according to any one of claims 141 to 148, wherein the spraying step is performed by a humidifier or a spray bottle.

150. The method according to any one of claims 141 to 149, wherein spraying about 0.01 milliliters (ml) to about 20 ml of the composition per area of ​​about 1 square meter neutralizes the allergenicity of the antigen.

151. A method for preparing an allergen-binding protein according to any one of claims 1 to 140 or a composition according to any one of claims 71 to 140, comprising the step of collecting the allergen-binding protein from an engineered microorganism or from a secretion by the microorganism, wherein the microorganism contains a heterologous nucleic acid encoding the allergen-binding protein.

152. The method according to claim 151, further comprising the step of incorporating the heterologous nucleic acid encoding the allergen-binding protein into a cell-free protein expression system.

153. The method according to claim 151, wherein the microorganism includes yeast or bacteria.

154. The method according to claim 153, wherein the microorganism includes bacteria, and includes Escherichia coli (E. coli).

155. The method according to claim 153, wherein the microorganism includes yeast and includes Pichia pastoris.

156. The method according to claim 151, wherein the allergen-binding protein is purified or concentrated from the secretions of the microorganism.

157. The method according to claim 154, wherein the purification or concentration includes a filtration step.

158. The method according to claim 157, wherein the filtration step includes passing the secretion through a filter of about 0.01 nm, 0.1 nm, 1 nm, 5 nm, or larger.

159. The method according to any one of claims 151 to 158, wherein the centrifugal separation step is not included.

160. A method for treating an allergy in a subject requiring allergy treatment, comprising the step of administering to the subject an allergen-binding protein according to any one of claims 1 to 70 or a composition according to any one of claims 71 to 140.

161. A method for treating an allergy in a subject requiring allergy treatment, comprising the step of administering to the subject a therapeutically effective amount of a microorganism manipulated to produce an allergen-binding protein according to any one of claims 1 to 70 or a composition according to any one of claims 71 to 140.

162. The method according to claim 161, wherein the microorganism is a yeast or a bacterium.

163. The method according to claim 162, wherein the microorganism includes bacteria, and includes Escherichia coli (E. coli).

164. The method according to claim 162, wherein the microorganism includes yeast and includes Pichia pastoris.

165. The method according to any one of claims 161 to 162, wherein the microorganism is administered orally to the subject.

166. The method according to any one of claims 161 to 163, wherein the microorganism, upon ingestion, produces the allergen-binding protein or the composition.

167. A method for neutralizing an allergen, comprising: (i) aerosolizing an allergen-binding protein according to any one of claims 3 to 70 or a composition according to any one of claims 73 to 140 into a mist; and (ii) contacting the mist with a surface containing an allergen.

168. The method according to claim 167, wherein the aerosolization step is performed by an aerosolization machine.

169. The method according to claim 168, wherein the aerosolizing machine is worn by the subject.

170. The method according to any one of claims 167 to 169, wherein the surface includes a pet accessory or an area / product on which an animal can sit or walk.

171. A method for neutralizing an allergen, comprising: (i) aerosolizing an allergen-binding protein according to any one of claims 3 to 70 or a composition according to any one of claims 73 to 140 into a mist; and (ii) contacting the mist with a food product.

172. The method according to claim 171, wherein the food is pet food.

173. The method according to claim 171, wherein the allergen is a pet allergen.

174. The method according to any one of claims 171 to 173, wherein the mist is applied as a topping on the food.

175. The method according to any one of claims 171 to 173, wherein the mist is mixed into the food.

176. A method for neutralizing an allergen, comprising the step of bringing an allergen-binding protein according to any one of claims 1 to 70 or a composition according to any one of claims 71 to 140 into contact with food.

177. The method according to claim 176, wherein the food is pet food.

178. The method according to claim 176, wherein the allergen is a pet allergen.

179. The method according to any one of claims 176 to 178, wherein the allergen-binding protein or the composition is applied as a topping on the food.

180. The method according to any one of claims 176 to 178, wherein the allergen-binding protein or the composition is mixed into the food.

181. A method for treating an allergy to a subject requiring allergy treatment, comprising the step of aerosolizing an allergen-binding protein according to any one of claims 3 to 70 or a composition according to any one of claims 73 to 140 into a mist.

182. The method according to claim 171, wherein the aerosolization step is performed by an aerosolization machine.

183. The method according to claim 171 or 182, wherein the aerosolizing machine is worn.

184. The method according to claim 183, wherein the aerosolizing machine is worn by the subject.

185. The method according to any one of claims 171 to 184, further comprising inhaling the mist by the subject.

186. The method according to any one of claims 171 to 185, wherein the subject is a human.

187. The method according to any one of claims 171 to 185, wherein the subject is a non-human animal.

188. (i) an allergen-binding protein according to any one of claims 1 to 70 or a composition according to any one of claims 71 to 140, and (ii) a pharmaceutically acceptable excipient.

189. An allergen-binding protein containing one of the sequences from sequence numbers 1-57 or 67-73.

190. An allergen-binding protein containing the sequence of Sequence ID No.

3.

191. An allergen-binding protein containing the sequence of SEQ ID NO:

16.

192. An allergen-binding protein containing the sequence of SEQ ID NO:

27.

193. An allergen-binding protein containing the sequence of SEQ ID NO:

28.

194. An allergen-binding protein containing the sequence of Sequence ID No.

49.

195. An allergen-binding protein containing the sequence of Sequence ID No.

57.

196. An allergen-binding protein containing the sequence of SEQ ID NO: 67.

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