Fusion Protein Formulations and Uses

Abstract

The present invention provides a solid formulation comprising microparticles comprising a fusion protein and at least one carrier, a method for preparing the solid formulation, and a method for treating allergic rhinitis in an individual, the method comprising administering to the individual a therapeutically effective amount of a solid formulation of the fusion protein.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, and in particular to solid formulations of fusion proteins, methods for preparing the solid formulations, and methods for treating allergic rhinitis in individuals with the solid formulations. [Background technology]

[0002] The immune system is constantly working to fight off parasites, fungi, viruses, and bacteria. However, the immune system can treat harmless substances (called allergens) as unwanted invaders and try to fight them. This overreaction of the body's immune system to normally harmless substances is called an allergic reaction.

[0003] There are a limited number of bioactive macromolecules for treating allergic reactions. Omalizumab is the only antibody approved by the FDA to target and block immunoglobulin E (IgE).

[0004] Studies have shown that by conjugating the FcεRI receptor and the FcγRII receptor in a fusion protein, the fusion protein can regulate the IgE-mediated signal transduction pathway and suppress the release of active mediators, thus inhibiting allergic reactions (WO2005085291A1).

[0005] However, preservation and storage of large proteins, including such fusion proteins, presents concerns due to their high degradability resulting from chemical instability (e.g., any process involving protein modification by bond formation or cleavage to generate new chemical entities) or physical instability (e.g., changes in the protein's higher-order conformation). Chemical instability can result from racemization, hydrolysis, oxidation, or beta-elimination, while physical instability can result from denaturation, aggregation, precipitation, or adsorption. A commercially available protein formulation must be administered safely and maintained to remain physically, chemically, and biologically stable within its recommended shelf life.

[0006] Allergic rhinitis, also known as "hay fever," is inflammation of the nose that occurs when the immune system overreacts to airborne allergens.

[0007] The present invention surprisingly reveals that the solid formulation of the present application can be administered to mammals via nasal administration, preferably via inhalation, and can effectively suppress or alleviate the symptoms of allergic rhinitis. Summary of the Invention

[0008] It is an object of the present invention to provide a solid formulation of a fusion protein that is stable within its recommended shelf life, preferably for one year at room temperature, and that comprises substantially spherical fusion protein particles dispersed in at least one carrier and that can be administered intranasally, preferably by inhalation.

[0009] Another object of the present invention is to provide a method for preparing a solid formulation.

[0010] It is yet another object of the present invention to provide a method for treating allergic rhinitis with a solid formulation.

[0011] In one aspect, the present invention provides a solid formulation comprising microparticles comprising a fusion protein and at least one carrier. Suitable carriers for the present invention include polyols, carbohydrates, and sugars. The carrier may be selected from the group consisting of sucrose, trehalose, sorbitol, glycerol, mannitol, lactose, xylitol, arabitol, erythritol, lactitol, maltitol, glucose, raffinose, maltose, dextran, inositol, or a combination thereof.

[0012] In a preferred embodiment, the microparticles comprise a fusion protein and two or more carriers, preferably two carriers, which are lactose and mannitol.

[0013] The fusion protein is a polypeptide comprising the amino acid sequence of SEQ ID No. 2 or an amino acid sequence containing deletions, substitutions, additions, or insertions of one or several amino acid residues in the amino acid sequence of SEQ ID No. 2.

[0014] In a preferred embodiment, the fusion protein comprises: 1) the DNA sequence of SEQ ID No. 1; 2) the amino acid sequence of SEQ ID No. 2, and 3) an amino acid sequence having at least 95% identity with SEQ ID No. 2, and a DNA sequence that further encodes the same functional protein sequence.

[0015] In a preferred embodiment, the DNA sequence of SEQ ID NO:1 consists of 1665 base pairs, and the reading frame is from the 1st base pair to the 1665th base pair starting from the 5' end.

[0016] Extensions of any fragment of the fusion protein are also included within the scope of the fusion protein.

[0017] The fusion protein may be one disclosed in WO2005085291A1.

[0018] In a preferred embodiment, the fusion protein comprises the amino acid sequence of SEQ ID No. 2 or has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 2.

[0019] In a preferred embodiment, the fusion protein is FP4 having the amino acid sequence of SEQ ID No. 2.

[0020] The present inventors have surprisingly discovered that the biological activity of solid formulations remains substantially unchanged after storage at room temperature for one year, and furthermore, the solid formulations have a substantially spherical shape and a substantially uniform diameter, and are suitable for administration by inhalation.

[0021] In another aspect, the invention provides a method of treating allergic rhinitis comprising administering to an individual a solid formulation comprising a therapeutically effective amount of a fusion protein.

[0022] In another aspect, the present invention provides the use of a solid formulation comprising a fusion protein for preparing a medicament for treating allergic rhinitis in an individual.

[0023] In a preferred embodiment, the fusion protein comprises the amino acid sequence of SEQ ID No. 2 or has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 2.

[0024] In a preferred embodiment, the fusion protein is FP4 having the amino acid sequence of SEQ ID No. 2.

[0025] In one aspect, the present invention relates to a solid formulation of a fusion protein, said solid formulation consisting of microparticles comprising said fusion protein and at least one carrier, and wherein said fusion protein comprises the amino acid sequence of SEQ ID No. 2 or is a protein having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 2.

[0026] In a preferred embodiment, the present invention relates to a solid formulation, wherein said microparticles comprise a fusion protein and one or more carriers.

[0027] In a preferred embodiment, the present invention relates to a solid formulation wherein said carrier is selected from the group consisting of polyols, carbohydrates and sugars.

[0028] In a preferred embodiment, the present invention relates to a solid formulation wherein the carrier is selected from the group consisting of sucrose, trehalose, sorbitol, glycerol, mannitol, lactose, xylitol, arabitol, erythritol, lactitol, maltitol, glucose, raffinose, maltose, dextran, inositol or combinations thereof.

[0029] In a preferred embodiment, the present invention relates to a solid formulation wherein the microparticles have a 50% cumulative volume diameter (D50) of 5 μm or less, preferably 4.5 μm or less, more preferably 3.5 to 4.5 μm, and a 90% cumulative volume diameter (D90) of 35 μm or less, preferably 30 μm or less, more preferably 28 to 30 μm.

[0030] In another preferred embodiment, the present invention relates to a solid formulation, wherein the microparticles have a 50% cumulative diameter by volume (D50) of 5 μm to more than 12 μm, preferably 8 to 12 μm, more preferably 8 to 10 μm, even more preferably about 9 μm or about 10 μm, and most preferably about 10 μm, and a 90% cumulative diameter by volume (D90) of 40 μm or less, preferably 38 μm or less, more preferably 34 to 36 μm.

[0031] In a preferred embodiment, the present invention relates to a solid formulation that is substantially free of surfactants, preferably a neat formulation.

[0032] In a preferred embodiment, the present invention relates to a solid formulation having a shelf life of at least six months, preferably at least one year.

[0033] In a preferred embodiment, the present invention relates to solid formulations that are inhalable.

[0034] In a preferred embodiment, the present invention relates to a solid formulation comprising 0.1 to 5 wt %, preferably about 0.5 to 4 wt %, more preferably 1 to 4 wt % of the fusion protein.

[0035] In another aspect, the present invention relates to a method for preparing said solid formulation, comprising the steps of spraying a suspension or solution of a fusion protein into a stream of droplets, entraining the stream of droplets in a stream of coolant to freeze the droplets into frozen microparticles, and drying such microparticles to form said solid formulation.

[0036] In another aspect, the invention relates to a method for treating allergic rhinitis in an individual comprising administering to the individual a therapeutically effective amount of the above-described solid formulation.

[0037] In a preferred embodiment, the present invention relates to a method for treating allergic rhinitis in an individual, wherein said solid formulation is administered by inhalation, preferably by a nasal spray device. [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 shows the particle size of one exemplary solid formulation of the present invention. [Figure 2] FIG. 2 shows the particle size of another exemplary solid formulation of the present invention. [Figure 3] FIG. 3 shows the biological activity and stability of the fusion protein FP4 present in the solid formulation of the present invention. [Figure 4]FIG. 4 shows that FP4 fusion proteins block allergic responses in transgenic mice. DETAILED DESCRIPTION OF THE INVENTION

[0039] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The incorporation of technology used herein is intended to refer to technology commonly understood in this field, and includes obvious modifications or equivalent substitutions made by those skilled in the art to the technology. Although it is believed that those skilled in the art will be familiar with the following terms, the following definitions are provided to better understand the present invention.

[0040] As used herein, the terms "including," "comprising," "having," "containing," or "comprising," and other variations thereof, are intended to be inclusive or open-ended and do not exclude other elements or method steps not listed.

[0041] The term "amino acid" refers to any compound containing both an amino group and a carboxylic acid group. The amino group is most commonly located adjacent to the carboxy functionality, but the amino group can be located anywhere within the molecule. Amino acids may further contain additional functional groups such as amino, thio, carboxyl, carboxamide, or imidazole groups. Amino acids may be synthetic or naturally occurring and may be used in either their racemic or optically active (D- or L-) form, including various ratios of stereoisomers.

[0042] A "neat" formulation according to the present invention refers to a solid formulation consisting of microparticles or a powder containing the fusion protein and at least one carrier (typically, the fusion protein comprises 0.1-5% by weight of the total mass of the fusion protein and at least one carrier), which is substantially free of additional excipients, i.e., contains less than about 1% by weight of other excipients.

[0043] "Fast-release" powders or microparticles are powders or microparticles that do not have controlled or sustained release properties when administered by inhalation.

[0044] A formulation consisting of fine particles or powder generally refers to a powder formulation containing less than about 10% moisture, preferably less than about 7% moisture, more preferably less than about 5-6% moisture, even more preferably less than about 3% moisture, even more preferably less than about 2% moisture, and most preferably less than about 1% moisture, depending on the type of active agent in the formulation.

[0045] An "inhalable" formulation "suitable for nasal delivery" refers to a formulation that contains solid (i.e., non-liquid) particles that (i) are readily dispersed within or by an inhalation device, and (ii) can be inhaled by a subject such that at least a majority of the particles reach the nasal cavity. Such powders are considered "respirable" or "inhalable."

[0046] In the context of the present invention, "surfactant-free" refers to a formulation that contains less than about 0.1% surfactant by weight.

[0047] When "about" is followed by a value or ratio, it means ±10%, preferably ±5%, and more preferably ±1% of such value or ratio.

[0048] "Substantially" has the meaning commonly known in the art. For example, "substantially no X is present (does not contain X)" means that X is present in an amount of 10 wt% or less, preferably 5 wt% or less, and more preferably 1 wt% or less, and "substantially unchanged" means that the decomposition rate is 10% or less, preferably 5% or less, and more preferably 1% or less.

[0049] "50% volume cumulative diameter (D50) of m or less" means that particles with a diameter of m or less account for 50% by volume of all particles. Similar meanings apply to 10% volume cumulative diameter (D10), 90% volume cumulative diameter (D90), etc. Particle sizes reported herein are measured by laser diffraction, although any commonly used technique (e.g., electron microscopy, light scattering, centrifugal sedimentation) can be used.

[0050] As used herein, the term "solvate" refers to a substance formed by combining, physically associating, and / or solvating a compound of the present invention with solvent molecules, e.g., a disolvate, a monosolvate, or a hemisolvate, in which the ratio of solvent molecules to the compound of the present invention is about 2:1, about 1:1, or about 1:2, respectively. Such physical association involves varying degrees of ionization and covalent bonding (including hydrogen bonding). In some cases (e.g., when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid), the solvate may be separated. Thus, solvates include both solution-phase and separable solvates. The compounds of the present invention may be in solvated forms with pharmaceutically acceptable solvents (e.g., water, methanol, ethanol, etc.), and the present application is intended to encompass both solvated and unsolvated forms of the compounds of the present invention.

[0051] One type of solvate is a hydrate. "Hydrate" refers to a specific subset of solvates in which the solvent molecule is water. Solvates generally function as pharmacological equivalent forms. The preparation of solvates is known in the art; see, for example, M. Caira et al., J. Pharmaceut. Sci., 93(3):601-611 (2004), which describes the preparation of a solvate of fluconazole with ethyl acetate and water. Similar methods for preparing solvates, hemisolvates, hydrates, etc. are described in van Tonder et al., AAPS Pharm. Sci. Tech., 5(1):Article 12 (2004) and A.L. Bingham et al., Chem. Commun. 603-604 (2001). A representative, non-limiting method for preparing a solvate includes dissolving a compound of the invention in a desired solvent (organic solvent, water, or a mixture thereof) at a temperature above 20° C. to about 25° C., then cooling the solution at a rate sufficient to form crystals, which are isolated by known methods such as filtration. Analytical techniques such as infrared spectroscopy can be used to confirm the presence of solvent in the solvate crystals.

[0052] In the context of the present invention, a "pharmaceutically acceptable carrier," "carrier," or "excipient" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered and which is suitable for contact with the tissues of humans and / or other animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. Further, in the context of the present invention, a "pharmaceutically acceptable carrier," "carrier," or "excipient" refers to an excipient that may optionally be included in a formulation of the present invention and which enters the nasal cavity or lungs and has no significant adverse toxicological effects on the subject, particularly the nasal cavity or lungs of the subject.

[0053] Pharmaceutically acceptable carriers or excipients that can be used in the pharmaceutical formulations of the present invention include, but are not limited to, sterile liquids such as water and oils, including those derived from petroleum, animal, vegetable, or synthetic sources (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Other pharmaceutical carriers or excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, nonfat dry milk, glycerin, propylene glycol, mannitol, water, ethanol, and the like. Pharmaceutical formulations may further contain pH buffering agents, if desired. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).

[0054] The solid formulations of the invention can act systemically and / or locally. To this end, they can be administered via a suitable route, for example, intranasally, transmucosally, topically, or by inhalation.

[0055] For these administration routes, the solid formulation of the present invention can be administered in an appropriate dosage form.

[0056] Dosage forms include, but are not limited to, pulvis or sprays.

[0057] As used herein, the term "effective amount" refers to that amount of active ingredient which, when administered, will relieve to some extent one or more of the symptoms of the condition being treated.

[0058] As used herein, an "individual" includes a human or a non-human animal. Exemplary human individuals include human individuals (referred to as patients) suffering from a disease (such as a disease described herein) or normal individuals. "Non-human animals" in the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, e.g., non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0059] Solid Formulations To better treat nasal disorders, inhalation administration can be advantageous because it allows the drug to reach the nasal cavity or lungs directly, resulting in rapid onset, improved bioavailability, and reduced dosage requirements. Furthermore, inhalation administration reduces systemic exposure, reduces drug toxicity, thereby reducing side effects, and is more convenient for patients.

[0060] However, formulating protein drugs into stable powders or microparticles remains a challenge.

[0061] Freeze-drying and spray-drying are commonly used to formulate protein drugs into powders. Currently, freeze-drying is the most widely used. Freeze-drying is applicable to heat-sensitive proteins, but it is not suitable for producing uniform powders with diameters of several micrometers to tens of micrometers, which can be easily inhaled. Freeze-drying also tends to concentrate proteins between ice crystals upon cooling. This concentration causes rapid changes in the pH and ionic strength surrounding the protein, leading to denaturation and precipitation (Schwartz, P.L. et al., Endocrinology, 92(6):1795, 1973; Koseki, T. et al., J. Biochem., 107:389, 1990). In contrast, spray-drying involves atomizing a continuous stream of liquid sample to form microscopically dispersed droplets, which are then instantly dried with hot air. Spray-drying has already been used to formulate a variety of drugs. Spray-drying has the advantage of producing powders whose particle size is suitable for drug delivery to the airways and lungs. Proteins are generally not stable to heat, so there are not many cases of protein pharmaceutical preparations being spray-dried with hot air.

[0062] Although some prior art powder or particulate solid formulations of proteins do not change their physical state (do not form lumps) within a given time at room temperature, it is most difficult for such solid formulations to maintain their biological activity over an extended period of time. Alternatively, such solid formulations may not be in particulate form or suitable for inhalation.

[0063] Furthermore, in the present invention, it has been found that a solid formulation having a D50 particle size of about 10 μm is most suitable for treating allergic rhinitis via nasal administration. The solid formulation has a particle size of about 10 μm, e.g., 8-12 μm, 5-15 μm, or 2-20 μm, which is the optimal particle size for adsorption to the nasal mucosa to improve the bioavailability of the fusion protein.

[0064] The present invention surprisingly reveals that fusion proteins can be sufficiently dispersed in lactose and mannitol to form stable, substantially uniform microparticles at room temperature, and the biological activity of the fusion protein contained therein remains substantially unchanged over a period of one year, making them particularly suitable for administration by inhalation. The mass percentage of the fusion protein and the carrier, such as lactose and mannitol, can be varied as long as the fusion protein remains stable in the solid formulation.

[0065] In a preferred embodiment, the solid formulation of the present invention is substantially free of surfactants, more preferably a neat formulation.

[0066] In a preferred embodiment, the solid formulations of the present invention are prepared by an atmospheric pressure freeze-drying process. In another aspect, the present invention provides a method for preparing a solid formulation of a fusion protein, said method comprising spraying a suspension or solution of the fusion protein into a stream of droplets, entraining the stream of droplets in a stream of coolant to freeze the droplets into frozen microparticles, and drying such microparticles to form said solid formulation.

[0067] Treatment Methods and Uses The present invention discloses that a solid formulation comprising a fusion protein formed by conjugating an FcεRI receptor and an FcγRII receptor (preferably comprising the amino acid sequence of SEQ ID No. 2 or having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 2, more preferably the fusion protein is FP4 having the amino acid sequence of SEQ ID No. 2) is particularly effective for the treatment of allergic rhinitis. Preferably, the solid formulation of the present invention can be administered to mammals via nasal administration, preferably by inhalation, and can effectively suppress or alleviate the symptoms of allergic rhinitis. [Example]

[0068] In order to clarify the purpose and technical solution of the present invention, the present invention will be further described below by specific examples. It should be understood that these examples are not intended to limit the scope of the present invention. Furthermore, specific experimental methods not mentioned in the following examples are carried out according to conventional experimental methods.

[0069] material and method Based on WO2005085291A1, a fusion protein (called FP4) having the amino acid sequence of SEQ ID No. 2 is prepared and purified.

[0070] Other reagents, including mannitol, lactose, PBS buffer and Evans blue, were purchased from Fisher Scientific.

[0071] CHO3D10 cells transfected with FcεRIα were purchased from ATCC.

[0072] Transgenic mice were purchased from Jackson Laboratory.

[0073] Example 1: Preparation and characterization of solid formulations FP4 is thoroughly mixed with mannitol and lactose in water, and the mixture is subjected to atmospheric freeze-drying process to obtain a solid formulation. Specifically, the mixture is sprayed into a liquid particle stream, which forms frozen particles at a temperature sufficiently reduced to entrain the frozen particles in a coolant stream and form frozen particles, and these frozen particles are dried at a relatively low temperature (which may be higher than the reduced temperature) to form the solid formulation.

[0074] The particle sizes of such particles were measured using a SympaTEC HELOS / BR device, and exemplary results are shown in Figure 1. It can be seen that particles with a diameter of 1.09 μm or less account for 10% by volume, particles with a diameter of 1.44 μm or less account for 16% by volume, particles with a diameter of 4.02 μm or less account for 50% by volume, particles with a diameter of 13.41 μm or less account for 84% by volume, particles with a diameter of 29.05 μm or less account for 90% by volume, and particles with a diameter of 134.82 μm or less account for 99% by volume.

[0075] The experiment was repeated multiple times. Another exemplary particle size distribution is shown in Figure 2. It can be seen that particles with a diameter of 1.33 μm or less account for 10% by volume, particles with a diameter of 2.03 μm or less account for 16% by volume, particles with a diameter of 8.87 μm or less account for 50% by volume, particles with a diameter of 24.56 μm or less account for 84% by volume, particles with a diameter of 34.76 μm or less account for 90% by volume, particles with a diameter of 48.21 μm or less account for 95% by volume, particles with a diameter of 61.42 μm or less account for 98% by volume, and particles with a diameter of 70.04 μm or less account for 99% by volume. Compared to the results shown in Figure 1, the median particle size is shifted to a relatively higher value, and the distribution has a relatively narrower span.

[0076] Example 2: Activity and stability testing of solid formulations The solid formulations were stored at room temperature for 1 month. The biological activity of the solid formulations was determined according to the following procedure.

[0077] The solid preparation prepared in Example 1, with a D50 of 8.87 μm, and stored as described above, was dissolved in PBS buffer and then incubated with CHO3D10 cells transfected with human FcεRIα. After 1 hour, the cells were washed and stained with PE-conjugated anti-human IgE. The binding ability was analyzed by flow cytometry (see the "Sample" row in Figure 3). Purified FP4 protein was used as a positive control (see the "PC" column in Figure 3). Cells alone and antibody ("Ab") alone were used as negative controls (see the "NC" row in Figure 3).

[0078] FIG. 3 reveals that the biological activity of FP4 remains substantially unchanged when the solid formulation containing FP4 is stored at room temperature for one month.

[0079] After storing the solid formulation at room temperature for a longer period, for example, 2 months, 3 months... 1 year, the biological activity of the solid formulation is measured according to the above procedure, and the test results reveal that when the solid formulation containing FP4 is stored at room temperature for up to 1 year, the biological activity of FP4 does not change substantially.

[0080] Example 3: Biological studies of solid formulations Transgenic mice expressing the human FcεRI α-chain and knockout of the mouse FcεRI α-chain were primed intradermally with 250 ng of NP-specific recombinant human IgE in 50 μl of saline. Different doses of FP4 fusion protein were simultaneously injected into individual sites. Four hours later, the mice were challenged intravenously with 1.5 mg / ml NP-BSA + 1% Evans blue in 300 μl of saline. Cutaneous anaphylaxis was assessed visually by the blue dye seeping from the blood vessels into the skin. In the event of an allergic reaction, the local skin color turned blue due to the dye seeping from the blood vessels. As shown in Figure 4, the addition of FP4 fusion protein partially blocked the allergic reaction at 0.1 μg and 1 μg doses, and completely blocked it at 10 μg doses (no blue local skin color). The results of this experiment further demonstrated that the FP4 fusion protein inhibited allergic reactions in vivo.

[0081] Example 4: Treatment of allergic rhinitis with a solid formulation Volunteers diagnosed with allergic rhinitis participated in a study to test the therapeutic effect of the solid formulation of the present invention. 50 volunteer subjects with different demographic characteristics (e.g., gender, age, weight) participated in the study. Participants self-evaluated the following nasal symptoms based on the criteria in Table 1 below, taking into account the symptoms of sneezing, runny nose, nasal congestion, and nasal itching. Before and after treatment, each participant scored themselves 0, 1, 2, or 3.

[0082] [Table 1]

[0083] During the treatment period, each participant was treated with 2 mg of the solid formulation with a D50 of 8.87 μm (FP4 fusion protein accounted for approximately 1 wt% of the total mass, i.e., a dose of approximately 20 μg) prepared in Example 1, inhaled into each nostril twice daily for three days. The scores reported by participants before and after treatment are shown in Table 2 below.

[0084] [Table 2]

[0085] As can be seen from the above results, after treatment, the volunteers experienced significant relief from symptoms of nasal congestion, sneezing, and / or runny nose, or complete recovery from these symptoms.

[0086] [Table 3]

[0087] Sequence Listing SEQ ID NO:1

[0088] SEQ ID NO:2 FTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGKGSEPKSCDKTHTCPPCPAPELLGGPSVPLPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKPNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGPYPSDIAVEWESNGQPENNYKTTPPVLDSDGSPPLYSKLTVDKSRWQQGNVPSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. 1. A solid formulation of a fusion protein, comprising: The solid formulation comprises microparticles comprising the fusion protein and at least one carrier, and the fusion protein comprises the amino acid sequence of SEQ ID No. 2 or is a protein having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID No.

2.

2. 2. The solid formulation of claim 1, wherein the fusion protein is FP4 having the amino acid sequence of SEQ ID No.

2.

3. 10. The solid formulation of claim 1, wherein the microparticles comprise a fusion protein and one or more carriers.

4. The solid formulation according to any one of claims 1 to 3, wherein the carrier is selected from the group consisting of polyols, carbohydrates and sugars.

5. 5. The solid formulation of claim 1, wherein the carrier is selected from the group consisting of sucrose, trehalose, sorbitol, glycerol, mannitol, lactose, xylitol, arabitol, erythritol, lactitol, maltitol, glucose, raffinose, maltose, dextran, inositol, or a combination thereof.

6. 6. A solid formulation according to any one of claims 1 to 5, wherein the microparticles have a 50% volume cumulative diameter (D50) of 5 μm or less, preferably 4.5 μm or less, more preferably 3.5 to 4.5 μm, and a 90% volume cumulative diameter (D90) of 35 μm or less, preferably 30 μm or less, more preferably 28 to 30 μm.

7. 6. The solid formulation according to any one of claims 1 to 5, wherein the microparticles have a 50% cumulative diameter by volume (D50) of from 5 μm to more than 12 μm, preferably from 8 to 12 μm, more preferably from 8 to 10 μm, even more preferably from about 9 μm to about 10 μm, and most preferably about 10 μm, and a 90% cumulative diameter by volume (D90) of not more than 40 μm, preferably not more than 38 μm, more preferably from 34 to 36 μm.

8. A solid formulation according to any one of claims 1 to 7, wherein the solid formulation is substantially free of surfactants, preferably a neat formulation.

9. 9. The solid formulation according to any one of claims 1 to 8, which has a shelf life of at least 6 months, preferably at least 1 year.

10. The solid formulation according to any one of claims 1 to 9, wherein the solid formulation is inhalable.

11. 11. The solid formulation according to any one of claims 1 to 10, wherein the solid formulation comprises 0.1 to 5 wt%, preferably about 0.5 to 4 wt%, more preferably 1 to 4 wt% of the fusion protein.

12. 12. A method for preparing a solid formulation according to any one of claims 1 to 11, said method comprising the steps of spraying a suspension or solution of a fusion protein into a stream of droplets, entraining the stream of droplets in a stream of coolant to freeze the droplets into frozen microparticles, and drying such microparticles to form said solid formulation.

13. A method for treating allergic rhinitis in an individual, comprising administering to said individual a therapeutically effective amount of a solid formulation according to any one of claims 1 to 11.

14. 14. The method of claim 13, wherein the solid formulation is administered by inhalation, preferably by a nasal spray device.

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