DRY POWDER FORMULATIONS OF THYMIC STROMAL LYMPHOPOIETY LEUKEMIA (TSLP) BINDING ANTIBODIES AND METHODS OF USE THEREOF

Abstract

The present technology relates generally to dry powder formulations of antibodies specific for thymic stromal lymphopoietin (TSLP) and methods of using the dry powder formulations to treat asthma, preferably via pulmonary delivery.

Description

[Technical field]

[0001] The present technology relates generally to dry powder formulations of antigen-binding fragments derived from antibodies specific for Thymic Stromal Lymphocyte Poisoning Protein (TSLP) and methods of using the dry powder formulations via pulmonary delivery to treat asthma, including mild, moderate and severe asthma, eosinophilic asthma and non / hypoeosinophilic asthma. The dry powder formulations include a mixture of leucine and trileucine, which makes the formulation particularly suitable for delivery of antigen-binding fragments derived from anti-TSLP antibodies via inhalation. [Background technology]

[0002] Asthma affects an estimated 300 million people worldwide, across all age groups, and places a serious burden on health systems and society through lost productivity at work and family disruption (“Pocket Guide for Asthma Management and Prevention,” Global Initiative for Asthma; 2019). Asthma causes symptoms such as wheezing, shortness of breath, chest tightness and coughing, the occurrence, frequency and intensity of which vary over time. Symptoms are often associated with bronchoconstriction, thickening of airway walls and increased mucus production. Asthma has a range of symptoms and can be well-controlled or poorly controlled based on the number and severity of attacks.

[0003] Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine produced in response to environmental and proinflammatory stimuli, activating multiple inflammatory cells and downstream pathways. TSLP is increased in the airways of asthmatic patients and correlates with Th2 cytokine and chemokine expression and disease severity. TSLP is central to Th2-type immune regulation, but may also play an important role in other inflammatory pathways and thus may be associated with multiple asthma phenotypes.

[0004] Delivery of antibodies against TSLP to patients, particularly by inhalation, may provide improved therapy for asthma patients, including those with mild asthma who may require daily low doses. Summary of the Invention

[0005] In view of the above, in one aspect, provided herein is a dry powder formulation comprising a plurality of microparticles, the microparticles comprising leucine, about 1% to about 10% by weight of trileucine, and an antigen-binding fragment of a thymic stromal lymphopoietin (TSLP) antibody.

[0006] In some embodiments, the antigen-binding fragment of an anti-thymic stromal lymphopoietin (TSLP) antibody comprises a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:1, a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:2, and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:3, wherein any of the heavy chain CDR1, 2, or 3 optionally comprises a heavy chain variable domain comprising a single amino acid substitution; and a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:5, a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:6, and a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:7, wherein any of the light chain CDR1, 2, or 3 optionally comprises a light chain variable domain comprising a single amino acid substitution; and the leucine and trileucine are present in a concentration ratio of about 0.1:1 to about 30:1 leucine:trileucine.

[0007] In another embodiment, there is provided a method of treating asthma in a patient comprising administering by inhalation a dry powder formulation of the first embodiment.

[0008] In another aspect, there is provided a dry powder formulation according to the first aspect for use in a method of treatment, wherein the formulation is administered by inhalation. In some embodiments, the formulation is for use in the treatment of asthma.

[0009] The foregoing and other features and aspects of the present technology may be better understood from the following description of embodiments, as illustrated in the accompanying drawings, which are incorporated in and form a part of this specification and further serve to illustrate the principles of the present technology. The drawings are not necessarily to scale. [Brief description of the drawings]

[0010] [Figure 1] Binding of Fab1 to human TSLP as measured by KinExA is shown. [Diagram 2] Binding of Fab1 to cynomolgus TSLP as measured by KinExA is shown. [Diagram 3] 1 shows competitive binding of Fab1 to human TSLP as measured using an HTRF assay. [Figure 4] 1 shows that Fab1 inhibits CCL17 release from TSLP-stimulated PBMCs. [Diagram 5] 1 shows that Fab1, Fab2 and Fab3 inhibit TSLP-induced CCL17 release from PBMCs. [Figure 6] AC show serum, BAL, and ELF PK profiles of Fab1 after single (groups 1 and 2) and multiple ascending dose (group 3) inhalation. [Figure 7] 1 shows microparticles of a dry powder formulation according to an embodiment. [Figure 8A] 1 shows the results of compressed bulk density as a function of leucine and trileucine in dry powder formulations. [Figure 8B] 1 shows the filling of capsules with a dry powder formulation described herein. [Figure 9] The specific surface area of ​​the microparticles of the dry powder formulation according to the present embodiments was measured using BET and is shown in m2 / g. [Figure 10] 1 shows an indirect correlation between moisture content and leucine concentration. [Figure 11] A to D show the surface rugosity of the microparticles detected by SEM. [Figure 12]1 shows the correlation between mass median aerodynamic diameter (MMAD) and wt% of leucine and trileucine. [Figure 13] 4 shows the correlation between device deposition and wt% values ​​of leucine and trileucine. [Figure 14] 1 shows the correlation between fine particle fraction (FPF) and wt% of leucine and trileucine. [Figure 15A] The number of subvisible particles is shown after reconstitution of a formulation containing 40% (w / w) Fab1 and various concentrations of polysorbate-80 (PS-80) to a solution concentration of 30 mg / ml Fab1 (in the figure, "≧" includes an upper size limit of 200 μm). [Figure 15B] The number of subvisible particles is shown after reconstitution of formulations containing 40% (w / w) Fab1 and various concentrations of PS-80 to a solution concentration of 2.5 mg / ml Fab1 (in the figure, "≧" includes an upper size limit of 200 μm). [Figure 16A] The number of subvisible particles is shown after reconstitution of formulations containing 40% (w / w) Fab1 and various concentrations of poloxamer-188 to a solution concentration of 30 mg / ml Fab1 (in the figure, "≧" includes an upper size limit of 200 μm). [Figure 16B] The number of subvisible particles is shown after reconstitution of formulations containing 40% (w / w) Fab1 and various concentrations of poloxamer-188 to a solution concentration of 2.5 mg / ml Fab1 (in the figure, "≧" includes an upper size limit of 200 μm). [Figure 17A] 4 shows the moisture content of a formulation containing 40% (w / w) Fab1 and 1.1% PS-80 after storage at 40° C. and 75% relative humidity (40 / 75) for 1 month or 3 months and at 25° C. and 60% relative humidity (25 / 60) for 3 months. [Figure 17B] FIG. 1 shows particle size distribution (PSD) of a formulation containing 40% (w / w) Fab1 and 1.1% PS-80 after storage at 40° C. and 75% relative humidity (40 / 75) for 1 month or 3 months and at 25° C. and 60% relative humidity (25 / 60) for 3 months. [Figure 17C]FIG. 1 shows particle morphology of a formulation containing 40% (w / w) Fab1 and 1.1% PS-80 after storage at 40° C. and 75% relative humidity (40 / 75) for 1 month or 3 months and at 25° C. and 60% relative humidity (25 / 60) for 3 months. [Figure 18A] 4 shows the moisture content of a formulation containing 1% (w / w) Fab1 and 1.1% PS-80 after storage for 1 or 3 months at 40° C. and 75% relative humidity (40 / 75) and 3 months at 25° C. and 60% relative humidity (25 / 60). [Figure 18B] FIG. 1 shows particle size distribution (PSD) of a formulation containing 1% (w / w) Fab1 and 1.1% PS-80 after storage at 40° C. and 75% relative humidity (40 / 75) for 1 month or 3 months and at 25° C. and 60% relative humidity (25 / 60) for 3 months. [Figure 18C] FIG. 1 shows particle morphology of a formulation containing 1% (w / w) Fab1 and 1.1% PS-80 after storage at 40° C. and 75% relative humidity (40 / 75) for 1 month or 3 months and at 25° C. and 60% relative humidity (25 / 60) for 3 months. [Figure 19A] A formulation containing 40% Fab1 and 1.1% PS-80 (w / w) was reconstituted to a solution concentration of 30 mg / ml Fab1, and the number of subvisible particles after storage for 1 or 3 months at 40 / 75 and for 3 months at 25 / 60 is shown. [Figure 19B] A formulation containing 1% Fab1 and 0.75% PS-80 (w / w) was reconstituted to a solution concentration of 30 mg / ml Fab1, and the number of subvisible particles after storage for 1 or 3 months at 40 / 75 and for 3 months at 25 / 60 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The dry powder formulations described herein address an unmet need by enabling the use of anti-TSLP antibody binding fragments for the treatment of asthma in a primary care setting. Subjects with asthma typically manage their asthma symptoms by self-delivering pharmaceutical compositions, such as long-acting beta agonists and / or glucocorticoids, via inhalation.

[0012] Existing biologic agents, both approved and clinically tested, offer new treatment paradigms for asthma patients, but they generally cannot be delivered to subjects via the familiar pulmonary route. Tezepelumab, a next-generation biologic agent, is a human immunoglobulin G2 (lgG2) monoclonal antibody (mAb) that binds to TSLP and inhibits its interaction with the TSLP receptor complex. In a recent phase 2 randomized, double-blind, placebo-controlled clinical trial, asthma subjects who received subcutaneous injections of tezepelumab had reduced rates of clinically significant asthma exacerbations compared to subjects who received a placebo (Corren et al (2017) NEJM 377:936-946).

[0013] The invention described herein combines the therapeutic benefits of next-generation biologic agents, such as tezepelumab, with a route of administration that is more accessible to subjects suffering from asthma. Thus, the invention enables such next-generation therapeutic agents to be administered in a primary care setting, expanding the availability of these agents to subjects who are underserved by specialized care.

[0014] In addition, the formulations described herein may be particularly useful for treating patients with less severe asthma, who are typically managed in a primary care setting. For example, patients with a Global Initiative for Asthma (GINA) scale of 3 or less, preferably a GINA scale of 2 or 3, may be particularly suitable for treatment with the formulations described herein. In certain embodiments, patients with a GINA score of 3 are suitable for treatment with the formulations described herein. In certain embodiments, patients with a GINA score of 2 are suitable for treatment with the formulations described herein. Furthermore, by delivering the biologic agent directly to the lungs, side effects associated with systemic administration, such as injection site inflammation, are reduced.

[0015] In addition, the formulation offers the possibility to treat patients with moderate-severe asthma who can be managed in a primary care setting or who have insufficient access to treatment through specialized care. For example, the formulation may be useful for treating patients with moderate-severe asthma with Global Initiative for Asthma (GINA) scale 4-5. Suitably, the formulation offers the possibility to treat uncontrolled moderate-severe asthma. Suitably, the formulation offers the possibility to treat moderate-severe asthma uncontrolled with medium to high doses of ICS:LABA with one or more exacerbations and frequent symptoms.

[0016] The term "about" is used herein to mean approximately, in the vicinity of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value with a variance of 10%.

[0017] As described herein, dry powder formulations are provided for the stabilization and delivery of pharma- ceutical active agents. Suitably, the dry powder formulations are formulated for pulmonary delivery, including by inhalation via a dry powder inhaler (DPI).

[0018] As used herein, a "dry powder formulation" refers to a formulation that includes a plurality of solid particulates in a powder composition that preferably contains less than about 20% moisture, more preferably less than 10% moisture, less than about 5-6% moisture, or less than about 3% moisture. As described herein, the dry powder formulation can be utilized for delivery by inhalation to a patient. In other embodiments, the dry powder formulation can be reconstituted and administered in liquid form either orally, intravenously, parenterally, etc. As described herein, one advantage of the dry powder formulations provided is increased throughput that improves manufacturability. An additional advantage is that the formulation platform described herein provides a high compressed bulk density. This means that more powder can be loaded per delivery unit (e.g., in a capsule). This means that a higher dose of active agent can be delivered to the subject per unit delivery. This surprising advantage may improve patient compliance by reducing the number of unit doses that need to be taken. In addition, the high compressed bulk density allows for a higher dose of active agent to be delivered, raising the upper end of the dose range that is administered. This may allow for delivery of active agents at therapeutically effective doses that were not previously possible.

[0019] As used herein, "microparticles" refers to solid particles having a mass mean diameter (MMD) size of less than 20 μm. Mass mean diameter is a measure of the average particle size of a microparticle and is measured using a suitable method, such as centrifugal sedimentation, electron microscopy, light scattering, laser diffraction, etc.

[0020] The dry powder formulations described herein preferably contain a plurality of microparticles. As used herein, "plurality" refers to two or more, preferably 5 or more, 10 or more, 50 or more, 100 or more, 500 or more, 1000 or more, etc.

[0021] In embodiments, the dry powder formulation comprises a plurality of microparticles, preferably comprising leucine, about 1% to about 10% by weight trileucine, and an anti-TSLP antibody-binding fragment as defined herein. Unless otherwise indicated, "active agent" refers to an antigen-binding fragment derived from an anti-TSLP antibody as defined herein.

[0022] 7 shows a scanning electron micrograph of microparticles of an exemplary dry powder formulation provided herein. In a further embodiment, the dry powder formulation comprising a plurality of microparticles preferably comprises about 1% to about 25% leucine, about 1% to about 10% trileucine, and an active agent.

[0023] As used herein, "leucine" refers to the amino acid leucine (C 6 H 13 NO 2 ), either in its racemic mixture or in its D- or L-form, as well as modified forms of leucine (i.e., those in which one or more atoms of leucine have been replaced with another atom or functional group). The chemical structure of leucine is shown below: [ka]

[0024] As used herein, "tri-leucine" refers to the compound C in which three leucine molecules are linked together in a peptide, such as leucine-leucine-leucine (Leu-Leu-Leu). 18 H 35 N 3 O 4 The chemical structure of trileucine is shown below. [ka]

[0025] The amounts of leucine and trileucine provided herein are given as a percentage by weight (wt%) of the formulation unless otherwise indicated. Dry powder formulations contain substantially no water, so the weight components of the dry powder formulations are the dry weight percentages of the final formulation.

[0026] In embodiments of the formulations comprising leucine, trileucine, and an antigen-binding fragment, the leucine and trileucine are maintained in a desired ratio range that provides the improved packed bulk density characteristics described herein, and further provides desired microparticle characteristics that may improve storage and delivery. In embodiments, the weight ratio of leucine to trileucine in the microparticles, i.e., leucine:trileucine, is about 0.1:1 to about 30:1. In further embodiments, the leucine and trileucine are present in a weight ratio of about 0.1:1 to about 25:1, about 0.5:1 to about 20:1, about 1:1 to about 20:1, about 1:1 to about 15:1, about 1:1 to about 12:1, about 1:1 to about 10:1, about 1:1 to about 7:1, about 1:1 to about 6:1, or about 1:1:, about 2:1, about 3:1, about 4:1, about 5:1, about 5.1:1:, about 5.2:1 about 5.25:1, about 5.3:1, about 5.4:1, about 5.5:1, about 5.75:1, or about 6:1 leucine:trileucine.

[0027] Unless otherwise indicated, the ratios described herein are expressed by weight % ratio (w / w - also referred to as "weight ratio"), i.e., weight of leucine:weight of trileucine in the formulations described herein. This ratio is achieved by providing the desired mg / mL concentrations of leucine and trileucine in a feedstock, followed by drying to remove the feedstock solvent, resulting in sprayed microparticles in which the starting concentration ratio (expressed in mg / mL) is maintained as a final weight ratio of leucine:trileucine.

[0028] Exemplary weight percentages of leucine and trileucine that can be utilized in the dry powder formulation to achieve these ratios are described herein. Preferably, the dry powder formulation comprises about 5% to about 15% leucine and about 1% to about 5% trileucine. In embodiments, the dry powder formulation comprises about 8% to about 11% leucine and about 2% to about 4% trileucine, and in embodiments, the dry powder formulation comprises about 10.5% leucine and about 2% trileucine.

[0029] In exemplary embodiments, the dry powder formulation comprises about 1% to about 10% by weight of trileucine, more preferably about 1% to about 9% by weight, about 1% to about 8% by weight, about 1% to about 7% by weight, about 1% to about 6% by weight, about 1% to about 5% by weight, about 2% to about 10% by weight, about 2% to about 9% by weight, about 2% to about 8% by weight, about 2% to about 7% by weight, about 2% to about 6% by weight, about 2% to about 5% by weight, about 2% to about 4% by weight, or about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, or about 6% by weight of trileucine.

[0030] In exemplary embodiments, the dry powder formulation comprises about 1% to about 25% by weight of leucine, more preferably about 2% to about 20% by weight, about 3% to about 20% by weight, about 4% to about 20% by weight, about 5% to about 20% by weight, about 5% to about 15% by weight, about 7% to about 12% by weight, about 8% to about 11% by weight, about 9% to about 11% by weight, about 10% to about 11% by weight, or about 5%, about 6%, about 7%, about 8%, about 8.5%, about 9%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12.5%, or about 13% by weight of leucine.

[0031] In a preferred embodiment, the dry powder formulation comprises about 8% to about 11% by weight leucine and about 2% to about 4% by weight trileucine, more preferably about 9% to about 11% by weight leucine and about 2% to about 3% by weight trileucine. In an exemplary embodiment, the dry powder formulation comprises about 10.5% by weight leucine and about 2% by weight trileucine.

[0032] As described herein, it has been surprisingly found that the combined use of leucine and trileucine in dry powder formulations reduces the total amount of leucine and trileucine required to prepare microparticles, compared to dry powder formulations that contain only one of these components, and still provides desired stability.In certain embodiments, the formulations of the present invention have increased compacted bulk density compared to the formulations of the art, which may allow for higher concentrations of active agent to be delivered to the lungs of patients after inhalation.These improved characteristics are believed to be related to the incorporation of leucine and trileucine into microparticles.

[0033] An exemplary process for preparing a dry powder formulation according to the present embodiment may be carried out as follows: A liquid feedstock containing the desired final components of the dry powder formulation is atomized using an atomizer into a fine mist. The mist is then dried as described herein. The atomized droplets contain the dissolved components initially as liquid droplets. As the droplets are dried, the various components of the formulation begin to saturate and precipitate at different rates. As described herein, a shell begins to form around the outer surface of the microparticles of the dry powder formulation. This shell preferably includes leucine and trileucine components on the outer surface of the shell. It should be noted that leucine and trileucine will be preferentially located on the outer surface of the microparticle, although small amounts of leucine and trileucine may be present throughout the microparticle. In an embodiment, a higher concentration of leucine and trileucine is present at or near the surface of the microparticle, rather than near the center of the microparticle. In an embodiment, the core of the microparticle contains a significant amount of active agent, preferably in amorphous form, along with other excipient components as described herein. As used herein, a "substantial amount" of active agent means that at least about 60% of the active agent (i.e., of the total active agent in the formulation) is located at or near the center of the microparticle, and preferably at least about 70%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, and in embodiments, between about 95% and 100% of the active agent is located at or near the center of the microparticle.

[0034] In further embodiments, the microparticles contain leucine and trileucine located substantially throughout the microparticle, but in greater amounts at or near the surface of the microparticle. As used herein, "substantially throughout the microparticle" means that the leucine and / or trileucine is located in a gradient from the outer surface of the microparticle toward the center of the microparticle, but preferably the amount of leucine and / or trileucine decreases toward the center, and in embodiments, there is no leucine or trileucine in the center of the microparticle where the active agent is located. In other embodiments, the amount of leucine and trileucine may be substantially uniform across the cross-section of the microparticle.

[0035] In an embodiment, substantially each of the microparticles of the dry powder formulation comprises leucine and trileucine. That is, preferably, at least about 60% of the microparticles contain leucine and trileucine, or alternatively, at least about 70%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, and in embodiments, about 95% to 100% of the microparticles contain leucine and trileucine. In an embodiment, each of the microparticles of the dry powder formulation comprises leucine and trileucine.

[0036] In additional embodiments, leucine and / or trileucine may be present in the dry powder formulation, but is not contained in or bound to microparticles of the formulation.Thus, in embodiments, free leucine and / or trileucine that is not bound to microparticles may be present in the dry powder formulation.However, in general, the amount of free leucine and / or trileucine (i.e., not bound to microparticles) is equal to less than about 10%, less than about 5%, less than about 1%, more preferably less than about 0.1% of the total amount of leucine and / or trileucine in the formulation.

[0037] In certain embodiments, the dry powder formulations described herein have a compressed bulk density that allows for the delivery of large amounts of active agent. "Compressed bulk density" refers to the mass per unit volume of a powder (preferably in g / cm) when measured under the following conditions: 3 ). A suitable assay for measuring the compressed bulk density (cBD) is described in the Examples (see, e.g., Example 6). Preferably, the compressed bulk density (CBD) of a powder is measured using a density analyzer such as a GeoPyc® Model 1360 Density Analyzer (Micromeritics, Norcross, GA). A powder sample is preferably prepared in a low humidity environment (<5% RH) and then transferred to the sample chamber of the density analyzer, which is purged with nitrogen gas. The net weight of the powder sample is recorded and then a compressive force of 10-14 N, preferably 12 N, is applied to the sample by the plunger at a rate of 250-350 compaction steps per second, preferably 300 compaction steps per second. The linear distance traveled by the plunger at each compaction step is then converted to the volumetric displacement of the powder sample. The average of the measurements for each compaction step is then calculated in g / cm. 3 The bulk density of the dry powder formulation is calculated as follows:

[0038] Suitably, the dry powder formulations described herein have a compressed bulk density of at least 0.4 g / cm 3 , preferably about 0.4 g / cm 3 ~Approx. 1.0g / cm 3 and more preferably between about 0.4 and 0.9 gm / cm 3 , about 0.4~0.8gm / cm 3 , about 0.5~0.8gm / cm 3 , about 0.6~0.8gm / cm 3 , or about 0.4 gm / cm 3 , about 0.5gm / cm 3 , about 0.6gm / cm 3 , about 0.7gm / cm 3 , or about 0.8 gm / cm 3 In certain embodiments, the compressed bulk density of the dry powder formulations described herein is about 0.4 gm / cm 3 ~about 0.9gm / cm 3In certain embodiments, the compressed bulk density of the dry powder formulations described herein is about 0.5 gm / cm 3 ~about 0.8gm / cm 3 It is.

[0039] FIG. 8A shows the compressed bulk density results for leucine and trileucine in the dry powder formulations described herein. Each column represents the amount of trileucine in the formulation. Within each column, the amount of leucine increases from about 1% to about 20%. As shown, increasing the amount of trileucine decreases the compressed bulk density, and within each group, increasing leucine decreases the compressed bulk density. Approximately 0.5 g / cm 3 ~about 0.8g / cm 3 In order to achieve a compacted bulk density between 0.1 and 0.5 wt. %, the amount of tri-leucine needs to be kept below 4 wt.

[0040] The formulations described herein comprise an antigen-binding fragment of an anti-thymic stromal lymphopoietin (anti-TSLP) antibody. Advantageously, the inventors have found that the formulations described herein allow for direct delivery of the antigen-binding fragment to the lungs by inhalation. Delivery of a therapeutically active antigen-binding fragment of an anti-TSLP antibody by inhalation advantageously allows for the use of biologic agents for the treatment of asthma in a primary care setting.

[0041] The sequence of the TSLP polypeptide is shown below. Met Phe Pro Phe Ala Leu Leu Tyr Val Leu Ser Val Ser Phe Arg Lys Ile Phe Ile Leu Gln Leu Val Gly Leu Val Leu Thr Tyr Asp Phe Thr Asn Cys Asp Phe Glu Lys Ile Lys Ala Ala Tyr Leu Ser Thr Ile Ser Lys Asp Leu Ile Thr Tyr Met Ser Gly Thr Lys Ser Thr Glu Phe Asn Asn Thr Val Ser Cys Ser Asn Arg Pro His Cys Leu Thr Glu Ile Gln Ser Leu Thr Phe Asn Pro Thr Ala Gly Cys Ala Ser Leu Ala Lys Glu Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile Trp Cys Pro Gly Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met Lys Lys Arg Arg Lys Arg Lys Val Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu Gln Gly Leu Trp Arg Arg Phe Asn Arg Pro Leu Leu Lys Gln Gln (SEQ ID NO: 27)

[0042] The term "antibody" as used herein refers to a protein comprising at least two heavy chains and two light chains connected by disulfide bonds. The term "antibody" includes naturally occurring antibodies as well as all recombinant forms of antibodies, such as humanized antibodies, fully human antibodies and chimeric antibodies. Each heavy chain usually consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain usually consists of a light chain variable region (VL) and a light chain constant region (CL). The term "antibody" however also includes other types of antibodies, such as single domain antibodies, heavy chain antibodies, i.e. antibodies consisting only of one or more, in particular two, heavy chains, and nanobodies, i.e. antibodies consisting only of a single monomeric variable domain.

[0043] Antibody-binding fragments include (i) Fab fragments, which are monovalent fragments consisting of the variable regions of each heavy and light chain and the first constant domain, (ii) F(ab)2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bridges in the hinge region, (iii) Fd fragments consisting of the variable region of the heavy chain and the first constant domain CH1, (iv) Fv fragments consisting of the heavy and light chain variable regions of a single arm of an antibody, (v) scFv fragments, which are Fv fragments consisting of a single polypeptide chain, (vi) (Fv)2 fragments consisting of two Fv fragments covalently linked to each other, (vii) a heavy chain variable domain, and (viii) a multibody consisting of a heavy chain variable region and a light chain variable region covalently linked to each other such that the binding of the heavy and light chain variable regions occurs only intermolecularly, not intramolecularly. In an embodiment, the antibody-binding fragment of the present invention is selected from Fab, Fab', F(ab')2, scFv, minibody, or diabody. In a particular embodiment, the antibody-binding fragment is a Fab. In some embodiments, the anti-TSLP antibody from which the antigen-binding fragment is derived is an IgG1.

[0044] Exemplary Fab sequences of the invention (referred to herein as Fab 1 ), which includes:

[0045] HCDR1 FAB1 Thr Tyr Gly Met His (SEQ ID NO: 1)

[0046] HCDR2 FAB1 Val Ile Trp Tyr Asp Gly Ser Asn Lys His Ala Tyr Asp Ser Val Lys Gly (SEQ ID NO: 2)

[0047] HCDR3 FAB1 Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile (SEQ ID NO: 3)

[0048] Heavy chain VH FAB1 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Asn Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser (SEQ ID NO: 4)

[0049] LCDR1 FAB1 Gly Gly Asn Asn Leu Gly Ser Lys Ser Val His (SEQ ID NO: 5)

[0050] LCDR2 FAB1 Asp Asp Ser Asp Arg Pro Ser (SEQ ID NO: 6)

[0051] LCDR3 FAB1 Gln Val Trp Asp Ser Ser Ser Asp His Val Val (SEQ ID NO: 7)

[0052] Light chain VL FAB1 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu(SEQ ID NO:8)

[0053] FAB1 Variable heavy chain VH (nucleic acid) cagatgcagt tggttgaatc tggtggcggc gtggtgcagc ctggcagatc tctgagactg 60 tcttgtgccg cctccggctt caccttcaga acctacggaa tgcactgggt ccgacaggcc 120 cctggcaaag gattggaatg ggtcgccgtg atttggtacg acggctccaa caagcactac 180 gccgactccg tgaagggcag attcaccatc accagagaca actccaagaa caccctgaac 240 ctgcagatga actccctgag agccgaggac accgccgtgt actattgtgc tagagcccct 300 cagtgggaac tcgtgcatga ggcctttgac atctggggcc agggaacaat ggtcaccgtc 360 tcctca 366 (SEQ ID NO: 9)

[0054] FAB1 variable light chain VL (nucleic acid) tcatatgttc ttacacaacc accgtcggtt tcggttgctc caggacaaac agctcgaatt 60 acatgcggag gaaacacacct cggatcgaag tcggttcact ggtatcaaca aaagccagga 120 caagctccag ttctcgtggt gtacgatgat tcagatcgac catcatggat ccgagcga 180 ttctcaggat caaactcggg aaatactgcc acgctcacaa tttcacgcgg agaagcggga 240 gatgaagctg attactattg ccaagtgtgg gactcgtcgt cagatcatgt tgttttcgga 300 ggtggaacaa agctcacagt gctc 324 (SEQ ID NO: 10)

[0055] FAB1 heavy chain (polypeptide) Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Asn Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys (SEQ ID NO: 28)

[0056] FAB1 light chain (polypeptide) Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser (SEQ ID NO: 29)

[0057] FAB1 heavy chain (nucleic acid) cagatgcagt tggttgaatc tggtggcggc gtggtgcagc ctggcagatc tctgagactg 60 tcttgtgccg cctccggctt caccttcaga acctacggaa tgcactgggt ccgacaggcc 120 cctggcaaag gattggaatg ggtcgccgtg atttggtacg acggctccaa caagcactac 180 gccgactccg tgaagggcag attcaccatc accagagaca actccaagaa caccctgaac 240 ctgcagatga actccctgag agccgaggac accgccgtgt actattgtgc tagagcccct 300 cagtgggaac tcgtgcatga ggcctttgac atctggggcc agggaacaat ggtcaccgtc 360 tcctcagcct ccaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 420 tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480 gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540 tcctcaggac tctactccct cagcagcgtg gtgacagtgc cctccagcag cttgggcacc 600 cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagagagtt 660 gagcccaaat cttgtgacaa a 681(SEQ ID NO: 30)

[0058] FAB1 light chain (nucleic acid) tcatatgttc ttacacaacc accgtcggtt tcggttgctc caggacaaac agctcgaatt 60 acatgcggag gaaacacacct cggatcgaag tcggttcact ggtatcaaca aaagccagga 120 caagctccag ttctcgtggt gtacgatgat tcagatcgac catcatggat ccgagcga 180 ttctcaggat caaactcggg aaatactgcc acgctcacaa tttcacgcgg agaagcggga 240 gatgaagctg attactattg ccaagtgtgg gactcgtcgt cagatcatgt tgttttcgga 300 ggtggaacaa agctcacagt gctcggtcag cccaaggctg ccccctcggt cactctgttc 360 ccgccctcct ctgaggagct tcaagccaac aaggccacac tggtgtgtct cataagtgac 420 ttctacccgg gagccgtgac agtggcctgg aaggcagata gcagccccgt caaggcggga 480 gtggagacca ccacaccctc caaacaaagc aacaacaagt acgcggccag cagctatctg 540 agcctgacgc ctgagcagtg gaagtcccac agaagctaca gctgccaggt cacgcatgaa 600 gggagcaccg tggagaagac agtggcccct acagaatgtt ca 642 (SEQ ID NO: 31)

[0059] The dry powder formulations provided herein comprise a plurality of microparticles comprising leucine, about 1% to about 10% by weight of trileucine, and an antigen-binding fragment of an anti-thymic stromal lymphopoietin (TSLP) antibody, wherein the leucine and trileucine are present in a concentration ratio of about 0.1:1 to about 30:1 leucine:trileucine.

[0060] In certain embodiments, the antigen-binding fragment in the dry powder formulation comprises: a. a heavy chain variable domain comprising a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:1, a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:2, and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:3, wherein any of heavy chain CDR1, 2 or 3 optionally comprises a single amino acid substitution; b. a light chain variable domain comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:5, a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:6, and a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:7, wherein any of light chain CDR1, 2 or 3 optionally comprises a single amino acid substitution.

[0061] In certain embodiments, the antigen-binding fragment in the dry powder formulation comprises a heavy chain variable domain comprising a light chain CDR1 sequence having the amino acid sequence set forth in SEQ ID NO:1, a heavy chain CDR2 sequence having the amino acid sequence set forth in SEQ ID NO:2, and a heavy chain CDR3 sequence having the amino acid sequence set forth in SEQ ID NO:3, and a light chain CDR1 sequence having the amino acid sequence set forth in SEQ ID NO:5, a light chain CDR2 sequence having the amino acid sequence set forth in SEQ ID NO:6, and a light chain CDR3 sequence having the amino acid sequence set forth in SEQ ID NO:7.

[0062] In an additional embodiment, the antigen-binding fragment used in the dry powder formulation comprises a heavy chain variable domain comprising SEQ ID NO: 4 and a light chain variable domain comprising SEQ ID NO: 8. In an additional embodiment, the antigen-binding fragment used in the dry powder formulation comprises a heavy chain having the sequence set forth in SEQ ID NO: 28 and a light chain having the sequence set forth in SEQ ID NO: 29.

[0063] In additional embodiments, the antigen-binding fragment used in the dry powder formulation comprises a heavy chain variable domain that is a sequence of amino acids at least 95%, 90%, 85% or 80% identical to SEQ ID NO:4, and a light chain variable domain that is a sequence of amino acids at least 95%, 90%, 85% or 80% identical to SEQ ID NO:8.

[0064] In additional embodiments, the antigen-binding fragment for use in the dry powder formulation comprises (a) a heavy chain variable domain that is a sequence of amino acids at least 95%, 90%, 85% or 80% identical to SEQ ID NO:4 or a sequence of amino acids encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO:30; (b) a light chain variable domain that is a sequence of amino acids at least 95%, 90%, 85% or 80% identical to SEQ ID NO:8 or a sequence of amino acids encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO:31; or the heavy chain variable domain of (a) and the light chain variable domain of (b).

[0065] Further light chain CDR (LCDR), light chain variable domain (VL), heavy chain CDR (HCDR) and heavy chain variable domain (VH) sequences of the antigen-binding fragment of the invention include the following:

[0066] LCDR1 FAB2 Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His (SEQ ID NO: 11)

[0067] Light chain VL FAB2 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO: 12)

[0068] LCDR1 FAB3 Gly Gly Asn Asn Val Gly Ser Lys Ser Val His (SEQ ID NO: 13) Light chain VL FAB3 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Val Gly Ser Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO: 14)

[0069] HCDR2 FAB4 Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Glu Ser Val Lys Gly (SEQ ID NO: 15)

[0070] Heavy chain VH FAB4

[0071] Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Asn Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser(SEQ ID NO: 16)

[0072] HCDR2 FAB5 Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val Lys Ala(SEQ ID NO: 17)

[0073] Heavy chain VH FAB5

[0074] Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val Lys Ala Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Asn Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser (SEQ ID NO: 18)

[0075] LCDR1 FAB6 Gly Gly Gln Asn Leu Gly Ser Lys Ser Val His (SEQ ID NO: 19)

[0076] Light chain VL FAB6

[0077] Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Gln Asn Leu Gly Ser Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu(SEQ ID NO: 20)

[0078] LCDR1 FAB7

[0079] Gly Gly Asn Gln Leu Gly Ser Lys Ser Val His(SEQ ID NO: 21)

[0080] Light chain VL FAB7

[0081] Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Gln Leu Gly Ser Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO: 22)

[0082] LCDR3 FAB8 Gln Val Trp Asp Thr Ser Ser Asp His Val Val (SEQ ID NO: 23)

[0083] Light chain VL FAB8 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu(SEQ ID NO: 24)

[0084] LCDR3 FAB9 Gln Val Trp Asp Ser Thr Ser Asp His Val Val(SEQ ID NO: 25)

[0085] Light chain VL FAB9

[0086] Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Thr Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO: 26).

[0087] In certain embodiments, the heavy and light chain variable domains of the antigen-binding fragment of the invention comprise any of the combinations of CDR sequences shown in the table below. [Table 1]

[0088] The formulations disclosed herein may be administered in combination with additional active agents used in the treatment of asthma.Exemplary active agents that can be administered in combination with the dry powder formulations described herein include, but are not limited to, inhaled corticosteroids (ICS), bronchodilators (including long-acting beta agonists (LABA), long-acting antimuscarinic agonists (LAMA), short-acting beta agonists (SABA), and muscarinic β2 agonists (MABA)), antihistamines, anti-leukotrienes, PDE-4 inhibitors, Janus kinase inhibitors, and phosphoinositide 3-kinase inhibitors.In certain embodiments, additional active agents are incorporated into the formulations of the present invention together with the anti-TSLP antibody binding fragments disclosed herein.

[0089] In a preferred embodiment, the dry powder formulations described herein further comprise a glass stabilizer to aid in the stabilization of the formulation, particularly the stabilization of the active agent. "Glass stabilizer" refers to an excipient that stabilizes the active agent (preferably a polypeptide) in the dry powder formulation, preferably by substituting water at the surface of the active agent upon drying or otherwise preventing the degradation process, forming an amorphous solid that includes the active agent. Examples of glass stabilizers include amorphous sugars, polymeric sugars, buffers, salts, or synthetic polymers (e.g., poly-L-glycolic acid), and mixtures of such components. In a preferred embodiment, the glass stabilizer is an amorphous sugar. In additional embodiments, the glass stabilizer is a buffer. In yet further embodiments, the formulations described herein may include both an amorphous sugar and a buffer, which may act as glass stabilizers together or separately.

[0090] Exemplary amorphous sugars for use in the formulations described herein include, but are not limited to, trehalose, sucrose, raffinose, inulin, dextran, mannitol, and cyclodextrin. Suitably, the amorphous sugar is present at about 30% to about 70% (weight percentage) of the dry powder formulation. In further embodiments, the amorphous sugar is present at about 30% to about 65%, about 35% to about 65%, about 35% to about 60%, about 40% to about 60%, about 30% to about 50%, or about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%. Preferably, the amorphous sugar is trehalose and is present in the formulation at about 30% to 60%, more preferably about 35% to 55%, or about 35%, about 40%, about 45% or about 50% by weight of the dry powder formulation.

[0091] Exemplary buffers that may be included in the dry powder formulation, preferably as glass stabilizers, include various citrate buffers (such as sodium citrate), phosphate buffers, histidine buffers, glycine buffers, acetate buffers, and tartrate buffers, as well as combinations of such buffers. The amount of buffer that may be included in the dry powder formulation may range from about 0.1% to about 20%, more preferably from about 0.5% to about 15%, from about 1% to about 10%, from about 2% to about 8%, from about 3% to about 7%, or from about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0092] The buffering agent also provides for adjustment of the pH of the dry powder formulation, suitably maintaining a pH between about pH 5 and about 8, e.g., between about pH 5 and about pH 6, or between about pH 5.5 and about pH 6.5, or between about pH 6 and about pH 7, or between about pH 6.5 and about pH 7.5, or between about pH 7 and about pH 8.

[0093] In an additional embodiment, a dry powder formulation is provided comprising about 30%-50% trehalose, about 10%-11% leucine, about 1%-3% trileucine, about 8%-9% citrate buffer and active agent, more preferably about 39% trehalose, about 10.5% leucine, about 2% trileucine, about 8.5% citrate buffer and active agent.

[0094] In additional embodiments, a dry powder formulation is provided consisting essentially of about 30%-50% amorphous sugar, leucine, about 1%-10% trileucine, about 1%-10% buffer, and an active agent, where the leucine and trileucine are present in a concentration ratio of about 0.1:1 to about 30:1 leucine:trileucine. In additional embodiments, a dry powder formulation is provided consisting essentially of about 30%-50% amorphous sugar, about 8%-11% leucine, about 2%-4% trileucine, about 1%-10% buffer, and an active agent. An additional dry powder formulation is provided consisting essentially of about 35%-45% trehalose, about 9%-11% leucine, about 2%-3% trileucine, about 2%-85% citrate buffer, and an active agent. In a further embodiment, the dry powder formulation consists essentially of about 39% trehalose, about 10.5% leucine, about 2% trileucine, about 8.5% citrate buffer, and the active agent.

[0095] In compositions and formulations "consisting essentially" of the recited ingredients, such compositions and formulations contain the recited ingredients and ingredients that do not significantly affect the basic and novel characteristics of the claimed formulation. Ingredients that do not significantly affect the basic and novel characteristics of the claimed formulation are those that do not limit the ability of leucine and trileucine to stabilize dry powder formulations. Suitably, compositions and formulations consisting essentially of the recited ingredients specifically exclude other amino acids or tripeptide amino acids, but may contain additional sugars, buffers, and the like.

[0096] In an exemplary embodiment, a dry powder formulation is provided that comprises about 30-50% trehalose, about 10-11% leucine, about 1-3% trileucine, about 8-9% citrate buffer, and about 30-50% anti-TSLP antibody fragment, more preferably about 39% trehalose, about 10.5% leucine, about 2% trileucine, about 8.5% citrate buffer, and about 40% anti-TSLP antibody fragment.

[0097] In a further exemplary embodiment, a dry powder formulation is provided consisting essentially of about 30-50% trehalose, about 10-11% leucine, about 1-3% trileucine, about 8-9% citrate buffer, and about 30-50% anti-TSLP antibody fragment, more preferably about 39% trehalose, about 10.5% leucine, about 2% trileucine, about 8.5% citrate buffer, and about 40% active agent.

[0098] The microparticles constituting the dry powder formulations described herein, when provided in aerosol form, preferably have a predetermined mass median aerodynamic diameter (MMAD). The microparticles may also have a predetermined optical volume equivalent mean diameter (oVMD). The oVMD may also be referred to as particle size distribution (PSD or pPSD).

[0099] As used herein, "mass median aerodynamic diameter" or "MMAD" is a measure of the aerodynamic size of a dispersed microparticle. Aerodynamic diameter is used to describe an aerosolized powder in terms of settling behavior and is the diameter of a sphere of unit density that has the same settling velocity in air as the microparticle. Aerodynamic diameter encompasses the particle shape, density, and physical size of the microparticle. As used herein, MMAD refers to the midpoint or median of the aerodynamic size distribution of an aerosolized powder as determined by cascade impaction, unless otherwise specified. Preferably, the microparticles of the dry powder formulations provided herein have a mass median aerodynamic diameter (MMAD) of about 1 μm to about 10 μm, more preferably about 2 μm to about 8 μm, about 2 μm to about 7 μm, about 2 μm to about 6 μm, about 2 μm to about 5 μm, about 2 μm to about 4 μm, about 3 μm to about 7 μm, about 4 μm to about 7 μm, about 3 μm to about 6 μm, or about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, or about 7 μm.

[0100] Preferably, the fine particle fraction (fraction of particles emitted from an inhalation device having an aerodynamic size of less than 5 μm) of the dry powder formulations described herein is ≧50%, more preferably ≧60%. This fine particle fraction (FPF) contributes to the low retention of the dry powder formulation in the device, where less than 20%, preferably less than 15%, less than 10%, or less than 5% may remain in the device after delivery to a patient.

[0101] In additional embodiments, the microparticles preferably have an optical volume equivalent mean diameter r (oVMD) of about 0.5 μm to about 7 μm. Optical volume equivalent mean diameter (oVMD) refers to the average diameter of a sphere that best approximates a particular optical interaction of the microparticle with light, where half of the microparticles best approximate an equivalent sphere that is smaller than the average, and half of the microparticles best approximate an equivalent sphere that is larger than the average, when measured using a suitable optical technique. In exemplary embodiments, the microparticles have an optical volume equivalent mean diameter (oVMD) of about 0.5 μm to about 6 μm, or about 1 μm to about 5 μm, or about 1 μm to about 4 μm, or about 2 μm to about 4.5 μm, or about 2.5 μm to about 4 μm, or about 2 μm to about 4 μm, or about 2 μm to about 3 μm, or about 2 μm to about 3.5 μm, or about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, or about 5 μm.

[0102] As described herein, high compacted bulk density allows the same delivery volume to be utilized to deliver large amounts of active agent.Certain biological preparations may require delivery amounts of 50mg / dose or more for effective treatment.As shown in Fig. 8B, by combining leucine and trileucine, dry powder preparations with higher bulk density can be obtained, and therefore, for the same amount of fill weight, the volume is substantially smaller.

[0103] Exemplary platform formulations shown in Figure 8B are as follows: LTC indicates a formulation without trileucine (TLeu) but containing leucine, trehalose and citrate buffer, TTC indicates a formulation without leucine (Leu) but containing trileucine, trehalose and citrate buffer, and TLTC indicates both leucine and trileucine, as well as trehalose and citrate buffer. Cit refers to citrate buffer. Tre refers to trehalose. [Table 2]

[0104] Capsules for each formulation (size 3 capsules) are shown at each respective fill weight in Figure 8B. As illustrated, for the TLTC formulation, the combination of trileucine and leucine allows 100 mg of dry powder formulation to be filled into a capsule and still have some space within the capsule. The other formulations could not be filled beyond about 70-80 mg fill weight. This represents a dramatic improvement provided by using leucine and trileucine in combination to prepare a formulation with high compacted bulk density, which allows for higher fill weights.

[0105] As described herein, the use of leucine and trileucine in dry powder formulations also results in microparticles having the desired size (MMAD) and desired specific surface area (SSA) and roughness, resulting in microparticles that have suitable flowability and can be delivered to the lungs using a variety of inhalation platforms.

[0106] The specific surface area (SSA) of a particulate is defined as the total surface area of ​​the particulate per unit mass (preferably m 2 SSA is expressed in units of 1 / g. Methods for measuring SSA are known in the art and include, for example, Brunauer-Emmett-Teller (BET) measurements using evaluation of the specific surface area of ​​a material by nitrogen adsorption measured as a function of relative pressure. Surface area is determined by calculating the amount of adsorbed gas that corresponds to a monolayer on the surface of the particulate. This technique measures the external area and an evaluation of any pore area to determine the total specific surface area. Instruments for measuring BET are known in the art.

[0107] In embodiments, the specific surface area (SSA) of the particulates of the dry powder formulation is about 3 m 2 / g~about 8m 2 In a preferred embodiment, the SSA of the plurality of particulates is about 3.5 m 2 / g~7.5m 2 / g, or about 4m 2 / g~7m 2 / g, or about 4.5m2 / g~7m 2 / g, or about 5m 2 / g~7m 2 / g, or about 4.5m 2 / g~6m 2 / g, or about 5m 2 / g~6m 2 / g, or about 4m 2 / g, approx. 4.5m 2 / g, approx. 5m 2 / g, approx. 5.5m 2 / g, approx. 6m 2 / g, approx. 6.5m 2 / g, or about 7m 2 / g.

[0108] Figure 9 shows the results of specific surface area measured using BET in m 2 The results are shown in terms of weight percent trileucine / g. Each column in FIG. 9 represents a different amount of trileucine in the formulation. Within each column, the amount of leucine increases from about 1% to about 20%. The inset photomicrographs show the physical appearance of microparticles with low SSA (bottom left) and high SSA (top right). As shown, at the lower wt% trileucine, the SSA is approximately 5mM. 2 / g, but increases with increasing leucine. Above about 2% trileucine, the SSA is 3.0m 2 / g and increases with increasing proportion of leucine. 2 / g, 7.0m 2 SSA values ​​approaching 1000 mg / g are achieved with trileucine levels above about 4%. 2 The desired range of specific surface area per gram can be readily achieved using amounts of trileucine between about 1-6% and leucine between about 1-20%. As shown, by utilizing amounts of trileucine below about 6%, the amount of leucine can be reduced to less than 10% or even less than 5% while still maintaining microparticles with the desired SSA and surface roughness. The photomicrograph at the top left shows the shape of microparticles of the dry powder formulation described herein, exhibiting the desired size, specific surface area, and surface roughness.

[0109] In certain embodiments, the dry powder formulation has a density of about 0.4-1.0 g / cm 3 Preferably, the dry powder formulation has a compressed bulk density of about 0.5 to 0.8 g / cm. 3 In embodiments, the compressed bulk density of the dry powder formulations described herein is about 0.4 to 0.9 gm / cm 3 , about 0.4~0.8gm / cm 3 , about 0.5~0.8gm / cm 3 , about 0.6~0.8gm / cm 3 , or about 0.4 gm / cm 3 , about 0.5gm / cm 3 , about 0.6gm / cm 3 , about 0.7gm / cm 3 , or about 0.8 gm / cm 3 In certain embodiments, the compressed bulk density of the dry powder formulations described herein is about 0.4 gm / cm 3 ~about 0.9gm / cm 3 In certain embodiments, the compressed bulk density of the dry powder formulations described herein is about 0.5 gm / cm 3 ~about 0.8gm / cm 3 It is.

[0110] Dry powder formulations preferably include a glass stabilizer as described herein, including the use of an amorphous sugar or a buffer, or both an amorphous sugar and a buffer. Exemplary amorphous sugars include those described herein, including trehalose, sucrose, raffinose, inulin dextran, and cyclodextrin. Preferably, the amorphous sugar is present at about 30% to about 70%, and in embodiments is trehalose, preferably present at about 35% to 60, or 35% to 55%.

[0111] Exemplary buffers used in the dry powder formulation are described herein and include citrate buffers, phosphate buffers, and tartrate buffers. Suitably, the buffer is present at about 1% to about 10%, and in embodiments is a citrate buffer. In certain embodiments, the pH of the citrate buffer is about pH 5.5 to about pH 6.5, such as about pH 5.5, about pH 5.6, about pH 5.7, about pH 5.8, about pH 5.9, about pH 6.0, about pH 6.1, about pH 6.2, about pH 6.3, about pH 6.4, or about pH 6.5. In certain embodiments, the pH of the citrate buffer is about pH 6.4.

[0112] In certain embodiments, the dry powder formulations described herein include a surfactant. As defined herein, "surfactant" refers to a molecule or compound that reduces particle aggregation, adhesion of particles to the surface of a capsule, the container wall or valve components of an inhalation delivery device. Surfactants have also been found to reduce the formation of subvisible particles (SVPs) upon reconstitution of the formulation. Removal or reduction of SVP formation simplifies analytical characterization of the formulation by reducing the burden of tracking SVP formation during manufacturing. Analytical characterization of SVPs may require the development of techniques with different principles to identify and quantify SVPs for quality control purposes. Thus, removing SVPs or reducing SVPs to acceptable levels removes the need for this characterization step from the manufacturing process, making manufacturing more efficient. Also, since the drug release kinetics from SVPs is unknown, removing SVPs may allow for more predictable dose ranges. Furthermore, removal of SVPs may increase the amount of active agent available to participate in pharmacological activity after reconstitution, meaning that not only can higher delivered doses be achieved, but more accurate predictions of delivered doses can be calculated. Additionally, delivering larger doses can be beneficial to the patient, for example by allowing a reduction in the number or frequency of doses that must be delivered to obtain pharmacological benefit.

[0113] "Subvisible particles" ("SVPs") are particles that are invisible to the naked eye between about 1 μm and about 200 μm. The presence of subvisible particles can be determined by reconstituting the dry powder formulation and the cloudy liquid. To actually determine the presence of SVPs, techniques such as microflow imaging can be used. Microflow imaging (or MFI) combines microfluidic flow microscopy and high-resolution image particle analysis to quantify SVP counts. MFI can be performed by binning the number of particles that are in the size ranges, for example, about 1 to about 200 μm, about 2 μm to about 200 μm, about 5 μm to about 200 μm, about 10 μm to about 200 μm, and about 25 μm to about 200 μm. The examples show that the inclusion of a surfactant in a dry powder formulation reduces the presence of SVPs in each particle size range compared to a control formulation in which no surfactant is present (e.g., FIG. 15A). Thus, in certain embodiments, the dry powder formulations disclosed herein include a surfactant to reduce the number of sub-visible particles in the formulation upon reconstitution, hi some embodiments, the number of sub-visible particles is reduced compared to a comparable formulation without the surfactant.

[0114] In certain embodiments, the number of SVPs with a size of about 25 μm to about 200 μm is reduced to less than 30,000 particles / ml, for example, 25,000 particles / ml, 20,000 particles / ml, 15,000 particles / ml, 10,000 particles / ml, or 5,000 particles / ml. In certain embodiments, the number of SVPs with a size of about 25 μm to about 200 μm is reduced to less than 1,000 particles / ml. In certain embodiments, the number of SVPs with a size of about 25 μm to about 200 μm is reduced to less than 1,000 particles / ml. In certain embodiments, the number of SVPs with a size of about 25 μm to about 200 μm is reduced to less than 100 particles / ml.

[0115] In certain embodiments, the number of SVPs with a size of about 10 μm to about 200 μm is reduced to less than 100,000 particles / ml, for example, 90,000 particles / ml, 80,000 particles / ml, 70,000 particles / ml, 60,000 particles / ml, 50,000 particles / ml, 40,000 particles / ml, or 30,000 particles / ml. In certain embodiments, the number of SVPs with a size of about 10 μm to about 200 μm is reduced to less than 10,000 particles / ml. In certain embodiments, the number of SVPs with a size of about 10 μm to about 200 μm is reduced to less than 1,000 particles / ml. In certain embodiments, the number of SVPs with a size of about 10 μm to about 200 μm is reduced to less than 100 particles / ml.

[0116] In certain embodiments, the number of SVPs with a size of about 5 μm to about 200 μm is reduced to less than 200,000 particles / ml, for example, 180,000 particles / ml, 170,000 particles / ml, 160,000 particles / ml, 150,000 particles / ml, or 140,000 particles / ml. In certain embodiments, the number of SVPs with a size of about 5 μm to about 200 μm is reduced to less than 50,000 particles / ml. In certain embodiments, the number of SVPs with a size of about 5 μm to about 200 μm is reduced to less than 10,000 particles / ml. In certain embodiments, the number of SVPs with a size of about 5 μm to about 200 μm is reduced to less than 2,000 particles / ml.

[0117] In certain embodiments, the number of SVPs with a size of about 2 μm to about 200 μm is about 1×10 6 Less than 0.8×10 particles / ml 6 particles / ml, 0.7×10 6 particles / ml, 0.6×10 6 particles / ml or 0.5 x 10 6particles / ml. In certain embodiments, the number of SVPs between about 2 μm and about 200 μm in size is reduced to less than 100,000 particles / ml. In certain embodiments, the number of SVPs between about 2 μm and about 200 μm in size is reduced to less than 50,000 particles / ml. In certain embodiments, the number of SVPs between about 2 μm and about 200 μm in size is reduced to less than 10,000 particles / ml.

[0118] In certain embodiments, the number of SVPs with a size of about 1 μm to about 200 μm is about 2×10 6 Less than 1.8 x 10 particles / ml 6 particles / ml, 1.7×10 6 particles / ml, 1.6×10 6 particles / ml or 1.5 x 10 6 In certain embodiments, the number of SVPs between about 1 μm and about 200 μm in size is reduced to less than 200,000 particles / ml. In certain embodiments, the number of SVPs between about 1 μm and about 200 μm in size is reduced to less than 150,000 particles / ml.

[0119] In certain embodiments, the number of SVPs between about 25 μm and about 200 μm in size is reduced by more than 2-fold, e.g., more than 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, more than 7-fold, more than 8-fold, or more than 9-fold, upon reconstitution, compared to a reference control. In certain embodiments, the number of SVPs between about 25 μm and about 200 μm in size is reduced by more than 10-fold, upon reconstitution, compared to a reference control.

[0120] In certain embodiments, the number of SVPs between about 10 μm and about 200 μm in size is reduced by more than 2-fold, e.g., more than 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, more than 7-fold, more than 8-fold, or more than 9-fold upon reconstitution compared to a reference control. In certain embodiments, the number of SVPs between about 10 μm and about 200 μm in size is reduced by more than 10-fold upon reconstitution compared to a reference control.

[0121] In certain embodiments, the number of SVPs between about 5 μm and about 200 μm in size is reduced by more than 2-fold, e.g., more than 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, more than 7-fold, more than 8-fold, or more than 9-fold upon reconstitution compared to a reference control. In certain embodiments, the number of SVPs between about 5 μm and about 200 μm in size is reduced by more than 10-fold upon reconstitution compared to a reference control.

[0122] In certain embodiments, the number of SVPs with a size of about 2 μm to about 200 μm is reduced by more than 2-fold, e.g., more than 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, more than 7-fold, more than 8-fold, or more than 9-fold, upon reconstitution, compared to the reference control. In certain embodiments, the number of SVPs with a size of about 2 μm to about 200 μm is reduced by more than 10-fold, upon reconstitution, compared to the reference control. In certain embodiments, the number of SVPs with a size of about 2 μm to about 200 μm is reduced by more than 100-fold, upon reconstitution, compared to the reference control.

[0123] In certain embodiments, the number of SVPs between about 1 μm and about 200 μm in size is reduced by more than 2-fold, e.g., more than 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, more than 7-fold, more than 8-fold, or more than 9-fold upon reconstitution compared to a reference control. In certain embodiments, the number of SVPs between about 1 μm and about 200 μm in size is reduced by more than 10-fold upon reconstitution compared to a reference control.

[0124] In certain embodiments, the reference control is an equivalent formulation that does not contain surfactant. In some embodiments, the formulation is reconstituted with water. In some embodiments, the formulation is reconstituted to an active agent concentration of 30 mg / ml. In some embodiments, the formulation is reconstituted to an active agent concentration of 2.5 mg / ml. In some embodiments, the number of SVPs is determined by microflow imaging (MFI). In certain embodiments, the number of SVPs is determined by microflow imaging (MFI) using the method defined in the Examples.

[0125] Exemplary surfactants suitable for use in the dry powder formulations described herein include, but are not limited to, polysorbate-20 (PS-20), polysorbate-40 (PS-40), polysorbate-60 (PS-60), polysorbate-80 (PS-80), and poloxamer-188. In certain embodiments, the formulations described herein include PS-80 at a concentration ranging from about 0.27% to about 2.7% by weight, preferably from about 0.27% to about 1.33% by weight, preferably from about 0.67% to about 1.33% by weight. In certain embodiments, the formulations include PS-80 at a concentration ranging from about 0.3% to about 3% by weight. In certain embodiments, the formulations include PS-80 at a concentration ranging from about 0.3% to about 2.5% by weight. In certain embodiments, the formulations include PS-80 at a concentration ranging from about 0.5% to about 2.5% by weight. In certain embodiments, the formulation comprises PS-80 at a concentration ranging from about 0.5% to about 2% by weight. In certain embodiments, the formulation comprises PS-80 at a concentration ranging from about 0.5% to about 1.5% by weight.

[0126] In an exemplary embodiment, the formulation comprises PS-80 at a concentration ranging from about 0.67% to about 1.33%.

[0127] In exemplary embodiments, the formulation comprises PS-80 at a concentration of about 0.7% (w / w), about 0.8% (w / w), about 0.9% (w / w), about 1.0% (w / w), about 1.1% (w / w), about 1.2% (w / w), or about 1.3% (w / w). In some embodiments, the formulation comprises PS-80 at a concentration of about 1.1% (w / w).

[0128] In exemplary embodiments, the composition comprises PS-80 at a concentration of 0.7%±0.35 (w / w), about 0.8%±0.4 (w / w), about 0.9%±0.45 (w / w), about 1.0%±0.5 (w / w), about 1.1%±0.55 (w / w), about 1.2%±0.6 (w / w), about 1.3%±0.65 (w / w), about 1.4%±0.7 (w / w), about 1.5%±0.75 (w / w), about 1.6%±0.8 (w / w), or about 1.7%±0.75 (w / w). In some embodiments, the formulation comprises PS-80 at a concentration of 1.1%±0.55 (w / w).

[0129] In certain embodiments, the formulations described herein include poloxamer-188, preferably at a concentration ranging from about 1% to about 10% by weight. In exemplary embodiments, the formulation includes poloxamer-188 (P188) at a concentration ranging from about 0.67% to about 2.67%. In certain embodiments, the formulation includes P188 at a concentration ranging from about 0.3% to about 3% by weight. In certain embodiments, the formulation includes P188 at a concentration ranging from about 0.3% to about 2.5% by weight. In certain embodiments, the formulation includes P188 at a concentration ranging from about 0.5% to about 2.5% by weight. In certain embodiments, the formulation includes P188 at a concentration ranging from about 0.5% to about 2% by weight. In certain embodiments, the formulation includes P188 at a concentration ranging from about 0.5% to about 1.5% by weight.

[0130] In an exemplary embodiment, the formulation comprises P188 at a concentration ranging from about 0.67% to about 1.67%.

[0131] In exemplary embodiments, the formulation comprises P188 at a concentration of about 0.7% (w / w), about 0.8% (w / w), about 0.9% (w / w), about 1.0% (w / w), about 1.1% (w / w), about 1.2% (w / w), about 1.3% (w / w), about 1.4% (w / w), about 1.5% (w / w), about 1.6% (w / w) or about 1.7% (w / w).

[0132] In an exemplary embodiment, the dry powder formulation comprises about 39% trehalose, about 10.5% leucine, about 2% trileucine, about 8.5% citrate buffer and an active agent.

[0133] Suitable sizes for the microparticles of the dry powder formulation are described herein, and in embodiments, the plurality of microparticles, when provided in aerosol form, have a mass median aerodynamic diameter (MMAD) of about 2 μm to about 4 μm. Suitable specific surface areas (SSA) of the microparticles are described herein, for example, about 4 to 7 m. 2 / g. Preferably, the microparticles have an optical volume mean diameter (oVMD) of about 1 μm to about 5 μm.

[0134] In further embodiments, methods of preparing dry powder formulations are provided herein. In embodiments, the methods include preparing a liquid feedstock, preferably comprising leucine, about 0.1 mg / mL to about 6 mg / mL trileucine, an activator, and preferably further comprising a glass stabilizer. The glass stabilizer described herein can be omitted from the dry powder formulation, if desired. The liquid feedstock can also include a surfactant. The liquid feedstock is prepared by combining these components in a liquid solvent to create a feedstock in which each of the components is dissolved. If desired or necessary, heat may be applied to increase the solubility of the various components forming the liquid feedstock. Exemplary liquid solvents include water, including deionized water, and dilute solutions of alcohol and water. In embodiments, the activator is preferably added to the liquid feedstock after the addition and dissolution of the remaining components of the feedstock.

[0135] In a preferred embodiment of the method of preparation, leucine and trileucine are present in the liquid feedstock in a concentration ratio of leucine:trileucine of about 0.1:1 to about 30:1. When preparing the liquid feedstock as described herein, the leucine and trileucine are provided in mg / mL amounts. Thus, in such an embodiment, the concentration ratio of leucine:trileucine of about 0.1:1 to about 30:1 in a set amount of liquid feedstock corresponds to the weight ratio of leucine:trileucine in the liquid feedstock. In further embodiments, the leucine and trileucine are present in the liquid feedstock in a concentration ratio of about 0.1:1 to about 25:1, about 0.5:1 to about 20:1, about 1:1 to about 20:1, about 1:1 to about 15:1, about 1:1 to about 12:1, about 1:1 to about 10:1, about 1:1 to about 7:1, about 1:1 to about 6:1, or about 1:1:, about 2:1, about 3:1, about 4:1, about 5:1, about 5:1:1:, about 5.2:1, about 5.25:1, about 5.3:1, about 5.4:1, about 5.5:1, about 5.75:1, or about 6:1 of leucine:trileucine.

[0136] The liquid feedstock may then be atomized. In certain embodiments, the liquid feedstock is filtered before being atomized. In certain embodiments, the liquid feedstock is filtered through a 0.22 micron filter. In certain embodiments, the liquid feedstock containing leucine and trileucine is filtered before adding the activator. In certain embodiments, the liquid feedstock is filtered and atomized after adding the activator. Atomization preferably involves the use of pressurized gas (CO 2, or inert gas, etc.) to convert a liquid feedstock into fine droplets. Exemplary devices for producing atomized liquid feedstock are known in the art and include the use of various atomizing nozzles having the desired size and flow characteristics. Exemplary parameters for atomization include an outlet temperature of about 50° C.-90° C., preferably about 60° C.-80° C., or about 70° C., a feedstock feed rate of about 8-15 ml / min, preferably about 9-14 ml / min, about 10-13 ml / min, or about 12 ml / min, an atomizing gas flow rate of about 9-15 kg / hour (hr. or h), preferably about 10-14 kg / hour, about 12-14 kg / hour, or about 13 kg / hour, and a drying gas flow rate of about 60-100 kg / hour, preferably about 60-90 kg / hour, about 70-90 kg / hour, or about 80 kg / hour.

[0137] The atomized liquid feedstock can then be dried, preferably under heat and in combination with an air flow to aid in drying. Drying results in a plurality of microparticles. Drying temperatures are typically in the range of about 50-100°C, or about 60-100°C, or about 70-90°C, and air flow rates are typically in the range of about 10-40 m3. 3 / hours.

[0138] Exemplary glass stabilizers, including amorphous sugars and buffers, are described herein, as are suitable amounts of glass stabilizers.Suitable amounts of leucine and trileucine are also described throughout this specification.The final dry powder formulation must contain the recited amounts of leucine and trileucine (and other components), so the amounts are also used in the liquid feedstock.As a result of the drying process after atomization, any liquid solvent is removed, so the total original dry weight of the components represents the final dry weight of the compound in the dry powder formulation.Exemplary active agents are also described herein.

[0139] The methods of preparing the dry powder formulations described herein preferably provide microparticles having the desired physical characteristics as indicated, including the desired compacted bulk density, specific surface area, and size. Exemplary sizes are described herein, for example, as is the exemplary SSA, which is approximately 10 m 2 / g, preferably about 4 to 7 m 2 Preferably, the method comprises the steps of: (a) providing a granular material having an optical volume equivalent mean diameter (oVMD) of about 1 μm to about 5 μm, and when provided in aerosol form, a mass median aerodynamic diameter (MMAD) of about 2 μm to about 4 μm, and a specific surface area of ​​about 0.4 g / cm. 3 ~0.8g / cm 3 A plurality of particulates having a compressed bulk density of

[0140] The advantage of the method for preparing dry powder formulations described herein relates to the high throughput of the process. For example, when the nebulizer flow rate was set to 20 ml / min, the following throughput (grams / hour) was determined: [Table 3]

[0141] As shown, a maximum solids loading of 25 mg / mL was reached (related to maximum solubility) when only trileucine was used in the feedstock at a maximum trileucine concentration of 5 mg / mL. This resulted in a total of 30 g / hour. A maximum solids loading of 33 mg / mL and a throughput of 40 g / hour was reached when leucine alone was used at 60% and a maximum leucine concentration of 20 mg / mL. Further results using low amounts of leucine and trileucine are also shown. In contrast, when three feedstocks containing both leucine and trileucine were tested, a maximum solids loading of 250 mg / mL and a throughput of 300 g / hour was reached using only 8% leucine and 2% trileucine. This was a surprising and unexpected discovery of the advantages of the methods and formulations disclosed herein, which are not only capable of providing dispersible particles using relatively small amounts of leucine and trileucine, but also allow for large amounts of throughput. Such high throughput has a significant impact on the ability to scale up the production of the dry powder formulations described herein, where large amounts of the formulation are required.

[0142] The methods and formulations described herein allow for the manufacture of capsules, blister packs, and other suitable containers suitable for dry powder formulations. Such containers can be manufactured with 10-200 mg of dry powder, preferably 10-100 mg, or 25-75 mg or 50 mg of dry powder formulation. Such containers can preferably deliver 0.1-10 mg of dry powder formulation to the lungs of a patient.

[0143] In some embodiments, use of the methods described herein provides dry powder formulations that can reduce the total number of capsules required for use in an inhalation device. For example, the volume required to deliver 50-100 mg of active agent can be reduced from two large size 00 capsules to one size 3 capsule.

[0144] The methods described herein also provide a mechanism for increasing the compressed bulk density and specific surface area of ​​dry powder formulations containing multiple particulates. As described throughout this specification, the incorporation of leucine and trileucine into dry powder formulations can increase the compressed bulk density and specific surface area to approximately 0.4-1.0 g / cm. 3 (Preferably about 0.5 to 0.8 g / cm 3 ) can easily achieve a compacted bulk density of about 5 to 10 m 2 / g (preferably about 5m 2 / g ~ approx. 7m 2 Specific surface areas of up to about 100 μm / g can also be achieved. In additional embodiments, the size of the microparticles can be formed in the ranges described herein, including microparticles having a mass median aerodynamic diameter (MMAD) of about 2 μm to about 4 μm when provided in aerosol form.

[0145] Methods of making dry powder formulations in aerosol form are known in the art and include, for example, the use of inhalation devices such as dry powder inhalers (DPIs) (e.g., Monodose RS01 DPI by PLASTIAPE, Osnago, Italy). The dry powder formulations described herein are exhaled into a gas stream by either a passive or active inhalation device, allowing at least a portion of the fine particles to be inhaled by the patient, thereby remaining suspended in the gas for a sufficient time for some of the fine particles to reach the lungs.

[0146] Also provided herein is a method of treating a medical condition in a mammalian patient, comprising administering to the patient by inhalation (including by a dry powder inhaler) a dry powder formulation described herein.

[0147] Medical conditions that can be treated using the methods described herein include those affecting the nervous system, endocrine system, muscular system, cardiovascular system, digestive system, respiratory system (especially the lungs), hormonal system, immune system, reproductive system, etc.

[0148] In embodiments, provided herein is a method for treating a TSLP-associated inflammatory condition in a patient.The TSLP-associated inflammatory condition can be induced by an allergic reaction or an environmental irritant or trigger.In some embodiments, the TSLP-associated inflammatory condition can be asthma, chronic obstructive pulmonary disease, allergic rhinitis, allergic rhinosinusitis, allergic conjunctivitis, atopic dermatitis, or eosinophilic esophagitis.

[0149] In some embodiments, the TSLP-associated inflammatory condition is asthma and the method of treatment comprises administering to a patient by inhalation a dry powder formulation comprising a therapeutically effective amount of an anti-TSLP antibody or antibody fragment variant. In certain embodiments, the patient is an adult. In certain embodiments, the patient is a child or adolescent.

[0150] As described herein, preferably, the dry powder formulation comprises a plurality of microparticles, the microparticles comprising leucine, about 1% to about 10% by weight of trileucine, and an anti-thymic stromal lymphopoietin (anti-TSLP) antibody or antibody variant, wherein the leucine and trileucine are present in a concentration ratio of about 0.1:1 to about 30:1 leucine:trileucine. The dry powder formulation has a concentration of about 0.3 to 1.0 g / cm. 3 Exemplary ingredients and amounts thereof for inclusion in the formulations are described throughout the specification.

[0151] As described herein, the ability to deliver anti-thymic stromal lymphopoietin (anti-TSLP) antibodies or antibody variants by inhalation provides a delivery mechanism more suitable for use in primary care settings.

[0152] In embodiments of the method of treating asthma, the dry powder formulation is administered at a lower dose and more frequently than a systemically administered anti-TSLP agent. In some embodiments, the formulation may be administered daily. Such embodiments may be more convenient for the subject or patient. Additionally, such embodiments may reduce side effects that may occur with systemic administration.

[0153] Suitably, the antigen-binding fragment of an antibody for use in the method of treatment comprises: A heavy chain CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO:1; A heavy chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO:2; A heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:3. A heavy chain variable domain; a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; and comprising a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and a light chain variable domain.

[0154] In an additional embodiment of the method of treatment, the antigen-binding fragment comprises a heavy chain variable domain comprising SEQ ID NO:4 and a light chain variable domain comprising SEQ ID NO:8.

[0155] In some embodiments, forms of asthma suitable for treatment with the formulations of the present invention include mild asthma, moderate asthma, severe asthma, non-eosinophilic asthma, low eosinophilic asthma and high eosinophilic asthma. In certain embodiments, the formulations of the present invention may be for use in the treatment of mild asthma. In certain embodiments, the formulations of the present invention may be for use in the treatment of moderate asthma. In certain embodiments, the formulations of the present invention may be for use in the treatment of severe asthma. In certain embodiments, the formulations of the present invention may be for use in the treatment of non-eosinophilic asthma. In certain embodiments, the formulations of the present invention may be for use in the treatment of low eosinophilic asthma. In certain embodiments, the formulations of the present invention may be for use in the treatment of high eosinophilic asthma.

[0156] As used herein, the terms "mild asthma" and "moderate asthma" refer to asthma with a Global Initiative for Asthma (GINA) scale of 3 or less, preferably a GINA scale of 2 or 3. The GINA scale measures the severity of asthma based on the following criteria (see "Pocket Guide for Asthma Management and Prevention," Global Initiative for Asthma; 2019):

[0157] The term "severe asthma" as used herein refers to asthma that requires intensive treatment (e.g., GINA Steps 4 and 5) to maintain good control or asthma where good control is not achieved despite intensive treatment (GINA, Global Strategy for Asthma Management and Prevention. Global Initiative for Asthma (GINA) December 2012). The term "severe asthma" also encompasses moderate-severe asthma. Moderate-severe asthma suitable for treatment with the formulations described herein may be uncontrolled with medium to high doses of ICS:LABA, with one or more exacerbations and frequent symptoms. In certain embodiments, severe asthma is further defined as severe asthma with type 2 inflammation characterized by blood eosinophilia (i.e., blood eosinophil counts of 150 cells / μL or more) and / or elevated FeNO (i.e., FeNO≧20 ppb).

[0158] The term "FENO" refers to the rate of exhaled nitric oxide and is a biomarker for bronchial or airway inflammation. FENO is produced by epithelial cells of the airways in response to inflammatory cytokines such as TSLP, IL-4 and IL-13. In healthy adults, FENO concentrations are between 2 and 30 ppb (parts per billion). An exemplary assay to measure FENO involves the subject inhaling to total lung capacity with a NIOX MINO® Airway Inflammation Monitor and then exhaling at 50 ml / sec for 10 seconds (assisted by visual and auditory cues).

[0159] As used herein, the term "hypereosinophilic asthma" refers to asthma patients with screening blood eosinophil counts of 250 cells / μL or greater.

[0160] In particular, the formulation offers the potential to treat patients with less severe asthma, typically managed in primary care settings, such as those with a Global Initiative for Asthma (GINA) scale of 3 or less, preferably a GINA scale of 2 or 3. The GINA scale measures the severity of asthma based on the following criteria (see "Pocket Guide for Asthma Management and Prevention," Global Initiative for Asthma; 2019): daytime asthma symptoms more than twice a week; Nighttime awakenings due to asthma; Use of asthma relief medications more than twice a week; and

[0161] Activity limitations due to asthma. A score of 0 on these criteria is considered "well controlled." A score of 1-2 on these criteria is considered "partially controlled." A score of 3-4 on these criteria is considered "poorly controlled."

[0162] In some embodiments, the formulation provides the possibility to treat patients with moderate-severe asthma who can be managed in a primary care setting or who have insufficient access to treatment through specialized care. For example, the formulation may be useful for treating patients with moderate-severe asthma with Global Initiative for Asthma (GINA) scale 4-5. Suitably, the formulation provides the possibility to treat uncontrolled moderate-severe asthma. Suitably, the formulation provides the possibility to treat uncontrolled moderate-severe asthma with medium to high doses of ICS:LABA with one or more exacerbations and frequent symptoms.

[0163] Additional Exemplary Embodiments Embodiment 1 is a dry powder formulation comprising a plurality of microparticles, the microparticles comprising leucine, about 1% by weight to about 10% by weight of trileucine, and an antigen-binding fragment of an anti-thymic stromal lymphopoietin (TSLP) antibody, the antigen-binding fragment comprising a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:1, a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:2, and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:3, wherein any of heavy chain CDR1, 2, or 3 optionally comprises a single amino acid substitution; and b. an antigen-binding fragment comprising a heavy chain variable domain, and a light chain variable domain comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:5, a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:6, and a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:7, wherein any of the light chain CDR1, 2 or 3 optionally comprises a single amino acid substitution, wherein the leucine and tri-leucine are present in a concentration ratio of about 0.1:1 to about 30:1 of leucine:tri-leucine.

[0164] Embodiment 2 is the dry powder formulation of embodiment 1, wherein the dry powder formulation has a compressed bulk density of about 0.4-1.0 g / cm3.

[0165] Embodiment 3 is a dry powder formulation of any of the preceding embodiments, further comprising a glass stabilizer.

[0166] Embodiment 4 is a dry powder formulation of embodiment 3, wherein the glass stabilizer is an amorphous sugar or a buffering agent.

[0167] Embodiment 5 is a dry powder formulation of embodiment 3, wherein the glass stabilizer comprises an amorphous sugar and a buffering agent.

[0168] Embodiment 6 is the dry powder formulation of embodiment 4 or embodiment 5, wherein the amorphous sugar is selected from the group consisting of trehalose, sucrose, raffinose, inulin, dextran, mannitol, and cyclodextrin.

[0169] Embodiment 7 is a dry powder formulation according to any one of embodiments 4 to 6, wherein the buffer is selected from the group consisting of citrate buffer, phosphate buffer, histidine buffer, glycine buffer, acetate buffer and tartrate buffer.

[0170] Embodiment 8 is a dry powder formulation of any one of embodiments 4-7, wherein the amorphous sugar is present at about 30% to about 70% by weight.

[0171] Embodiment 9 is a dry powder formulation of any one of embodiments 4 to 8, wherein the amorphous sugar is trehalose.

[0172] Embodiment 10 is the dry powder formulation of embodiment 9, wherein the trehalose is present at about 30% to 65% by weight.

[0173] Embodiment 11 is the dry powder formulation of any one of embodiments 4-10, wherein the buffering agent is present at about 1% to about 10% by weight.

[0174] Embodiment 12 is a dry powder formulation of any one of embodiments 1-11, wherein the concentration ratio of leucine:trileucine is from about 1:1 to about 12:1.

[0175] Embodiment 13 is a dry powder formulation of any one of embodiments 1-12, wherein the concentration ratio of leucine:trileucine is from about 1:1 to about 7:1.

[0176] Embodiment 14 is a dry powder formulation of any one of embodiments 1-13, wherein the concentration ratio of leucine:trileucine is about 5.25:1.

[0177] Embodiment 15 is a dry powder formulation of any one of embodiments 1-14 comprising about 1% to about 7% trileucine by weight.

[0178] Embodiment 16 is a dry powder formulation of any one of embodiments 1-15, comprising about 8% to about 11% by weight leucine and about 2% to about 4% by weight trileucine.

[0179] Embodiment 17 is a dry powder formulation of any one of embodiments 1-16 comprising about 10.5% by weight leucine and about 2% by weight trileucine.

[0180] Embodiment 18 is the dry powder formulation of any one of embodiments 1 to 17, optionally further comprising a surfactant selected from polysorbate-20 (PS-20), polysorbate-40 (PS-40), polysorbate-60 (PS-60), polysorbate-80 (PS-80), and poloxamer-188.

[0181] Embodiment 19 is a dry powder formulation of embodiment 18, wherein the surfactant is PS-80, and optionally, the PS-80 is present in a concentration ranging from about 0.27% to about 2.7% by weight.

[0182] Embodiment 20 is a dry powder formulation of embodiment 18, wherein the surfactant is poloxamer-188, and optionally, the poloxamer-188 is present in a concentration ranging from about 1% to about 10% by weight.

[0183] Embodiment 21 is a dry powder formulation of any one of embodiments 1-20, wherein the plurality of microparticles have an optical volume-equivalent mean diameter (oVMD) of about 1 μm to about 5 μm.

[0184] Embodiment 22 is a dry powder formulation of any one of embodiments 1 to 21, wherein the plurality of microparticles, when provided in aerosol form, have a mass median aerodynamic diameter (MMAD) of about 2 μm to about 4 μm.

[0185] Embodiment 23 is a dry powder formulation according to any one of embodiments 2 to 22, having a compressed bulk density of about 0.5 g / cm3 to about 0.8 g / cm3.

[0186] Embodiment 24 is a dry powder formulation of any one of embodiments 2-23 comprising about 39% trehalose, about 10.5% leucine, about 2% trileucine, and about 8.5% citrate buffer.

[0187] Embodiment 25 is a dry powder formulation of any one of embodiments 1-24, wherein the plurality of microparticles has a specific surface area of ​​less than about 10 m2 / g.

[0188] Embodiment 26 is the dry powder formulation of embodiment 25, wherein the plurality of particulates have a specific surface area of ​​about 4 mg2 / g to about 7 m2 / g.

[0189] Embodiment 27 is a dry powder formulation according to any of the preceding embodiments, wherein the heavy chain variable domain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, the heavy chain variable domain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, the heavy chain variable domain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:3, the light chain variable domain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:5, the light chain variable domain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:6, and the light chain variable domain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:7.

[0190] Embodiment 28 is a dry powder formulation of embodiment 27, wherein the antigen-binding fragment comprises a heavy chain variable domain comprising SEQ ID NO:4 and a light chain variable domain comprising SEQ ID NO:8.

[0191] Embodiment 29 is a dry powder formulation of any of the preceding embodiments, wherein the antigen-binding fragment is selected from a Fab, Fab', F(ab')2, scFv, a minibody, or a diabody.

[0192] Embodiment 30 is a dry powder formulation of embodiment 29, wherein the antigen-binding fragment is a Fab.

[0193] Embodiment 31 is a dry powder formulation of embodiment 30, wherein the Fab is human or humanized.

[0194] Embodiment 32 is a dry powder formulation of any of the preceding embodiments, wherein the anti-TSLP antibody from which the antigen-binding fragment is derived is an IgG1.

[0195] Embodiment 33 is a method of treating asthma in a patient comprising administering by inhalation a dry powder formulation according to any one of embodiments 1-29.

[0196] Embodiment 34 is the method of embodiment 33, wherein the asthma is mild asthma.

[0197] Embodiment 35 is the method of embodiment 33, wherein the asthma is moderate asthma.

[0198] Embodiment 36 is the method of embodiment 33, wherein the asthma is severe asthma.

[0199] Embodiment 37 is the method of embodiment 33, wherein the asthma is eosinophilic or non-eosinophilic asthma.

[0200] Embodiment 38 is the method of embodiment 33, wherein the asthma is hypoeosinophilic asthma.

[0201] Embodiment 39 is a method according to any one of embodiments 33-38, wherein the asthma is characterized by less than three of the following: daytime asthma symptoms more than twice a week; nighttime awakenings due to asthma; use of asthma relief medication more than twice a week; and activity limitations due to asthma.

[0202] Embodiment 40 is a dry powder formulation according to any one of embodiments 1 to 32 for use in a method of treatment, wherein the formulation is administered by inhalation.

[0203] Embodiment 41 is a dry powder formulation for use as described in embodiment 37 in a method for treating asthma.

[0204] Embodiment 42 is a dry powder formulation for use according to embodiment 38, wherein the asthma is mild asthma.

[0205] Embodiment 43 is a dry powder formulation for use according to embodiment 38, wherein the asthma is moderate asthma.

[0206] Embodiment 44 is a dry powder formulation for use according to embodiment 38, wherein the asthma is severe asthma.

[0207] Embodiment 45 is a dry powder formulation for use according to embodiment 38, wherein the asthma is eosinophilic asthma or non-eosinophilic asthma.

[0208] Embodiment 46 is a dry powder formulation for use according to embodiment 38, wherein the asthma is hypoeosinophilic asthma.

[0209] Embodiment 47 is a dry powder formulation for use according to embodiment 38, wherein the asthma is characterized by less than three of the following: daytime asthma symptoms more than twice a week; nighttime awakenings due to asthma; use of asthma relief medication more than twice a week; and activity limitations due to asthma. EXAMPLES

[0210] Example 1 - Generation of anti-TSLP Fab A series of antibody binding fragments (Fabs) derived from the anti-TSLP monoclonal antibody "A5" disclosed in WO2009 / 035577 (hereby incorporated by reference in its entirety) were generated using standard molecular biology and cloning techniques. Briefly, the CDR sequences of A5 were cloned into an IgG1 Fab scaffold to produce the Fabs described herein. 1 Alternatively, a Fab fragment designated Fab1 was obtained. 1 The VH and VL sequences of are disclosed in SEQ ID NOs: 4 and 8, respectively.

[0211] In addition, Fab containing mutations in the CDR region 2~9 Variants of Fab 1 The CDR combinations of VH and VL for each of Fab1 to 9 are shown in Table 3. [Table 4]

[0212] Fab 1 The purity, stability and aggregation of Fab were analyzed. 1was formulated in 30 mM sodium citrate, 105 mM trehalose, pH 6.0. Samples were placed in stability chambers at 40°C and 5°C for various times. At various time points, samples were tested by relevant analytical techniques such as high performance size exclusion chromatography (HP-SEC). An Agilent HPLC system equipped with a temperature-controlled autosampler, DAD or VWD, and Agilent ChemStation software / OpenLAB ECM CDS from Agilent Technologies (Santa Clara, CA, USA) was used. Guard columns included TSKgel columns (7.9 mm ID, Cat. No. 08543) and TSK-Gel G3000SWxl columns (5 μm, 250 Å, and 7.8×300 mm, Cat. No. 08541) from Tosoh Bioscience (Griesheim, Germany). The mobile phase used was 0.1 M anhydrous disodium phosphate, 0.1 M sodium sulfate, pH 6.8. The results of the stability and aggregation analyses are shown in Table 4. [Table 5]

[0213] Fab 1 The stability of Fab was also tested by differential scanning calorimetry (DSC). A MicroCal Capillary VP DSC from Malvern Panalytical (Malvern, UK) was used for the tests, and Origin 7.0 software (Northampton, MA, USA) was used for data analysis. 1 The samples were diluted to 5 mg / mL with formulation buffer (30 mM sodium citrate, 105 mM trehalose, pH 6.0). 1 500 μL of sample and reference (combined buffer) were injected into the DSC sample and reference cells by autosampler. The solutions were heated from 25° C. to 100° C. at a scan rate of 95° C. / hr. For baseline correction of the samples, a scan of the buffer (filled in both the sample and reference cells) was also obtained as a blank.

[0214] Fab 1 The charge profile of Fab1 was also determined by imaging isoelectric focusing (IEF) using an iCE3 analyzer. The iCE3 capillary IEF analyzer, PrinCE MicroInjector autosampler, and MicroInjection coated transfer capillary were all purchased and supplied by Protein Simple. Samples were analyzed using an FC Cartridge (part number 101701, Protein Simple) with fluorocarbon coated capillaries and a built-in electrolyte reservoir. The autosampler was maintained at 4°C during analysis. The pI range of Fab1 was determined to be 8.35-8.80.

[0215] Example 2 - Fab 1 binds to human TSLP and cynomolgus monkey TSLP with pM affinity Fab determined by BIAcore 1 Binding affinity of to TSLP Fabs against human TSLP and cynomolgus monkey TSLP expressed by recombinant mammalian cells 1 The specificity and affinity of was determined using a Biacore 8K SPR instrument (GE Healthcare, Little Chalfont, Bucks, UK).

[0216] S Series C1 biosensor chips, amine coupling kits, HEPES-buffered saline buffer and regeneration buffer were obtained from GE Healthcare and used according to the manufacturer's instructions. Streptavidin surfaces were prepared using lyophilized streptavidin reconstituted in D-PBS. Briefly, streptavidin was diluted to 4 μg mL-1 in 10 mM sodium acetate pH 4.5 and covalently immobilized onto two flow cell surfaces of an S Series C1 biosensor chip using standard amine coupling methods. Finally, a streptavidin surface of 170 response units (RU) was achieved. A control blank surface with no immobilized streptavidin was also prepared using amine coupling reagents to serve as a reference surface in each flow cell. N-terminally tagged biotinylated TSLP (human and cynomolgus monkey) was then titrated onto each streptavidin surface to achieve <100 RU of Fab1 binding at saturation (Rmax). The low level of analyte binding ensured that artifacts caused by mass transport were minimized, especially when combined with the relatively fast assay flow rate of 50 μL min-1 used during the kinetic measurement step. Dilutions of monomerized Fab1 (2-fold dilutions in HBS-EP+ buffer ranging from 1.25 to 20 nM) (Multi-Cycle Kinetics) were injected at an assay flow rate of 50 μL min-1, with association times of 2 min and dissociation times of 10 min. Multiple injections of buffer alone were performed under the same conditions throughout the experiment to allow for double referencing of the final set of sensorgrams.

[0217] The chip surface was fully regenerated by two 30-s pulses of 10 mM glycine (pH 1.7). Binding affinities and kinetics were determined using a 1:1 Langmuir model.

[0218] The results shown in Table 5 indicate that Fab 1 have been shown to bind immobilized human TSLP and cynomolgus TSLP with similar affinity (within 2-fold; 46 pM and 88 pM, respectively). [Table 6]

[0219] Binding affinity determined by equilibrium exclusion assay (KinExA) In addition, Fabs against human TSLP and cynomolgus monkey TSLP 1 Liquid-phase binding affinity (K D ) was also determined using a KinExA3200 instrument (Sapidyne Instruments, Boise, Idaho, USA) and the resulting data were processed using KinExA Pro software version 4.1.11. KinExA methodology has been reviewed (Darling and Brault, 2004).

[0220] Fab 1 was premixed with various concentrations of human TSLP and cynomolgus TSLP, respectively, until equilibrium was reached (at least 12 concentrations of human TSLP and cynomolgus TSLP, respectively, were prepared using two-fold serial dilutions). 1 The amount of Fab was analyzed using a KinExA instrument by capturing free Fab using human TSLP-coated beads, washing to remove unbound material, and capturing bound Fab using commercially available species-specific antibodies (Alexa Fluor 647-labeled mouse anti-human heavy and light chain specific antibodies (Jackson Immunoresearch 209-605-088)). 1 Fab against human TSLP was measured by fluorescent detection. 1 K D , with 1000 pM (filled diamonds), 500 pM (filled inverted triangles) or 40 pM (open squares) of immobilized Fab 1 The Fab fragments against cynomolgus TSLP were extracted by 1:1 global fitting to three data sets obtained from titration of human TSLP into a concentration solution (Figure 1). 1 K D , with 1000 pM (filled diamonds) or 40 pM (open squares) of immobilized Fab1 The α- and β-terminal tails were extracted by 1:1 global fitting to two data sets obtained from cynomolgus TSLP titration into the concentration solution (Figure 2).

[0221] Free Fab detected at each human TSLP and cynomolgus monkey TSLP concentration 1 The amount of Fab was plotted against the titrated concentration of TSLP (Figures 1 and 2, respectively). The equilibrium dissociation constant (KD) was calculated using KinExA software. The results, shown in Table 6, indicate that Fab 1 binds human TSLP with 1.7-fold higher affinity than it binds to cynomolgus TSLP in free solution. [Table 7]

[0222] Example 3 - Fab1 and tezepelumab bind to TSLP with similar binding properties Fab 1 The binding properties of tezepelumab for human TSLP were directly compared to tezepelumab, a human immunoglobulin G2 (IgG2) monoclonal antibody (mAb) that binds to TSLP and inhibits its interaction with the TSLP receptor complex. A proof-of-concept study in patients with mild atopic asthma demonstrated that tezepelumab inhibited early and late asthmatic responses and suppressed biomarkers of Th2 inflammation after inhaled allergen challenge. Tezepelumab is currently under clinical investigation as a specialty agent for the treatment of severe asthma.

[0223] Fab 1 The in vitro binding ability of was determined based on a TSLP:mAb binding assay using homogeneous fluorescence resonance energy transfer (FRET) homogeneous time-resolved fluorescence (HTRF®, Cisbio International). Biotinylated TSLP was detected using streptavidin cryptate. Briefly, unlabeled Fab was used. 1The samples were titrated into the HTRF assay to compete with DyLight-labeled tezepelumab for binding to biotinylated His-Avi human TSLP. As a positive control, a competition assay was also performed using unlabeled tezepelumab and DyLight-labeled tezepelumab.

[0224] The result was Fab 1 It has been shown that Fab competes with tezepelumab for binding to human TSLP and binds to human TSLP with similar potency as tezepelumab (IC50: 1 0.38 nM; tezepelumab 0.23 nM; Figure 3). Fab 2~9 HTRF assays using Fabs were also performed and show that each of these Fabs also compete with tezepelumab for binding to human TSLP and bind to human TSLP with potencies similar to tezepelumab (Table 7). [Table 8]

[0225] Example 4 - Fab 1 Neutralizes TSLP activity in peripheral blood mononuclear cell (PBMC) assays Next, Fab against TSLP 1 To determine whether the binding of Fab has functional blocking activity in primary cell assays, 1 The effect of TSLP on CCL17 release was determined by measuring TSLP-induced release of CCL17 from PBMCs upon treatment with

[0226] Blood was obtained from healthy donors under the blood donor program established at MedImmune, Cambridge, UK. Peripheral blood mononuclear cells were isolated by standard procedures using a Ficoll gradient. Briefly, 20 ml of blood diluted in PBS (10 ml blood: 30 ml PBS) was layered on 15 ml of Ficoll. The tubes were centrifuged at 400g for 40 minutes at room temperature without brake. The PBMC layer was collected and the cells were washed twice with 50 ml of PBS. PBMC were counted using a hemocytometer and trypan blue to exclude dead cells, and resuspended in culture medium (RPMI with 10% fetal bovine serum and 1% penicillin / streptomycin) before plating in 96-well plates. TSLP-binding antibody fragment Fab 1 Cells were stimulated with TSLP (0.5 ng / ml) for 48 hours in the presence of . The assay was also performed using the TSLP-binding antibody tezepelumab as a positive control. After 48 hours, supernatants were removed and assayed for CCL17 production using the R&D DuoSet ELISA according to the manufacturer's protocol. Experiments were performed using six donors in three independent studies.

[0227] The result was Fab 1 but suppressed CCL17 production from PBMCs with an IC of 1.39 nM 50 The results show that the Fab fragment inhibited the Fab fragment (Figure 4). 1 In addition, Fab 2 and Fab 3 The assay was repeated using (containing the variable heavy and variable light chain sequences outlined in Table 3) with similar results (Figure 5).

[0228] Example 5 - Determination of maximum tolerated dose and pharmacokinetics of inhaled Fab1 in cynomolgus monkeys The purpose of this study is to 1 The objective of this study was to determine the maximum tolerated dose (MTD) or maximum tolerated dose (MFD) and pharmacokinetics (PK) of cynomolgus monkeys following inhalation exposure via facemask.

[0229] Female cynomolgus monkeys were given 8- and 20-minute Fab 1A single inhalation of 1 mg / kg was administered (Groups 1 and 2, 3 animals per group). For Groups 1 and 2, the doses delivered to the lungs were 1 mg / kg and 2 mg / kg, based on 25% lung deposition. In Group 3, dose escalation was repeated. One female and one male cynomolgus monkey were treated as follows: 8 min inhalation / day for the first 2 days, 20 min inhalation / day for 2 days, and 60 min inhalation / day for 3 days. Serial blood samples were collected using Fab 1 Serum PK and urea concentrations were collected. Bronchoalveolar lavage (BAL) samples were collected for Fab 1 Samples were collected for PK and urea concentration. Epithelial lining fluid (ELF) was calculated from BAL using urea concentration as a dilution marker. A hybrid immunoaffinity LC-MS / MS method was used to detect Fab in serum and BAL sample matrices. 1 Concentrations were determined. The lower limits of quantification were 4 ng / mL in serum and 10 ng / mL in BAL. Noncompartmental analysis (NCA) was performed on individual plasma PK data using Phoenix WinNonlin (version 7.0, Certara, LP, St Louis, MO).

[0230] Fab 1 After inhalation of Fab, serum PK, BAL and ELF concentrations increased with dose, 1 There was a wide variation in the concentrations (Figures 6A-6C). The mean serum elimination half-life of Fab1 ranged from 9.75 to 13.6 hours. max T 2-4 hours after inhalation max The ELF concentration was much higher (>2000-fold higher) than in serum after inhalation, and the Fab 1 This suggests that distribution of the antibody in serum was low.

[0231] Example 6: Evaluation of the physical characteristics of spray-dried formulations containing leucine and trileucine The effect of the concentration ratio of tri-leucine to leucine on particle properties is evaluated by the following method.

[0232] A total of 24 different wt% trileucine, leucine, and trehalose (TLT) powders were spray dried at 10% total feedstock solids using the same process parameters in a pilot scale spray dryer. Because the feedstock was prepared at 10% total solids (100 mg / mL), all wt% values ​​in this study are the same as the concentration values ​​(mg / mL). The range of concentration values ​​for each particulate excipient is shown in Table 8. [Table 9]

[0233] Each feedstock (Table 9) was prepared by dissolving the excipients in water. Once all excipients were completely dissolved, the feedstocks were spray dried using the following process parameters: outlet temperature 70° C.; feedstock feed rate 12 ml / min; nebulizer gas flow rate 13 kg / hr; and drying gas flow rate 80 kg / hr. The parameters were selected to achieve the desired particle and aerosol characteristics for the dry powder formulations intended for inhalation. Each of the 24 formulations was manufactured in a batch size of 18 g to provide sufficient powder for characterization and product performance evaluation. Batches were randomized and manufactured over two days. [Table 10]

[0234] The following physical powder characteristics were tested for all formulations: [Table 11]

[0235] The compressed bulk density (CBD) of the powders was measured using a GeoPyc® Model 1360 density analyzer (Micromeritics, Norcross, GA). The powder samples were prepared in a low humidity environment (<5% RH) and then transferred to the nitrogen gas purged sample chamber of the density analyzer. The net weight of the powder sample was recorded and then a compressive force of 12 N was applied to the sample by the plunger at a rate of 300 compaction steps per second. The linear distance traveled by the plunger at each compaction step was converted to the volumetric displacement of the powder sample. The average of the measurements for each compaction step was then calculated in g / cm. 3 The calculated bulk density was calculated as:

[0236] The results show that the leucine and trileucine contents were found to have a significant impact on particle properties. Trileucine was identified as the primary factor with the greatest impact, and leucine was identified as a secondary factor with a significant impact as well. The results are summarized in Table 11. [Table 12]

[0237] Example 7 - Aerosol performance characteristics of leucine / trileucine formulations The following examples evaluate the aerosol performance of formulations containing leucine and trileucine in a dry powder inhalation device. Twenty of the 24 formulations listed in Table 4 were tested for the aerosol performance outputs listed in Table 7. All product performance characterization was completed using a Monodose RS01 device with size 3 capsules. Next Generation Impactor (NGI) analysis was performed at a flow rate of 60 L / min.

[0238] USP <601> A cascade impaction test was performed according to the USP41, Chapter 4 to measure the aerosol performance of the spray-dried formulation when delivered from a dry powder inhalation device. The cascade impactor used was the Next Generation Impactor (NGI; USP41, Chapter 4). <601> ). In the aerosol measurements made in these examples, one size 3 HPMC capsule containing the spray-dried powder formulation was dispensed from a dry powder inhalation device and delivered to the NGI under vacuum at 60 L / min according to USP methods. Samples from each stage of the NGI were collected and assayed for protein content by UV absorbance at 280 nm. The main aerosol performance parameters calculated from these measurements were a) fine particle fraction <5 μm (FPF<5 μm), defined as the fraction of particles emitted from the device that measure less than 5 μm in aerodynamic size, and b) mass median aerodynamic diameter MMAD. [Table 13]

[0239] The results of the aerosol analysis are summarized in Table 13. [Table 14]

[0240] Example 8 - Preparation of inhaled leucine / trileucine formulations containing anti-TSLP antibody binding fragments (Fab) The characteristics of different formulations containing different Fabs were tested. Anti-TSLP Fabs were used, derived from human IgG1 monoclonal antibodies that specifically bind to TSLP (thymic stromal lymphopoietin) (see sequences shown in SEQ ID NOs: 1-8 provided herein). Different formulations were made consisting of the mass concentrations outlined in Table 14. [Table 15]

[0241] First, the anti-TSLP Fab was placed in a liquid buffer containing 105 mM trehalose, 30 mM citric acid (pH 6.0). Leucine, trileucine, trehalose, and citric acid were dissolved in separate aqueous solutions, which were then added to the anti-TSLP Fab solution to generate the bulk liquid feedstock solution for spray drying. Table 15 summarizes the composition of the feedstocks prepared to achieve the desired powder formulation composition. The liquid feedstock solutions were then spray dried using the process parameters listed in Table 16. The parameters were selected to achieve the desired particle and aerosol properties for a dry powder formulation intended for inhalation. [Table 16] [Table 17]

[0242] The results of powder and aerosol performance characterization of the spray dried formulations are summarized in Table 17. For aerosol performance measurements, 20 mg of spray dried powder was loaded into a size 3 HPMC capsule and dispensed from a dry powder inhalation device and tested for all three formulations. [Table 18]

[0243] Of particular note, 50 mg of formulation #3 could be loaded into one size 3 HPMC capsule due to the high bulk density of the powder. The high bulk density (cBD) allows for very high payload delivery from one capsule (FPM<5 μm approx. 14 mg, FPF 82%, MMAD 2.4 μm).

[0244] In addition, formulation #3 showed similar cBD (0.58 g / cm3) and SSA (4.6 m2 / g) as anti-IL-4 Fab formulation #2 (cBD=0.59 g / cm3, SSA=4.5 m2 / g), suggesting that powder properties are transferable between pharmaceutical formulations containing different active ingredients.

[0245] Example 9 - Powder and aerosol properties of anti-TSLP formulations spray dried in three batch sizes This example provides an analysis of the powder and aerosol properties of an anti-TSLP Fab-leucine / tri-leucine formulation using larger batch sizes to enable non-GLP and GLP inhalation toxicity studies. Scale-up requires the use of alternative scale spray drying equipment and adjustments to the spray drying process parameters to account for the increased heat and mass flow rates in the system and the need for extended processing times.

[0246] Three batches of spray dried anti-TSLP Fab formulation were produced with increasing batch size. The batches contained 40% w / w anti-TSLP Fab, 39% w / w trehalose, 10.5% w / w leucine, 2% w / w trileucine, and 8.5% w / w citric acid pH 6.0. Selected process parameters for each batch are shown in Table 18. [Table 19]

[0247] Aerosol performance testing for batch #1 was performed with a fill mass of 50 mg powder in size 3 HPMC capsules, whereas batches #2 and #3 were tested with a fill mass of 20 mg. Increasing the batch size from 8.5 g to 1.2 kg batch size resulted in a slight increase in oVMD, ranging from 0.45 to 0.85 g / cm. 3 A compacted bulk powder density (cBD) of 0.01 mg / kg was achieved. The aerosol performance of the powder was also maintained regardless of batch size, with high payload delivery of anti-TSLP Fab from the capsule-type inhalation device, demonstrating the scalability of the formulation with minimal adjustments to the spray drying process. All results of the powder characterization and aerosol performance testing are summarized in Table 19. [Table 20]

[0248] Example 10 Further characterization of leucine / trileucine formulations containing surfactants Additional batches of tri-leucine / leucine formulations containing various amounts of PS-80 were made. The formulation composition and process parameters used to make each batch are shown in Table 20. Otherwise, the formulations were made as described in Example 6. [Table 21-1] [Table 21-2] The aerosol properties of the formulations in Table 20 were analyzed using the method disclosed in Example 7. The results of the analysis are shown in Table 21. [Table 22]

[0249] The aggregate content, oVMD, residual moisture content, Tg, cBD and SSA were also measured using the methods described in the previous examples. The results of the powder characterization are shown in Table 22. [Table 23]

[0250] Analysis shows that the powder properties are similar to the control formulation regardless of the % (w / w) amount of PS-80.

[0251] The formulations listed in Table 22 were then analyzed for subvisible particle (SVP) content. Subvisible particle (SVP) counts were measured using microflow imaging technology (MFI). MFI combines microfluidic flow microscopy and high resolution image particle analysis to quantify SVP counts and bin these counts across a range of particle sizes. Prior to testing, powder samples were dissolved in water and shaken with gentle swirling to ensure uniform particle distribution, then loaded onto a Protein Simple MFI 5200 (CA, USA). Results were reported as counts of different particle sizes (≦1 μm, ≦2 μm, ≦5 μm, ≦10 μm, and ≦25 μm) / ml. Figure 14A shows that the inclusion of 0.27% (w / w / ) PS-80 in the dry powder formulation reduces the absolute number of SVP / ml upon reconstitution. The reduction in SVP counts decreases with increasing concentrations of PS-80. The addition of 0.67% (w / w) PS-80 resulted in a significant reduction in SVPs, with negligible amounts of SVPs with particle sizes greater than 5 μm. This trend was consistent with the FAB concentration of 30 mg / ml. 1 or FAB at a concentration of 2.5mg / ml 1 This was observed when the ribosomal RNA was reconstituted into ribosomal RNA (Figure 14B).

[0252] Formulation characterization and SVP analysis for formulations containing a second excipient were performed as described above. In this study, poloxamer 188 was used as the excipient as opposed to PS-80.

[0253] Multiple % w / w amounts of Poloxamer 188 were tested. Each formulation batch was made with the formulation composition and process parameters listed for the PS-80 containing formulation in Table 20. The amount of trehalose was varied to compensate for the varying amounts of Poloxamer 188.

[0254] The aggregate content, oVMD, residual moisture content, Tg, cBD and SSA were also measured using the methods described in the previous examples. The results of the powder characterization are shown in Table 23. [Table 24]

[0255] The aerosol properties of the poloxamer-188 formulations were also analyzed using the method disclosed in Example 7. The results are shown in Table 24. [Table 25] The SVP content of P188 formulations was analyzed using the methods described above. Figure 15A shows that the inclusion of 0.67% (w / w / ) P188 in the dry powder formulation reduces the absolute number of SVP / ml upon reconstitution. This trend was consistent with the FAB concentration of 30 mg / ml. 1 (Figure 15A) or FAB at a concentration of 2.5 mg / ml 1 (Figure 15B).

[0256] Example 11. Characterization of Leucine / Trileucine Formulations Containing 1.1% (w / w) PS-80 In this example, either 1% or 40% (w / w) Fab 1 and 1.1% (w / w / ) PS-80 were analyzed for powder properties. The complete formulation composition is shown in Table 25. The formulation was prepared as described in Example 6. [Table 26]

[0257] The stability of the formulations was analyzed after storage for 1 or 3 months at either 40° C. and 75% relative humidity (40 / 75) or 25° C. and 60% relative humidity (25 / 60). Particle size distribution, moisture content and surface rugosity were examined. Figures 16A and 16B show the Fab solubility at 40% (w / w) 1 Figures 17A and 17B show that the moisture content and particle size distribution are stable over time for a formulation containing 1% (w / w) Fab. C shows that the particle morphology is constant over time. 1Figure 17C shows that the moisture content and particle size distribution are stable over time for the formulation containing . Figure 17C shows that the particle morphology is constant over time.

[0258] The formation of SVPs upon reconstitution after storage at either 40 / 75 for 1 or 3 months, or at 25 / 60 for 3 months was analyzed. The analysis was performed as described in Example 8. Figure 18A shows the results of the analysis of SVPs after storage at 40% (w / w) Fab 1 The formulation was 30 mg.ml of Fab 1 Figure 18B shows that when reconstituted at 1% (w / w) Fab, the amount of SVP formed under each condition remains unchanged. 1 The formulation was 0.75 mg / ml Fab 1 These results indicate that when reconstituted at the same concentration, the amount of SVP formed under each condition remains unchanged.

[0259] The aerosol characteristics after storage were also tested, and the results are shown in Tables 26 and 27. [Table 27] [Table 28]

[0260] The percent delivered dose (DD) after storage of each formulation under each condition was also evaluated, and the results are shown in Tables 26 and 27.

[0261] Each Fab of the formulations described in Table 25 1 The potency of was also tested after 1 or 3 months of storage at 40 / 75, or 3 months at 25 / 60.

[0262] The titers were determined using homogeneous time-resolved fluorescence (HTRF). HTRF combines the technique of fluorescence resonance energy transfer (FRET) with time-resolved measurement (TR). When two fluorophores, the donor and the acceptor, are in close proximity to each other, excitation of the donor leads to energy transfer to the acceptor, generating a FRET signal. In this assay, streptavidin-europium cryptate bound to biotinylated human TSLP is the donor and d2-labeled anti-TSLP mAb is the acceptor. FAB 1 binds to human TSLP and inhibits the binding of the labeled mAb, resulting in an increased distance between the donor and acceptor fluorophores and a decrease in the FRET signal.

[0263] After evaluating the parallelism between the reference standard and the assay control or the reference standard and the test samples, a constrained four-parameter logistic (4PL) curve fitting was performed to obtain the FAB ratio by dividing the IC50 value of the reference standard by the IC50 value of the assay control or each test sample and multiplying by 100%. 1 The relative potency of the assay control and test samples is calculated.

[0264] Fab 1 The titer levels of Fab immediately after reconstitution (i.e., t=0) from the equivalent formulation 1 The titers were 85-110% of those of the original antibody.

[0265] References: Darling RJ,Brault PA.Assay and Drug Development Technologies.2004;2:647-657

[0266] Gauvreau GM, O'Byrne PM, Boulet LP, et al.N Engl J Med 2014;370:2102-10

[0267] Tepper,JS,et al Int J Toxicol 2016;35:376-92

[0268] Rennard, SI, et al J Appl Physiol 1986;60:532-538

[0269] It will be readily apparent to those skilled in the relevant art that other suitable modifications and adaptations to the methods and applications described herein may be made without departing from the scope of any of the embodiments. .fruit Examples are included herein for purposes of illustration only and are not intended to be limiting. Embodiments of the present invention are further described in the following sections: [Section 1] 1. A dry powder formulation comprising a plurality of microparticles, the microparticles comprising: a. leucine, b. about 1% to about 10% by weight of trileucine; c. An antigen-binding fragment of an anti-thymic stromal lymphopoietin (TSLP) antibody, i. a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:1; ii. a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:2; and iii. a heavy chain variable domain comprising a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:3, wherein any of heavy chain CDR1, 2 or 3 optionally comprises a single amino acid substitution; and i. a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; ii. a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; and iii. the antigen-binding fragment comprises a light chain variable domain comprising a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:7, wherein any of the light chain CDR1, 2 or 3 optionally comprises a single amino acid substitution; The dry powder formulation, wherein the leucine and the trileucine are present in a concentration ratio of about 0.1:1 to about 30 leucine:trileucine. [Section 2] The dry powder formulation has a density of about 0.4 to 1.0 g / cm 3 Item 2. The dry powder formulation according to item 1, having a compressed bulk density of [Section 3] Item 11. The dry powder formulation of any one of the preceding items, further comprising a glass stabilizer. [Section 4] Item 4. The dry powder formulation according to item 3, wherein the glass stabilizer is an amorphous sugar or a buffering agent. [Section 5] 4. The dry powder formulation according to claim 3, wherein the glass stabilizer comprises an amorphous sugar and a buffering agent. [Section 6] 6. The dry powder formulation according to item 4 or 5, wherein the amorphous sugar is selected from the group consisting of trehalose, sucrose, raffinose, inulin, dextran, mannitol, and cyclodextrin. [Section 7] 7. The dry powder formulation according to any one of items 4 to 6, wherein the buffer is selected from the group consisting of a citrate buffer, a phosphate buffer, a histidine buffer, a glycine buffer, an acetate buffer, and a tartrate buffer. [Section 8] 8. The dry powder formulation according to any one of items 4 to 7, wherein the amorphous sugar is trehalose. [Section 9] 9. The dry powder formulation according to any one of items 1 to 8, wherein the concentration ratio of leucine:trileucine is about 1:1 to about 12:1, optionally about 1:1 to about 7:1, optionally about 5.25:1. [Section 10] 10. The dry powder formulation according to any one of items 1 to 9, comprising about 8% by weight to about 11% by weight of leucine and about 2% by weight to about 4% by weight of trileucine. [Section 11] 11. The dry powder formulation according to any one of items 1 to 10, comprising about 10.5% by weight of leucine and about 2% by weight of trileucine. [Section 12] 12. The dry powder formulation according to any one of items 1 to 11, further comprising a surfactant, optionally selected from polysorbate-20 (PS-20), polysorbate-40 (PS-40), polysorbate-60 (PS-60), polysorbate-80 (PS-80) and poloxamer-188. [Section 13] Item 13. The dry powder formulation of item 12, wherein the surfactant is PS-80, and optionally, PS-80 is present in a concentration ranging from about 0.27% to about 2.7% by weight, optionally from about 0.67% to about 1.33% by weight. [Section 14] 14. The dry powder formulation of claim 13, wherein the PS-80 is present in a concentration of about 1.1% by weight. [Section 15] 1. A dry powder formulation comprising a plurality of microparticles, the microparticles comprising: a. about 10.5% by weight of leucine; b. about 2% by weight of trileucine; c. about 1% by weight to about 40% by weight of an antigen-binding fragment of an anti-thymic stromal lymphopoietin (TSLP) antibody, i. a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:1; ii. a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:2; and iii. a heavy chain variable domain comprising a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:3, wherein any of heavy chain CDR1, 2 or 3 optionally comprises a single amino acid substitution; and i. a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; ii. a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; and iii. the antigen-binding fragment comprising a light chain variable domain comprising a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:7, wherein any of the light chain CDR1, 2 or 3 optionally comprises a single amino acid substitution; and d. about 1.1% by weight of polysorbate-80; and e. trehalose up to 100% by weight; The dry powder formulation comprising: [Section 16] The dry powder formulation of any one of the preceding claims, wherein the heavy chain variable domain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the heavy chain variable domain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, the heavy chain variable domain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, the light chain variable domain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5, the light chain variable domain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6, and the light chain variable domain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7. [Section 17] the antigen-binding fragment comprising: a. a heavy chain variable domain comprising SEQ ID NO:4; b. A light chain variable domain comprising SEQ ID NO:8; Item 17. The dry powder formulation according to item 16, comprising: [Section 18] Item 18. The dry powder formulation according to item 17, wherein the antigen-binding fragment comprises a heavy chain having the sequence shown in SEQ ID NO:28 and a light chain having the sequence shown in SEQ ID NO:29. [Section 19] The dry powder formulation of any one of the preceding claims, wherein the antigen-binding fragment is selected from a Fab, Fab', F(ab')2, scFv, minibody, or diabody. [Section 20] 20. The dry powder formulation according to item 19, wherein the antigen-binding fragment is a Fab. [Section 21] 21. The dry powder formulation of claim 20, wherein the Fab is human or humanized. [Section 22] The dry powder formulation of any one of the preceding claims, wherein the anti-TSLP antibody from which the antigen-binding fragment is derived is IgG1. [Section 23] 23. A method for treating asthma in a patient, comprising administering by inhalation the dry powder formulation according to any one of items 1 to 22 above. [Section 24] 24. The method according to item 23 above, wherein the asthma is mild asthma. [Section 25] 24. The method according to claim 23, wherein the asthma is moderate asthma. [Section 26] 24. The method according to item 23 above, wherein the asthma is severe asthma. [Section 27] 24. The method according to claim 23, wherein the asthma is eosinophilic or non-eosinophilic asthma. [Section 28] 28. The method according to item 27 above, wherein the asthma is hypoeosinophilic asthma. [Section 29] The asthma is less than three of the following: Daytime asthma symptoms more than twice a week, Asthma-related nighttime awakenings, Use of asthma relief medications more than twice a week, and Activity limitations due to asthma 24. The method according to claim 23, characterized in that [Section 30] 23. The dry powder formulation according to any one of the above paragraphs 1 to 22 for use in a method of treatment, wherein the formulation is administered by inhalation. [Section 31] 31. A dry powder formulation for use according to claim 30 in a method for treating asthma. [Section 32] Item 32. The dry powder formulation for use according to item 31, wherein the asthma is mild asthma, moderate asthma, severe asthma, eosinophilic asthma or non-eosinophilic asthma, or hypoeosinophilic asthma. [Section 33] The asthma is less than three of the following: Daytime asthma symptoms more than twice a week, Asthma-related nighttime awakenings, Use of asthma relief medications more than twice a week, and Activity limitations due to asthma Item 32. A dry powder formulation for use according to item 31 above, characterized in that:

[0270] Although certain embodiments have been illustrated and described herein, it is to be understood that the claims are not limited to the specific forms or combinations of parts described and shown. Although exemplary embodiments are disclosed herein and specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.

[0271] Although various embodiments have been described above, it should be understood that they are presented only as illustrations and examples of the present technology, and are not limiting. It will be apparent to those skilled in the relevant art that various changes in form and detail can be made without departing from the spirit and scope of the present technology. Thus, the breadth and scope of the present technology should not be limited by any of the above-described embodiments, but should be defined only according to the appended claims and their equivalents. It should also be understood that each feature of each embodiment discussed in this specification and each feature of each reference cited in this specification can be used in combination with the features of any other embodiment. All patents and publications discussed in this specification are incorporated herein by reference in their entirety.

Claims

1. 1. A dry powder formulation comprising a plurality of microparticles, the microparticles comprising: a. between 8% and 11% by weight of leucine, wherein the leucine is present as a single amino acid; b. 2% to 4% by weight of trileucine; c. An antigen-binding fragment of an anti-thymic stromal lymphopoietin (TSLP) antibody, i. a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:1; ii. a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:2; and iii. a heavy chain variable domain comprising a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; and i. a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; ii. a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; and and the antigen-binding fragment comprises a light chain variable domain, the light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:

7.

2. The dry powder formulation has a density of 0.4 to 1.0 g / cm ３ 2. The dry powder formulation of claim 1 having a compressed bulk density of

3. 3. The dry powder formulation of claim 1 or claim 2, further comprising a glass stabilizer, wherein (i) the glass stabilizer is an amorphous sugar or a buffering agent, or (ii) the glass stabilizer comprises an amorphous sugar and a buffering agent.

4. 4. The dry powder formulation of claim 3, wherein the amorphous sugar is selected from the group consisting of trehalose, sucrose, raffinose, inulin, dextran, mannitol, and cyclodextrin.

5. 4. The dry powder formulation of claim 3, wherein the buffer is selected from the group consisting of a citrate buffer, a phosphate buffer, a histidine buffer, a glycine buffer, an acetate buffer and a tartrate buffer.

6. 4. The dry powder formulation of claim 3, wherein the amorphous sugar is trehalose.

7. The dry powder formulation of claim 1, comprising 10.5% by weight leucine and 2% by weight trileucine.

8. 10. The dry powder formulation of claim 1, further comprising a surfactant.

9. The dry powder formulation of claim 8, wherein the surfactant is selected from polysorbate-20 (PS-20), polysorbate-40 (PS-40), polysorbate-60 (PS-60), polysorbate-80 (PS-80), and poloxamer-188.

10. 10. The dry powder formulation of claim 9, wherein the surfactant is PS-80.

11. The dry powder formulation of claim 10, wherein PS-80 is present at a concentration of 0.27% to 2.7% by weight.

12. The dry powder formulation of claim 11, wherein PS-80 is present at a concentration in the range of 0.67% to 1.33% by weight.

13. 13. The dry powder formulation of claim 12, wherein the PS-80 is present at a concentration of 1.1% by weight.

14. A dry powder formulation comprising a plurality of microparticles, the microparticles comprising: a. 10.5% by weight leucine, wherein the leucine is present as a single amino acid; b. 2% by weight of trileucine; c. a Fab consisting of a heavy chain having the sequence shown in SEQ ID NO:28 and a light chain having the sequence shown in SEQ ID NO:29; d. a buffer; e. trehalose, The dry powder formulation of any one of claims 1 to 13, comprising:

15. the antigen-binding fragment comprising: a. a heavy chain variable domain comprising SEQ ID NO:4; b. a light chain variable domain comprising SEQ ID NO:8; 2. The dry powder formulation of claim 1, comprising:

16. 16. The dry powder formulation of claim 15, wherein the antigen-binding fragment comprises a heavy chain having the sequence set forth in SEQ ID NO:28 and a light chain having the sequence set forth in SEQ ID NO:

29.

17. 2. The dry powder formulation of claim 1, wherein the antigen-binding fragment is a Fab.

18. 2. The dry powder formulation of claim 1, wherein the anti-TSLP antibody from which the antigen-binding fragment is derived is IgG1.

19. 15. The dry powder formulation of claim 1 or claim 14 for use in a method of treatment.

20. 20. The dry powder formulation of claim 19 for use in a method for treating asthma.

21. 21. The dry powder formulation of claim 20, wherein the asthma is mild asthma, moderate asthma, severe asthma, eosinophilic or non-eosinophilic asthma, or hypoeosinophilic asthma.

22. The asthma is less than three of the following: Daytime asthma symptoms more than twice a week, Asthma-related nighttime awakenings, Use of asthma relief medications more than twice a week; and Activity limitations due to asthma 21. The dry powder formulation of claim 20,

23. 20. The dry powder formulation of claim 19, wherein the formulation is administered by inhalation.

24. An antigen-binding fragment of an anti-TSLP antibody comprising a heavy chain having the sequence shown in SEQ ID NO:28 and a light chain having the sequence shown in SEQ ID NO:29, wherein the antigen-binding fragment of the anti-TSLP antibody is an IgG1-derived Fab.

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