Dry powder formulations of antibodies that bind thymic stromal lymphopoietin (tslp) 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, suitably via pulmonary delivery.

Description

Invention Field

[0001] This invention generally relates to dry powder formulations derived from antigen-binding fragments of antibodies specific to thymic stromal lymphopoietin (TSLP), and methods for treating asthma (including mild, moderate, and severe asthma, eosinophilic asthma, and non-eosinophilic / hypoeosinophilic asthma) via pulmonary delivery using said dry powder formulations. The dry powder formulation comprises a mixture of leucine and trileucine, which makes the formulation particularly suitable for delivery via inhalation of antigen-binding fragments derived from anti-TSLP antibodies. Background Technology

[0002] Asthma affects approximately 300 million people worldwide, across all age groups, and places a significant burden on healthcare systems and society as it reduces workplace productivity and disrupts families (“Pocket Guide for Asthma Management and Prevention,” Global Initiative for Asthma; 2019). Asthma causes symptoms such as wheezing, shortness of breath, chest tightness, and cough, the occurrence, frequency, and intensity of which can vary over time. Symptoms are typically associated with bronchoconstriction, thickening of the airway walls, and increased mucus production. Asthma can present with varying degrees of symptom severity and can be well-controlled or poorly controlled depending on the frequency and severity of attacks.

[0003] TSLP is an epithelial cell-derived cytokine produced in response to environmental and pro-inflammatory stimuli, leading to the activation of various inflammatory cells and downstream pathways. TSLP is increased in the airways of asthmatic patients and is associated with the expression of Th2 cytokines and chemokines, as well as disease severity. Although TSLP is extremely important for Th2 immune regulation, it may also play a key role in other inflammatory pathways and is therefore associated with multiple asthma phenotypes.

[0004] Delivering antibodies against TSLP (particularly via inhalation) to patients can provide improved treatment options for asthma patients, including those with mild asthma who may require low-dose daily administration. Summary of the Invention

[0005] In view of the foregoing, in one respect, this document provides 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 an anti-thymocyte stromal lymphopoietin (TSLP) antibody.

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

[0007] On the other hand, a method for treating a patient’s asthma is provided, the method comprising administering the dry powder formulation of the first aspect via inhalation.

[0008] On the other hand, a dry powder formulation according to the first aspect is provided for use in a treatment method, wherein the formulation is administered by inhalation. In some embodiments, the formulation is used to treat asthma. Attached Figure Description

[0009] The above and other features and aspects of the invention will be better understood through the following description of the embodiments, and as illustrated in the accompanying drawings. The drawings, which are incorporated herein and form a part of this specification, further illustrate the principles of the invention. The drawings are not necessarily to scale.

[0010] Figure 1 The binding of Fab1 to human TSLP, as measured by KinExA, is shown.

[0011] Figure 2 The binding of Fab1 with cynomolgus monkey TSLP, as measured by KinExA, is shown.

[0012] Figure 3 The competitive binding of Fab1 to human TSLP, as measured using HTRF, is shown.

[0013] Figure 4 This demonstrates that Fab1 inhibits the release of CCL17 from PBMCs excited by TSLP.

[0014] Figure 5The results show that Fab1, Fab2, and Fab3 inhibit TSLP-induced release of CCL17 from PBMCs.

[0015] Figures 6A to 6C The Fab1 serum, BAL, and ELF PK curves after single-dose escalation (Groups 1 and 2) and repeated-dose escalation (Group 3) inhalation are shown.

[0016] Figure 7 The image shows microparticles from a dry powder formulation according to an embodiment of the present invention.

[0017] Figure 8A The results show the compressed bulk density in dry powder formulations as a function of leucine and trileucine.

[0018] Figure 8B Capsules filled with the dry powder formulation described herein are shown.

[0019] Figure 9 The results of the specific surface area (in m²) of the particles of the dry powder formulation according to an embodiment of the present invention, measured using BET, are shown. 2 / g (calculated).

[0020] Figure 10 The indirect correlation between moisture content and leucine concentration is shown.

[0021] Figures 11A to 11D The surface wrinkles of the particles are shown as detected by SEM.

[0022] Figure 12 The correlation between mass median aerodynamic diameter (MMAD) and leucine and trileucine wt% values ​​is shown.

[0023] Figure 13 The correlation between device deposition and leucine and trileucine wt% values ​​is shown.

[0024] Figure 14 The correlation between fine particulate fraction (FPF) and wt% values ​​of leucine and trileucine is shown.

[0025] Figure 15A The number of subvisible particles is shown after reconstituted formulations containing 40% (w / w) Fab1 and different concentrations of polysorbate-80 (PS-80) to a solution concentration of 30 mg / ml Fab1 ("≥" in the figure includes the upper limit of 200 μm size).

[0026] Figure 15BThe number of subvisible particles is shown after reconstructing formulations containing 40% (w / w) Fab1 and different concentrations of PS-80 to a solution concentration of 2.5 mg / ml Fab1 (≥ in the figure includes the upper limit of 200 μm size).

[0027] Figure 16A The number of subvisible particles after reconstituted formulations containing 40% (w / w) Fab1 and different concentrations of poloxamer-188 to a solution concentration of 30 mg / ml Fab1 is shown ("≥" in the figure includes an upper limit of 200 μm in size).

[0028] Figure 16B The number of subvisible particles is shown after reconstituted formulations containing 40% (w / w) Fab1 and different concentrations of poloxamer-188 to a solution concentration of 2.5 mg / ml Fab1 ("≥" in the figure includes an upper limit of 200 μm in size).

[0029] Figure 17A The moisture content of the formulation containing 40% (w / w) Fab1 and 1.1% PS-80 is shown after storage at 40°C and 75% relative humidity (40 / 75) for 1 or 3 months and at 25°C and 60% relative humidity (25 / 60) for 3 months.

[0030] Figure 17B The particle size distribution (PSD) of formulations containing 40% (w / w) Fab1 and 1.1% PS-80 is shown after storage at 40°C and 75% relative humidity (40 / 75) for 1 or 3 months and at 25°C and 60% relative humidity (25 / 60) for 3 months.

[0031] Figure 17C The particle morphology of formulations containing 40% (w / w) Fab1 and 1.1% PS-80 is shown after storage at 40°C and 75% relative humidity (40 / 75) for 1 or 3 months and at 25°C and 60% relative humidity (25 / 60) for 3 months.

[0032] Figure 18A The moisture content of formulations containing 1% (w / w) Fab1 and 1.1% PS-80 after storage at 40°C and 75% relative humidity (40 / 75) for 1 or 3 months and at 25°C and 60% relative humidity (25 / 60) for 3 months is shown.

[0033] Figure 18B The particle size distribution (PSD) of formulations containing 1% (w / w) Fab1 and 1.1% PS-80 is shown after storage at 40°C and 75% relative humidity (40 / 75) for 1 or 3 months and at 25°C and 60% relative humidity (25 / 60) for 3 months.

[0034] Figure 18C The particle morphology of formulations containing 1% (w / w) Fab1 and 1.1% PS-80 is shown after storage at 40°C and 75% relative humidity (40 / 75) for 1 or 3 months and at 25°C and 60% relative humidity (25 / 60) for 3 months.

[0035] Figure 19A The number of subvisible particles is shown after reconstituted a formulation containing 40% Fab1 and 1.1% PS-80 (w / w) to a solution concentration of 30 mg / ml Fab1, and after storage at 40 / 75 for 1 or 3 months and at 25 / 60 for 3 months.

[0036] Figure 19B The number of subvisible particles is shown after reconstituted a formulation containing 1% Fab1 and 1.1% PS-80 (w / w) to a solution concentration of 0.75 mg / ml Fab1, and after storage at 40 / 75 for 1 or 3 months and at 25 / 60 for 3 months. Detailed Implementation

[0037] The dry powder formulation described herein enables the treatment of asthma in a primary care setting using anti-TSLP antibody-binding fragments, thereby addressing an unmet need. Subjects with asthma typically control their asthma symptoms via inhaled self-delivered drug compositions, such as long-acting beta-agonists and / or corticosteroids.

[0038] However, existing biologics (both approved and in clinical trials) offer new treatment paradigms for asthma patients, but these drugs are often not delivered to subjects via the familiar pulmonary route. Tezepelumab (a next-generation biologic) is a human immunoglobulin G2 (IgG2) monoclonal antibody (mAb) that binds to TSLP, thereby blocking its interaction with the TSLP receptor complex. In a recent phase 2, randomized, double-blind, placebo-controlled trial, asthma subjects receiving subcutaneous tezepelumab had a clinically significant lower rate of asthma exacerbations than those receiving placebo (Corren et al. (2017) NEJM 377:936-946).

[0039] The invention described herein combines the therapeutic advantages of next-generation biological drugs (such as terzurumab) with a route of administration more familiar to subjects with asthma. Therefore, this invention enables the administration of such next-generation therapies in primary care settings, thereby extending the availability of these drugs to subjects beyond the scope of specialized care.

[0040] Furthermore, the formulations described herein are particularly suitable for treating less severe asthma patients whose condition is typically controlled in a primary care setting. For example, patients with a Global Asthma Initiative (GINA) grade 3 or lower (appropriately GINA grade 2 or 3) are particularly well-suited for treatment with the formulations described herein. In some embodiments, patients with a GINA score of 3 are well-suited for treatment with the formulations described herein. In some embodiments, patients with a GINA score of 2 are well-suited for treatment with the formulations described herein. Additionally, by delivering the biological agent directly to the lungs, side effects associated with systemic administration (such as injection site inflammation) are reduced.

[0041] In addition, the formulation provides the possibility of treating patients with moderate-to-severe asthma who can be controlled in a primary care setting or who have moderate-to-severe asthma that is difficult to treat with specialized care. For example, the formulation can be used to treat patients with moderate-to-severe asthma at Global Asthma Initiative (GINA) level 4-5. Where appropriate, the formulation provides the possibility of treating uncontrolled moderate-to-severe asthma. Where appropriate, the formulation provides the possibility of treating uncontrolled moderate-to-severe asthma with moderate to high doses of ICS:LABA and one or more episodes and frequent symptoms.

[0042] This document uses the term "approximately" to mean approximately, in the region of, roughly, or around. When the term "approximately" is used with a numerical range, it modifies the range by extending the upper and lower limits of the listed values. Typically, this document uses the term "approximately" to modify values ​​above and below the stated value with a deviation of 10%.

[0043] As described herein, dry powder formulations are provided for stabilizing and delivering pharmaceutical active agents. Suitablely, dry powder formulations are formulated for pulmonary delivery, including pulmonary delivery via inhalation through a dry powder inhaler (DPI).

[0044] As used herein, "dry powder formulation" refers to a formulation comprising a plurality of solid microparticles in a powder composition, said powder composition suitably containing less than about 20% moisture, more suitably less than 10% moisture, less than about 5%-6% moisture, or less than about 3% moisture. As described herein, dry powder formulations can be used for delivery to patients via inhalation. In other embodiments, the dry powder formulation can be reconfigured and administered in liquid form via oral, intravenous, parenteral, or other routes. As described herein, the advantage of the provided dry powder formulation is increased production capacity to improve manufacturability. Another advantage is that the formulation platform described herein provides a high compressibility packing density. This means that a larger mass of powder can be packaged per delivery unit (e.g., within a capsule). This means that each unit delivery can deliver a high dose of active agent to the subject. This surprising advantage can improve patient compliance by reducing the number of unit doses required. Furthermore, the high compressibility packing density enables the delivery of even higher doses of active agent, thereby increasing the upper limit of the dosage range. This enables the delivery of active agent at previously impossible therapeutically effective doses.

[0045] As used herein, “microparticles” refers to solid particles with a mass-mean diameter (MMD) of less than 20 μm. The mass-mean diameter is a measure of the average particle size, which is measured using appropriate methods, including, for example, centrifugation, electron microscopy, light scattering, laser diffraction, etc.

[0046] The dry powder formulations described herein suitably contain a plurality of microparticles. As used herein, “a plurality of” means two or more items, and suitably means five or more, ten or more, fifty or more, one hundred or more, five hundred or more, one thousand or more, or so on.

[0047] In an embodiment, the dry powder formulation comprises a plurality of microparticles, said microparticles suitably containing leucine; about 1% to about 10% by weight of trileucine; and an anti-TSLP antibody binding fragment as defined herein. Unless otherwise stated, “active agent” means an antigen-binding fragment derived from an anti-TSLP antibody as defined herein.

[0048] Figure 7 Scanning electron micrographs of the microparticles of the exemplary dry powder formulation provided herein are shown. In another embodiment, the dry powder formulation comprising a plurality of microparticles suitably contains about 1% to about 25% leucine; about 1% to about 10% trileucine; and an active agent.

[0049] As used in this article, whether it exists as a single amino acid or as an amino acid component of a peptide, "leucine" refers to the amino acid leucine (C6H4O2). 13NO2, which can be a racemic mixture or in its D or L form, as well as a modified form of leucine (i.e., in which one or more atoms of leucine have been substituted by another atom or functional group). The chemical structure of leucine is provided below:

[0050]

[0051] As used in this article, "trileucine" refers to a chemical compound in which three leucine molecules are linked together in a peptide, such as leucine-leucine-leucine (Leu-Leu-Leu), C 18 H 35 N3O4. The chemical structure of trileucine is provided below:

[0052]

[0053] Unless otherwise stated, the amounts of leucine and trileucine provided herein are provided as a weight percentage (wt%) of the formulation. Since dry powder formulations contain virtually no water (or very little water even if present), the weight components of dry powder formulations are percentages of the dry weight of the final formulation.

[0054] In embodiments of formulations comprising leucine, trileucine, and an antigen-binding fragment, leucine and trileucine are maintained within a desired ratio range that provides the improved compressible packing density characteristics described herein, as well as the desired particle characteristics that allow for improved storage and delivery. In embodiments, the weight ratio of leucine to trileucine in the particles (i.e., leucine:trileucine) is from about 0.1:1 to about 30:1. In another embodiment, leucine and trileucine are present in a leucine:trileucine 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.

[0055] Unless otherwise stated, ratios described herein are expressed as a percentage by weight (w / w – also known as “weight ratio”), i.e., the weight of leucine : the weight of trileucine in the formulation described herein. These ratios are achieved by providing the desired mg / mL concentrations of leucine and trileucine in the feedstock, followed by drying to remove the feedstock solvent, thereby forming atomized particles, wherein the initial concentration ratio (expressed in mg / mL) is maintained as the final weight ratio of leucine : trileucine.

[0056] This document describes exemplary weight percentages of leucine and trileucine that can be used in dry powder formulations to achieve these ratios. Suitably, 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.

[0057] In an exemplary embodiment, the dry powder formulation comprises about 1% to about 10% of trileucine by weight, more suitably about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, 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% of trileucine by weight.

[0058] In an exemplary embodiment, the dry powder formulation comprises about 1% to about 25% leucine by weight, more appropriately about 2% to about 20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 5% to about 15%, about 7% to about 12%, about 8% to about 11%, about 9% to about 11%, about 10% to about 11%, 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% leucine by weight.

[0059] In suitable embodiments, the dry powder formulation comprises about 8% to about 11% leucine and about 2% to about 4% trileucine by weight, more preferably about 9% to about 11% leucine and about 2% to about 3% trileucine by weight. In an exemplary embodiment, the dry powder formulation comprises about 10.5% leucine and about 2% trileucine by weight.

[0060] As described herein, it has been surprisingly found that the use of a combination of leucine and trileucine in dry powder formulations allows for a reduction in the total amount of leucine and trileucine required to prepare the microparticles (compared to dry powder formulations containing only one of these components), while also providing the desired stability. In some embodiments, the formulations of the present invention have an increased compressive bulk density compared to formulations in the art, which enables the delivery of higher concentrations of the active agent to the patient's lungs upon inhalation. These improved features appear to be related to the incorporation of leucine and trileucine into the microparticles.

[0061] According to embodiments of the present invention, an exemplary method for preparing a dry powder formulation is as follows: A liquid feedstock containing the desired final components of the dry powder formulation is atomized into a fine mist using an atomizer. The mist is then dried as described herein. The atomized droplets contain dissolved components initially in droplet form. As the droplets dry, different 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 particles of the dry powder formulation. This shell suitably includes leucine and trileucine components on its outer surface. It should be noted that leucine and trileucine are preferentially located on the outer surface of the particles, while small amounts of leucine and trileucine may also be present throughout the particles. In embodiments, higher concentrations of leucine and trileucine are suitably present on or near the particle surface, rather than near the particle center. In embodiments, the center of the particles contains a large amount of active agent, as well as other excipient components as described herein, which are suitably in an amorphous form. As used herein, “a large amount” of an active agent means that at least about 60% of the active agent (i.e., the total active agent in the formulation) is located at or near the center of the microparticle, suitably at least about 70%, and more suitably 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%-100% of the active agent is located at or near the center of the microparticle.

[0062] In another embodiment, the microparticles contain leucine and trileucine, which are substantially distributed throughout the microparticle, but in higher concentrations on or near the microparticle surface. As used herein, "substantially distributed throughout the microparticle" means that leucine and / or trileucine are distributed in a gradient from the outer surface of the microparticle to the center, but the amount of leucine and / or trileucine appropriately decreases closer to the center, and in this embodiment, leucine or trileucine is not found 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 entire cross-section of the microparticle.

[0063] In the embodiments, substantially every particle of the dry powder formulation contains leucine and trileucine. That is, suitably at least about 60% of the particles contain leucine and trileucine, or at least about 70%, and more suitably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, and in the embodiments about 95%-100% of the particles contain leucine and trileucine. In the embodiments, each particle of the dry powder formulation contains leucine and trileucine.

[0064] In another embodiment, leucine and / or trileucine may be present in the dry powder formulation, but are not contained within the particles of the formulation or are not associated with the particles of the formulation. Therefore, in this embodiment, free leucine and / or trileucine that are not associated with the particles may be found in the dry powder formulation. However, typically, the amount of free (i.e., not associated with the particles) leucine and / or trileucine is approximately less than about 10%, less than about 5%, less than about 1%, and more suitably less than about 0.1% of the total amount of leucine and / or trileucine in the formulation.

[0065] In some embodiments, the dry powder formulations described herein have a compressed bulk density that allows for the delivery of large quantities of active agent. "Compressed bulk density" refers to the mass per unit volume of powder (appropriately, g / cm³) when measured under the following conditions. 3 The examples describe a suitable determination of compressible bulk density (cBD) (see, for example, Example 6). Appropriately, a density analyzer, such as... The compressive bulk density (CBD) of the powder was measured using a Model 1360 density analyzer (Micromeritics, Norcross, GA). Powder samples were prepared appropriately in a low-humidity environment (<5% RH) and then transferred to the sample chamber of the density analyzer, which had been purged with nitrogen. The net weight of the powder sample was recorded, and then a compressive force of 10⁻¹⁴ N (appropriately 12 N) was applied to the sample via a plunger at a rate of 250–350 consolidation steps per second (appropriately 300 consolidation steps per second). The linear distance traveled by the plunger in each consolidation step was converted into the volumetric displacement of the powder sample. The average of the measurements from each consolidation step was then converted into a calculated bulk density value for the dry powder formulation, in g / cm³. 3 express.

[0066] Appropriately, the compressed bulk density of the dry powder formulation described herein is at least 0.4 g / cm³. 3 And appropriately at about 0.4 g / cm 3 To approximately 1.0 g / cm³ 3 Between, and appropriately approximately 0.4-0.9 gm / cm 3 Approximately 0.4-0.8 gm / cm 3 Approximately 0.5-0.8 gm / cm 3 Approximately 0.6-0.8 gm / cm 3 or approximately 0.4 gm / cm 3 Approximately 0.5 gm / cm 3 Approximately 0.6 gm / cm 3 Approximately 0.7 gm / cm 3 or approximately 0.8 gm / cm 3In some embodiments, the compressed bulk density of the dry powder formulation described herein is about 0.4 gm / cm³. 3 Approximately 0.9 gm / cm 3 In some embodiments, the compressed bulk density of the dry powder formulation described herein is about 0.5 g m / cm³. 3 Approximately 0.8 gm / cm 3 .

[0067] Figure 8A The results show the compressed bulk density as a function of leucine and trileucine in the dry powder formulation described herein. Each column represents the amount of trileucine in the formulation. Within each column, the amount of leucine increases from approximately 1% to approximately 20%. As shown in the figure, increasing the amount of trileucine results in a lower compressed bulk density, while increasing leucine within each group also reduces the compressed bulk density. To achieve approximately 0.5 g / cm³... 3 Approximately 0.8 g / cm³ 3 The compressible packing density should keep the amount of trileucine below 4% by weight.

[0068] The formulations described herein contain an antigen-binding fragment of an anti-thymocyte-lymphopoietin (anti-TSLP) antibody. Advantageously, the inventors have discovered that the formulations described herein enable direct delivery of the antigen-binding fragment to the lungs via inhalation. Delivery of the therapeutically active antigen-binding fragment of the anti-TSLP antibody via inhalation advantageously allows for the use of biological drugs to treat asthma in primary care settings.

[0069] The sequence of the TSLP peptide is provided below:

[0070] Met Phe Pro Phe Ala Leu Leu Tyr Val Leu Ser Val Ser Phe Arg Lys IlePhe Ile Leu Gln Leu Val Gly Leu Val Leu Thr Tyr Asp Phe Thr Asn Cys Asp PheGlu Lys Ile Lys Ala Ala Tyr Leu Ser Thr Ile Ser Lys Asp Leu Ile Thr Tyr MetSer Gly Thr Lys Ser Thr Glu Phe Asn Asn Thr Val Ser Cys Ser Asn Arg Pro HisCys Leu Thr Glu Ile Gln Ser Leu Thr Phe Asn Pro Thr Ala Gly Cys Ala Ser LeuAla Lys Glu Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile Trp Cys Pro GlyTyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met Lys Lys Arg Arg Lys Arg LysVal Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu Gln Gly Leu Trp Arg ArgPhe Asn Arg Pro Leu Leu Lys Gln Gln (SEQ ID NO: 27)

[0071] As used herein, the term "antibody" refers to a protein comprising at least two heavy chains and two light chains linked 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 typically consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain typically consists of a light chain variable region (VL) and a light chain constant region (CL). However, the term "antibody" also includes other types of antibodies, such as single-domain antibodies, heavy chain antibodies (i.e., antibodies consisting of only one or more, especially two, heavy chains), and nanobodies (i.e., antibodies consisting of only a single monomeric variable domain).

[0072] The antibody-binding fragments include: (i) Fab fragments, i.e., monovalent fragments consisting of variable regions of the heavy chain and light chain, and a first constant domain; (ii) F(ab)2 fragments, i.e., divalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of variable regions of the heavy chain and the first constant domain CH1; (iv) Fv fragments consisting of variable regions of the heavy chain and light chain of a single arm of the antibody; (v) scFv fragments, i.e., Fv fragments consisting of a single polypeptide chain; (vi) (Fv)2 fragments consisting of two covalently linked Fv fragments; (vii) a heavy chain variable domain; and (viii) multiply fragments consisting of covalently linked heavy chain and light chain variable regions, wherein the covalent linkage allows association of the heavy chain and light chain variable regions to occur only intermolecularly, and not intramolecularly. In embodiments, the antibody-binding fragments of the present invention are selected from Fab, Fab′, F(ab′)2, scFv, microantibodies, or biantibodies. In some embodiments, the antibody-binding fragment is Fab. In some embodiments, the anti-TSLP antibody that derives the antigen-binding fragment is IgG1.

[0073] The sequence of an exemplary Fab (hereinafter referred to as Fab1) of the present invention includes:

[0074] HCDR1 FAB1

[0075] Thr Tyr Gly Met His (SEQ ID NO: 1)

[0076] HCDR2 FAB1

[0077] Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val Lys Gly (SEQ ID NO: 2)

[0078] HCDR3 FAB1

[0079] Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile (SEQ ID NO: 3)

[0080] Heavy chain VH FAB1

[0081] Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr Gly Met His TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp GlySer Asn Lys His Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Thr Arg Asp AsnSer Lys Asn Thr Leu Asn Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala ValTyr Tyr Cys Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile TrpGly Gln Gly Thr Met Val Thr Val Ser Ser(SEQ ID NO:4)

[0082] LCDR1 FAB1

[0083] Gly Gly Asn Asn Leu Gly Ser Lys Ser Val His(SEQ ID NO:5)

[0084] LCDR2 FAB1

[0085] Asp Asp Ser Asp Arg Pro Ser(SEQ ID NO:6)

[0086] LCDR3 FAB1

[0087] Gln Val Trp Asp Ser Ser SerAsp His Val Val(SEQ ID NO:7)

[0088] Light chain VL FAB1

[0089] Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln ThrAla Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val His Trp Tyr GlnGln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro SerTrp Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr IleSer Arg Gly Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser SerSer Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO: 8)

[0090] FAB1 variable heavy chain VH (nucleic acid)

[0091]

[0092] FAB1 variable light chain VL (nucleic acid)

[0093]

[0094] FAB1 heavy chain (peptide)

[0095] 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)

[0096] FAB1 light chain (polypeptide)

[0097] 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)

[0098] FAB1 heavy chain (nucleic acid)

[0099]

[0100] FAB1 light chain (nucleic acid)

[0101]

[0102] The dry powder formulation provided herein comprises a plurality of microparticles, said microparticles comprising: leucine; about 1% to about 10% by weight of trileucine; and an antigen-binding fragment of an anti-thymocyte stromal lymphopoietin (TSLP) antibody, wherein leucine and trileucine are present in a leucine:trileucine concentration ratio of about 0.1:1 to about 30:1.

[0103] In some embodiments, the antigen-binding fragment within the dry powder formulation contains

[0104] a. A heavy-chain variable structural domain, wherein the heavy-chain variable structural domain comprises:

[0105] The heavy chain CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO: 1, the heavy chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO: 2, and the heavy chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO: 3, wherein any one of the heavy chains CDR1, 2, or 3 may contain a single amino acid substitution as desired, and

[0106] b. A light chain variable structural domain, wherein the light chain variable structural domain comprises:

[0107] The light chain CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO: 5, the light chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO: 6, and the light chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO: 7; wherein any one of the light chains CDR 1, 2, or 3 optionally comprises a single amino acid substitution.

[0108] In some embodiments, the antigen-binding fragment within the dry powder formulation comprises a heavy chain variable domain, said heavy chain variable domain comprising a light chain CDR1 sequence having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 sequence having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 sequence having the amino acid sequence shown in SEQ ID NO: 3, as well as a light chain CDR1 sequence having the amino acid sequence shown in SEQ ID NO: 5, a light chain CDR2 sequence having the amino acid sequence shown in SEQ ID NO: 6, and a light chain CDR3 sequence having the amino acid sequence shown in SEQ ID NO: 7.

[0109] In another embodiment, the antigen-binding fragment for use in a 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 another embodiment, the antigen-binding fragment for use in a dry powder formulation 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.

[0110] In another embodiment, the antigen-binding fragment for use in a dry powder formulation comprises: a heavy chain variable domain, the heavy chain variable domain being an amino acid sequence having at least 95%, 90%, 85%, or 80% identity with SEQ ID NO: 4; and a light chain variable domain, the light chain variable domain being an amino acid sequence having at least 95%, 90%, 85%, or 80% identity with SEQ ID NO: 8.

[0111] In another embodiment, the antigen-binding fragment for use in a dry powder formulation comprises: (a) a heavy chain variable domain, said heavy chain variable domain being an amino acid sequence having at least 95%, 90%, 85%, or 80% identity with SEQ ID NO: 4, or an amino acid sequence encoded by a polynucleotide sequence having at least 80% identity with SEQ ID NO: 30; (b) a light chain variable domain, said light chain variable domain being an amino acid sequence having at least 95%, 90%, 85%, or 80% identity with SEQ ID NO: 8, or an amino acid sequence encoded by a polynucleotide sequence having at least 80% identity with SEQ ID NO: 31; or the heavy chain variable domain of (a) and the light chain variable domain of (b).

[0112] Other 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 present invention include:

[0113] LCDR1 FAB2

[0114] Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His (SEQ ID NO: 11)

[0115] Light chain VL FAB2

[0116] 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)

[0117] LCDR1 FAB3

[0118] Gly Gly Asn Asn Val Gly Ser Lys Ser Val His(SEQ ID NO:13)

[0119] Light chain VL FAB3

[0120] 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)

[0121] HCDR2 FAB4

[0122] Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Glu Ser Val Lys Gly(SEQ ID NO:15)

[0123] Heavy chain VH FAB4

[0124] Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr Gly Met His TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp GlySer Asn Lys His Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Thr Arg Asp AsnSer Lys Asn Thr Leu Asn Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala ValTyr Tyr Cys Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile TrpGly Gln Gly Thr Met Val Thr Val Ser Ser(SEQ ID NO:16)

[0125] HCDR2 FAB5

[0126] Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val Lys Ala(SEQ ID NO:17)

[0127] Heavy chain VH FAB5

[0128] Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr Gly Met His TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp GlySer Asn Lys His Tyr Ala Asp Ser Val Lys Ala Arg Phe Thr Ile Thr Arg Asp AsnSer Lys Asn Thr Leu Asn Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala ValTyr Tyr Cys Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile TrpGly Gln Gly Thr Met Val Thr Val Ser Ser(SEQ ID NO:18)

[0129] LCDR1 FAB6

[0130] Gly Gly Gln Asn Leu Gly Ser Lys Ser Val His(SEQ ID NO:19)

[0131] Light chain VL FAB6

[0132] 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)

[0133] LCDR1 FAB7

[0134] Gly Gly Asn Gln Leu Gly Ser Lys Ser Val His(SEQ ID NO:21)

[0135] Light chain VL FAB7

[0136] 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)

[0137] LCDR3 FAB8

[0138] Gln Val Trp Asp Thr Ser Ser Asp His Val Val(SEQ ID NO:23)

[0139] Light chain VL FAB8

[0140] Ser Tyr Val Leu Thr Gln Pro Pr0 Ser Val Ser Val Ala Pro Gly Gln ThrAla Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val His Trp Tyr GlnGln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro SerTrp Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr IleSer Arg Gly Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr SerSer Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu(SEQ ID NO:24)

[0141] LCDR3 FAB9

[0142] Gln Val Trp Asp Ser Thr Ser Asp His Val Val(SEQ ID NO:25)

[0143] Light chain VL FAB9

[0144] Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val A1a Pro Gly Gln ThrAla Arg Ile Thr Cys Gly Gly Gly Asn Asn Leu Gly Ser Lys Ser Val His Trp Tyr GlnGln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro SerTrp Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr IleSer Arg Gly Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser ThrSer Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO: 26).

[0145] In some embodiments, the heavy chain variable domain and light chain variable domain of the antigen-binding fragment of the present invention comprise any combination of the CDR sequences listed in the table below:

[0146] VH CDR 1, 2 and 3 VL CDR 1, 2 and 3 <![CDATA[Fab1]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 5, 6 and 7 <![CDATA[Fab2]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 11, 6 and 7 <![CDATA[Fab3]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 14, 6 and 7 <![CDATA[Fab4]]> SEQ ID NO: 1, 15 and 3 SEQ ID NO: 5, 6 and 7 <![CDATA[Fab5]]> SEQ ID NO: 1, 17 and 3 SEQ ID NO: 5, 6 and 7 <![CDATA[Fab6]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 19, 6 and 7 <![CDATA[Fab7]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 19, 6 and 7 <![CDATA[Fab8]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 5, 6 and 23 <![CDATA[Fab9]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 5, 6 and 25

[0147] The formulations disclosed herein can be administered in combination with other active agents for 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 β-agonists (LABA), long-acting antimuscarinic agonists (LAMA), short-acting β-agonists (SABA), and muscarinic β2 agonists (MABA)), antihistamines, antileukotrienes, PDE-4 inhibitors, Janus kinase inhibitors, and phosphoinositol 3-kinase inhibitors. In some embodiments, additional active agents are incorporated into the formulations of the present invention along with the anti-TSLP antibody-binding fragments disclosed herein.

[0148] In suitable embodiments, the dry powder formulations described herein also include glass stabilizers to help stabilize the formulation, particularly the active agent. A “glass stabilizer” is an excipient that stabilizes an active agent (suitably a polypeptide) in a dry powder formulation and forms an amorphous solid containing said active agent, said stabilization being suitably achieved by displacing water on the surface of said active agent during drying or otherwise preventing degradation processes. 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 suitable embodiments, the glass stabilizer is an amorphous sugar. In other embodiments, the glass stabilizer is a buffer. In still other embodiments, the formulations described herein may contain both amorphous sugars and buffers, which may act together or separately as glass stabilizers.

[0149] Exemplary amorphous sugars used 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 in the form of about 30% to about 70% (by weight) of the dry powder formulation. In further embodiments, the amorphous sugar is present in the form of 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% by weight of the dry powder formulation. Suitably, the amorphous sugar is trehalose and is present in the formulation in the form of about 30%-60%, more suitably about 35%-55%, or about 35%, about 40%, about 45%, or about 50% by weight of the dry powder formulation.

[0150] Exemplary buffers (suitably as glass stabilizers) that may be included in dry powder formulations 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 a dry powder formulation may range from about 0.1% to about 20%, more suitably 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%.

[0151] The buffer also provides pH control for the dry powder formulation, appropriately maintaining the pH between about pH 5 and about pH 8, for example, between about pH 5 and about pH 6, or about pH 5.5 and about pH 6.5, or about pH 6 and about pH 7, or about pH 6.5 and about pH 7.5, or about pH 7 and about pH 8.

[0152] In another embodiment, a dry powder formulation is provided, the dry powder formulation 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.

[0153] In another embodiment, a dry powder formulation is provided, which is substantially composed of about 30%-50% amorphous sugar, leucine, about 1% to about 10% trileucine, about 1% to about 10% buffer and active agent, wherein leucine and trileucine are present in a leucine:trileucine concentration ratio of about 0.1:1 to about 30:1. In another embodiment, a dry powder formulation is provided, which is substantially composed of about 30%-50% amorphous sugar, about 8% to about 11% leucine, about 2% to about 4% trileucine, about 1% to about 10% buffer and active agent. A further dry powder formulation is provided, which is substantially composed of about 35%-45% trehalose, about 9% to about 11% leucine, about 2% to about 3% trileucine, about 2% to about 85% citrate buffer and active agent. In another embodiment, the dry powder formulation consists essentially of about 39% trehalose, about 10.5% leucine, about 2% trileucine, about 8.5% citrate buffer, and an active agent.

[0154] In compositions and formulations consisting "substantially composed of" the listed ingredients, such compositions and formulations contain the listed components as well as those components that do not substantially affect the essential and novel characteristics of the claimed formulation. Components that do not substantially affect the essential and novel characteristics of the claimed formulation are those components that do not limit the ability of leucine and trileucine to stabilize dry powder formulations. Appropriately, compositions and formulations consisting "substantially composed of" the listed ingredients specifically exclude other amino acids or tripeptide amino acids, but may include additional sugars, buffers, etc.

[0155] In an exemplary 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 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.

[0156] In another exemplary embodiment, a dry powder formulation is provided that consists 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.

[0157] When the microparticles constituting the dry powder formulation described herein are provided in aerosol form, they suitably have a specified mass median aerodynamic diameter (MMAD). The microparticles may also have a specified equivalent optical volume average diameter (oVMD). oVMD may also be referred to as particle size distribution (PSD or pPSD).

[0158] As used herein, “mass median aerodynamic diameter” or “MMAD” is a measure of the aerodynamic particle size of dispersed particles. Aerodynamic diameter describes the settling behavior of atomized powder and is the diameter of a sphere of unit density in air with the same settling rate as the particles. Aerodynamic diameter encompasses the particle shape, density, and physical size. As used herein, unless otherwise specified, MMAD refers to the midpoint or median of the aerodynamic particle size distribution of atomized powder as determined by cascade impaction. Suitablely, the particles of the dry powder formulations provided herein have the following median mass aerodynamic diameter (MMAD): about 1 μm to about 10 μm, more suitably 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.

[0159] Appropriately, the fine particulate fraction (the fraction of particles with an aerodynamic diameter of less than 5 μm ejected from the inhalation device) of the dry powder formulation described herein is ≥50%, more appropriately ≥60%. This fine particulate fraction (FPF) can contribute to low device residue of the dry powder formulation after delivery to the patient, with less than 20%, appropriately less than 15%, less than 10%, or less than 5% remaining in the device.

[0160] In another embodiment, the particles suitably have an equivalent optical volume average diameter (oVMD) of about 0.5 μm to about 7 μm. The equivalent optical volume average diameter (oVMD) refers to the average diameter of the sphere that most closely approximates the specific optical interaction between the particle and light, wherein, when measured using suitable optical techniques, half of the particles are most closely approximate to an equivalent sphere smaller than the average, while the other half are most closely approximate to an equivalent sphere larger than the average. In an exemplary embodiment, the particles have an equivalent optical volume average 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.

[0161] As described herein, high compressible packing density allows for the delivery of larger quantities of active agent using the same delivery volume. Some biologics may require delivery of payloads of up to 50 mg / dose or higher for effective treatment. Figure 8B As shown in the figure, the combination of leucine and trileucine can produce a dry powder formulation with a higher bulk density, thus occupying a significantly smaller volume for the same amount of filler weight.

[0162] The following provides Figure 8B The exemplary platform formulations shown are as follows. LTC indicates a formulation that does not contain trileucine (TLeu) but contains leucine, trehalose, and citrate buffer; TTC indicates a formulation that does not contain leucine (Leu) but contains trileucine, trehalose, and citrate buffer; TLTC indicates a formulation containing both leucine and trileucine, as well as trehalose and citrate buffer. Cit refers to citrate buffer. Tre refers to trehalose.

[0163] Table 1: Exemplary Platform Formulations

[0164] platform %Tre %Leu %TLeu %Cit LTC 46 45 0 9 TTC 81 0 11.2 7.8 TLTC 79 10.5 2 8.5

[0165] Figure 8B The capsules for each formulation (capsule #3) are shown in the diagram with corresponding fill weights. As illustrated, for the TLTC formulation, the combination of trileucine and leucine allows for filling the capsule with 100 mg of dry powder while still leaving some space within the capsule. Other formulations cannot be filled with a fill weight exceeding approximately 70-80 mg. This represents a significant improvement provided by using the combination of leucine and trileucine to prepare formulations with high compressive packing density, thus allowing for higher fill weights.

[0166] As described herein, the use of leucine and trileucine in dry powder formulations also produces microparticles with desired size (MMAD) and desired specific surface area (SSA) and roughness, thereby producing microparticles that can flow appropriately and be delivered to the lungs using a variety of inhalation platforms.

[0167] The specific surface area (SSA) of a particle is defined as the total surface area of ​​particles per unit mass (appropriately expressed in m³). 2 Methods for measuring SSA are known in the art and include, for example, the Brunauer-Emmett-Teller (BET) measurement, which uses nitrogen adsorption as a function of relative pressure to assess the specific surface area of ​​a material. The surface area is determined by calculating the amount of adsorbed gas corresponding to a monolayer on the particle surface. This technique measures the external area and any pore area assessments to determine the total specific surface area. Instruments used for measuring BET are known in the art.

[0168] In the implementation scheme, the specific surface area (SSA) of the particles in the dry powder formulation is approximately 3 m². 2 / g to approximately 8m 2 / g. In a suitable implementation, the SSA of multiple particles is approximately 3.5m. 2 / g-7.5m 2 / g, or approximately 4m 2 / g-7m 2 / g, or approximately 4.5m 2 / g-7m 2 / g, or approximately 5mg 2 / g-7m 2 / g, or approximately 4.5m 2 / g-6m 2 / g, or approximately 5m 2 / g-6m 2 / g, or approximately 4m 2 / g, approximately 4.5m 2 / g, approximately 5m 2 / g, approximately 5.5m 2 / g, approximately 6m 2 / g, approximately 6.5m 2 / g, or approximately 7m 2 / g.

[0169] Figure 9 The specific surface area (in m²) measured using BET is shown. 2 The result ( / g calculation). Figure 9Each column represents a different amount of trileucine in the formulation. Within each column, the amount of leucine increases from approximately 1% to approximately 20%. The inset micrographs show the physical appearance of the particles at low SSA (bottom left) and high SSA (top right). As shown, at lower wt% trileucine, the SSA remains below approximately 5 μm. 2 / g, but it increases with increasing leucine content. For trileucine at approximately 2%, SSA increases to over 3.0m. 2 / g, and it also increases with the percentage of leucine. When the amount of trileucine is greater than about 4%, the achieved SSA value is greater than 5.5m. 2 / g and close to 7.0m 2 / g. Approximately 4-7 mg can be easily achieved using amounts of trileucine between approximately 1% and 6% and leucine between approximately 1% and 20%. 2 The desired range of specific surface area per g. As shown in the figure, by using an amount of trileucine below about 6%, the amount of leucine can be kept below 10%, or even below 5%, while still maintaining the desired SSA and particles with surface roughness. The micrograph in the upper left shows the particle shape of the dry powder formulation described herein, exhibiting the desired size, specific surface area, and surface roughness.

[0170] In some embodiments, the compressed bulk density of the dry powder formulation is about 0.4-1.0 g / cm³. 3 Appropriately, the compressed bulk density of dry powder formulations is approximately 0.5-0.8 g / cm³. 3 In the implementation scheme, the compressed bulk density of the dry powder formulation described herein is approximately 0.4-0.9 gm / cm³. 3 Approximately 0.4-0.8 gm / cm 3 Approximately 0.5-0.8 gm / cm 3 Approximately 0.6-0.8 gm / cm 3 or approximately 0.4 gm / cm 3 Approximately 0.5 gm / cm 3 Approximately 0.6 gm / cm 3 Approximately 0.7 gm / cm 3 or approximately 0.8 gm / cm 3 In some embodiments, the compressed bulk density of the dry powder formulation described herein is about 0.4 gm / cm³. 3 Approximately 0.9 gm / cm 3 In some embodiments, the compressed bulk density of the dry powder formulation described herein is about 0.5 g m / cm³. 3 Approximately 0.8 gm / cm 3 .

[0171] Dry powder formulations suitably include glass stabilizers as described herein, said glass stabilizers comprising amorphous sugars or buffers, or both amorphous sugars and buffers. Exemplary amorphous sugars include those described herein, including trehalose, sucrose, raffinose, inulin, dextran, and cyclodextrin. Suitably, the amorphous sugar is present in an amount of about 30% to about 70%; and in embodiments the amorphous sugar is trehalose, which is suitably present in an amount of about 35%-60%, or 35%-55%.

[0172] This document describes exemplary buffers for use in dry powder formulations, including citrate buffers, phosphate buffers, and tartrate buffers. Suitably, the buffer is present at about 1% to about 10%, and in some embodiments, the buffer is a citrate buffer. In some embodiments, the citrate buffer has a pH of 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 some embodiments, the citrate buffer has a pH of about pH 6.4.

[0173] In some embodiments, the dry powder formulations described herein contain surfactants. As defined herein, a "surfactant" is a molecule or compound that reduces particle aggregation or adhesion to the surface of capsules, container walls, or valve components of inhalable delivery devices. Surfactants have also been found to reduce the formation of subvisible particles (SVPs) after formulation remodeling. Removing or reducing SVP formation simplifies the analytical characterization of the formulation because it eliminates the burden of tracking SVP formation during manufacturing. Analytical characterization of SVPs may involve developing orthogonal techniques to identify and quantify SVPs for quality control purposes. Therefore, removing SVPs or reducing them to acceptable levels eliminates the need for this characterization step during manufacturing, thereby streamlining the manufacturing process. Since the drug release kinetics from SVPs are unknown, removing SVPs can also make the dose range more predictable. Furthermore, removing SVPs may increase the amount of active agent available to exert pharmacological activity after remodeling, which may mean not only achieving higher delivery doses but also calculating more accurate predicted delivery doses. Higher delivery doses can also benefit patients, for example, by potentially reducing the number or frequency of doses that must be delivered to extract the pharmacological benefit.

[0174] Sub-visible particles (“SVPs”) are particles of approximately 1 μm to approximately 200 μm that are invisible to the naked eye. The presence of SVPs can be determined by reconstructing dry powder formulations and liquids with turbidity. Techniques such as microfluidic imaging (MFI) can be used to confirm the actual presence of SVPs. Microfluidic imaging (MFI) combines microfluidic microscopy and high-resolution imaging particle analysis to quantify SVP counts. MFI can classify these counts across particle size ranges, for example, by classifying particle counts within size ranges of approximately 1 to approximately 200 μm, approximately 2 μm to approximately 200 μm, approximately 5 μm to approximately 200 μm, approximately 10 μm to approximately 200 μm, and approximately 25 μm to approximately 200 μm. Examples show that the inclusion of surfactants in dry powder formulations reduces the presence of SVPs in each particle size range compared to control formulations without surfactants (e.g., ...). Figure 15A Therefore, in some embodiments, the dry powder formulations disclosed herein contain a surfactant, wherein the number of subvisible particles in the formulation is reduced after reconfiguration. In some embodiments, the number of subvisible particles is reduced compared to an equivalent formulation without a surfactant.

[0175] In some 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, such as 25,000 particles / ml, 20,000 particles / ml, 15,000 particles / ml, 10,000 particles / ml, or 5,000 particles / ml. In some 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 some 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 some embodiments, the number of SVPs with a size of about 25 μm to about 200 μm is reduced to less than 100 particles / ml.

[0176] In some 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, such as 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 some 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 some 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 some embodiments, the number of SVPs with a size of about 10 μm to about 200 μm is reduced to less than 100 particles / ml.

[0177] In some 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, such as 180,000 particles / ml, 170,000 particles / ml, 160,000 particles / ml, 150,000 particles / ml, or 140,000 particles / ml. In some 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 some 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 some 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.

[0178] In some implementations, the number of SVPs with a size of about 2 μm to about 200 μm is reduced to less than 1 x 102 6 Particles / ml, e.g., 0.8 x 10⁻⁶ 6 Particles / ml, 0.7x10 6 Particles / ml, 0.6x10 6 1 particle / ml or 0.5 x 10 6 In some embodiments, the number of SVPs with a size of about 2 μm to about 200 μm is reduced to less than 100,000 particles / ml. In some embodiments, the number of SVPs with a size of about 2 μm to about 200 μm is reduced to less than 50,000 particles / ml. In some embodiments, the number of SVPs with a size of about 2 μm to about 200 μm is reduced to less than 10,000 particles / ml.

[0179] In some implementations, the number of SVPs with a size of about 1 μm to about 200 μm is reduced to less than 2 x 10⁻⁶. 6 Particles / ml, e.g., 1.8 x 10⁻⁶ 6 1.7 x 10⁻⁶ particles / ml 6 Particles / ml, 1.6 x 10 6 1 particle / ml or 1.5 x 10 6 Particles / ml. In some embodiments, the number of SVPs with a size of about 1 μm to about 200 μm is reduced to less than 200,000 particles / ml. In some embodiments, the number of SVPs with a size of about 1 μm to about 200 μm is reduced to less than 150,000 particles / ml.

[0180] In some embodiments, the number of SVPs with a size of about 25 μm to about 200 μm is reduced by more than 2 times, such as more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, or more than 9 times, after reconstruction compared to a reference control. In some embodiments, the number of SVPs with a size of about 25 μm to about 200 μm is reduced by more than 10 times after reconstruction compared to a reference control.

[0181] In some embodiments, the number of SVPs with a size of about 10 μm to about 200 μm is reduced by more than 2 times, such as more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, or more than 9 times, after reconstruction compared to a reference control. In some embodiments, the number of SVPs with a size of about 10 μm to about 200 μm is reduced by more than 10 times after reconstruction compared to a reference control.

[0182] In some embodiments, the number of SVPs with a size of about 5 μm to about 200 μm is reduced by more than 2 times, such as more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, or more than 9 times, after reconstruction compared to a reference control. In some embodiments, the number of SVPs with a size of about 5 μm to about 200 μm is reduced by more than 10 times after reconstruction compared to a reference control.

[0183] In some embodiments, the number of SVPs with a size of about 2 μm to about 200 μm is reduced by more than 2 times, such as more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, or more than 9 times, after reconstruction compared to a reference control. In some embodiments, the number of SVPs with a size of about 2 μm to about 200 μm is reduced by more than 10 times, compared to a reference control. In some embodiments, the number of SVPs with a size of about 2 μm to about 200 μm is reduced by more than 100 times, compared to a reference control.

[0184] In some embodiments, the number of SVPs with a size of about 1 μm to about 200 μm is reduced by more than 2 times, such as more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, or more than 9 times, after reconstruction compared to a reference control. In some embodiments, the number of SVPs with a size of about 1 μm to about 200 μm is reduced by more than 10 times after reconstruction compared to a reference control.

[0185] In some embodiments, the reference control is an equivalent formulation lacking the surfactant. In some embodiments, the formulation is reconstituted in water. In some embodiments, the formulation is reconstituted to an surfactant concentration of 30 mg / ml. In some embodiments, the formulation is reconstituted to an surfactant concentration of 2.5 mg / ml. In some embodiments, the number of SVPs is determined by microfluidic imaging (MFI). In some embodiments, the number of SVPs is determined by microfluidic imaging (MFI) using the method defined in the examples.

[0186] 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 some embodiments, the formulations described herein comprise PS-80 at concentrations ranging from approximately 0.27% to approximately 2.7% by weight, approximately 0.27% to approximately 1.33% by weight, or approximately 0.67% to approximately 1.33% by weight, appropriately by weight. In some embodiments, the formulation comprises PS-80 at concentrations ranging from approximately 0.3% to approximately 3% by weight. In some embodiments, the formulation comprises PS-80 at concentrations ranging from approximately 0.3% to approximately 2.5% by weight. In some embodiments, the formulation comprises PS-80 at a concentration ranging from about 0.5% to about 2.5% by weight. In some embodiments, the formulation comprises PS-80 at a concentration ranging from about 0.5% to about 2% by weight. In some embodiments, the formulation comprises PS-80 at a concentration ranging from about 0.5% to about 1.5% by weight.

[0187] In an exemplary embodiment, the formulation comprises PS-80 at a concentration ranging from about 0.67% to about 1.33%.

[0188] In exemplary embodiments, the formulation comprises PS-80 at concentrations of 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).

[0189] In exemplary embodiments, the composition comprises PS-80 at concentrations 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).

[0190] In some embodiments, the formulation described herein comprises poloxamer-188 at a suitable concentration range of about 1% to about 10% by weight. In exemplary embodiments, the formulation comprises poloxamer-188 (P188) at a concentration range of about 0.67% to about 2.67%. In some embodiments, the formulation comprises P188 at a concentration range of about 0.3% to about 3% by weight. In some embodiments, the formulation comprises P188 at a concentration range of about 0.3% to about 2.5% by weight. In some embodiments, the formulation comprises P188 at a concentration range of about 0.5% to about 2.5% by weight. In some embodiments, the formulation comprises P188 at a concentration range of about 0.5% to about 2% by weight. In some embodiments, the formulation comprises P188 at a concentration range of about 0.5% to about 1.5% by weight.

[0191] In an exemplary embodiment, the formulation comprises P188 at a concentration ranging from about 0.67% to about 1.67%.

[0192] In an exemplary embodiment, the formulation comprises P188 at concentrations 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).

[0193] 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.

[0194] This document describes suitable particle sizes for dry powder formulations, and in embodiments, multiple particles having a median mass aerodynamic diameter (MMAD) of about 2 μm to about 4 μm when provided in aerosol form. Suitable specific surface areas (SSA) of the particles are described herein and include, for example, about 4-7 m². 2 The specific surface area is approximately 1 μm to approximately 5 μm. The particles have an equivalent optical volume average diameter (oVMD) of approximately 1 μm to approximately 5 μm.

[0195] In another embodiment, this document provides a method for preparing a dry powder formulation. In this embodiment, the method suitably includes preparing a liquid raw material comprising leucine, about 0.1 mg / mL to about 6 mg / mL of trileucine, an active agent, and suitably also a glass stabilizer. If desired, the glass stabilizer as described herein may be omitted from the dry powder formulation. The liquid raw material may also contain a surfactant. The liquid raw material is prepared by combining these components in a liquid solvent to produce a raw material in which each component is dissolved. Heating may be added, as desired, to increase the solubility of the various components to form the liquid raw material. Exemplary liquid solvents include water (including deionized water) and diluted solutions of alcohol and water. In this embodiment, after the remaining components of the raw material are added and dissolved, the active agent is suitably added to the liquid raw material.

[0196] In a suitable embodiment of the preparation method, leucine and trileucine are present in the liquid feedstock at a leucine:trileucine concentration ratio of about 0.1:1 to about 30:1. As described herein, when preparing the liquid feedstock, leucine and trileucine are provided in amounts of mg / mL. Therefore, in such embodiments, a leucine:trileucine concentration ratio of about 0.1:1 to about 30:1 in a given volume of the liquid feedstock corresponds to a leucine:trileucine ratio by weight in the liquid feedstock. In another embodiment, leucine and trileucine are present in the liquid feedstock at 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.

[0197] The liquid feedstock can then be atomized. In some embodiments, the liquid feedstock is filtered before atomization. In some embodiments, the liquid feedstock is filtered through a 0.22-micron filter. In some embodiments, the liquid feedstock containing leucine and trileucine is filtered before the addition of the surfactant. In some embodiments, the liquid feedstock is filtered after the addition of the surfactant and before atomization. Atomization refers to the conversion of a liquid feedstock into fine droplets using a pressurized gas (such as CO2 or an inert gas). Exemplary apparatuses for producing atomized liquid feedstocks are known in the art and include the use of various atomizing nozzles having desired sizes and flow characteristics. Exemplary parameters for atomization include an outlet temperature of about 50°C-90°C, suitably about 60°C-80°C, or about 70°C; a feed rate of about 8-15 ml / min, suitably about 9-14 ml / min, about 10-13 ml / min, or about 12 ml / min; an atomizer gas flow rate of about 9-15 kg / h (hr. or h), suitably about 10-14 kg / h, about 12-14 kg / h, or about 13 kg / h; and a dry gas flow rate of about 60-100 kg / h, suitably about 60-90 kg / h, about 70-90 kg / h, or about 80 kg / h.

[0198] The atomized liquid raw material can then be dried, appropriately combined with heating and flowing air to aid the drying process. The drying process produces multiple particles. The drying temperature range is typically approximately 50°C-100°C, or approximately 60°C-100°C, or approximately 70°C-90°C; the airflow velocity can be approximately 10-40 m / s. 3 / Hour.

[0199] This document describes exemplary glass stabilizers, including amorphous sugars and buffers, and suitable amounts of the glass stabilizers. Furthermore, suitable amounts of leucine and trileucine are provided throughout the document. The final dry powder formulation should contain the listed amounts of leucine and trileucine (and other components), which are also used in the liquid feedstock. The result of the atomized drying process is that the liquid solvent is removed, so the total original dry weight of the components corresponds to the final dry weight of the compounds in the dry powder formulation. Exemplary surfactants are also described herein.

[0200] The methods for preparing dry powder formulations described herein appropriately provide microparticles with desired recorded physical characteristics, including desired compressible bulk density, specific surface area, and size. Exemplary sizes, such as exemplary SSAs, are described herein, including smaller than about 10 μm. 2 / g, approximately 4-7m 2The specific surface area is approximately 0.4 g / cm³. Suitablely, the method provides a plurality of particles having an equivalent optical volume average diameter (oVMD) of about 1 μm to about 5 μm, as described herein; a median mass aerodynamic diameter (MMAD) of about 2 μm to about 4 μm when provided in aerosol form; and approximately 0.4 g / cm³. 3 -0.8g / cm 3 The compressed packing density.

[0201] The advantages of the method for preparing the dry powder formulation described herein involve high-volume process properties. For example, if the atomization flow rate is set to 20 ml / min, the following production volume (in grams per hour) is determined.

[0202] Table 2: Effect of Concentration on Production Rate

[0203]

[0204] As described above, using only trileucine (with a maximum trileucine concentration of 5 mg / mL) in the feedstock resulted in a maximum solids loading of 25 mg / mL (related to maximum solubility). This led to a production rate of 30 g / h. Using only 60% leucine (with a maximum leucine concentration of 20 mg / mL), a maximum solids loading of 33 mg / mL and a production rate of 40 g / h were achieved. Further results using only leucine and trileucine are also shown. Conversely, for the three feedstocks tested containing both leucine and trileucine, using only 8% leucine and 2% trileucine resulted in a maximum solids loading of 250 mg / mL and a production rate of 300 g / h. This is a surprising and unexpected finding regarding the advantages of the methods and formulations disclosed herein, as dispersible particles can be provided using relatively small amounts of leucine and trileucine, yet still allow for high production volumes. This high production rate significantly impacts the ability to scale up the production of the dry powder formulations described herein, especially in cases requiring large-volume formulations.

[0205] The methods and formulations described herein allow for the production of capsules, blister packs, and other suitable containers for dry powder formulations. Such containers can be used to produce dry powder formulations of 10-200 mg (appropriately 10-100 mg, or 25-75 mg, or 50 mg). Such containers can appropriately deliver 0.1-10 mg of dry powder formulations to the patient's lungs.

[0206] In some embodiments, the methods described herein provide dry powder formulations that reduce the total volume of capsules required for an inhalation device. For example, the volume required to deliver 50-100 mg of active agent can be reduced from two larger 00 capsules to a single 3 capsule.

[0207] The methods described herein also provide a mechanism for increasing the compressive bulk density and specific surface area of ​​dry powder formulations containing multiple microparticles. As described throughout, approximately 0.4–1.0 g / cm³ can be readily achieved by incorporating leucine and trileucine into the dry powder formulation. 3 (Approximately 0.5-0.8 g / cm³) 3 The compressed packing density is approximately 5-10 m³. 2 / g (approximately 5mg) 2 / g to approximately 7m 2 The specific surface area is ( / g). In another embodiment, particle sizes within the range described herein may be formed, including, when provided in aerosol form, a mass median aerodynamic diameter (MMAD) of about 2 μm to about 4 μm.

[0208] Methods for producing aerosol forms of dry powder formulations are known in the art and include, for example, the use of inhaler devices such as dry powder inhalers (DPIs) (e.g., PLASTIAPE (Osnago, Italy) Monodose RS01 DPI). The dry powder formulations described herein can be dispersed into an airflow via a passive or active inhalation device and remain suspended in the gas for a sufficient amount of time to allow at least a portion of the particles to be inhaled by the patient, thereby enabling a portion of the particles to reach the lungs.

[0209] This article also provides methods for treating medical conditions in mammalian patients, methods comprising administering a dry powder formulation as described herein to the patient by inhalation (including via a dry powder inhaler).

[0210] Medical conditions that can be treated using the methods described herein include those affecting the nervous, endocrine, muscular, cardiovascular, digestive, respiratory (especially the lungs), hormonal, immune, and reproductive systems.

[0211] In this implementation, a method for treating a patient with TSLP-related inflammatory disorders is provided. TSLP-related inflammatory disorders may be caused by allergic reactions or environmental irritants or stimulants. In some implementations, TSLP-related inflammatory disorders may be asthma, chronic obstructive pulmonary disease, allergic rhinitis, allergic sinusitis, allergic conjunctivitis, atopic dermatitis, or eosinophilic esophagitis.

[0212] In some embodiments, the TSLP-related inflammatory condition is asthma, and the treatment method includes administering, via inhalation, a dry powder formulation containing a therapeutically effective amount of an anti-TSLP antibody or antibody fragment variant to the patient. In some embodiments, the patient is an adult. In some embodiments, the patient is a child or adolescent.

[0213] As described herein, dry powder formulations suitably comprise a plurality of microparticles, said microparticles comprising: leucine; about 1% to about 10% by weight of trileucine; and an anti-thymocyte stromal lymphopoietin (anti-TSLP) antibody or antibody variant, wherein leucine and trileucine are present in a leucine:trileucine concentration ratio of about 0.1:1 to about 30:1. The dry powder formulation may contain about 0.3-1.0 g / cm³. 3 The compressed bulk density. The full text describes exemplary components contained in the formulation and their amounts.

[0214] As described in this article, the ability to deliver anti-thymocyte stromal lymphopoietin (anti-TSLP) antibodies or antibody variants via inhalation provides a delivery mechanism more suitable for use in primary care settings.

[0215] In embodiments of methods for treating asthma, the dry powder formulation is administered frequently at a lower dose compared to systemically administered anti-TSLP drugs. In some embodiments, the formulation may be administered daily. Such embodiments are more convenient for subjects or patients. Furthermore, such embodiments reduce the side effects that may occur with systemic administration.

[0216] Appropriately, the antigen-binding fragment of the antibody used in the treatment method contains

[0217] Heavy chain variable structural domain, the heavy chain variable structural domain comprising:

[0218] The heavy chain CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO: 1;

[0219] The heavy chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO: 2;

[0220] The heavy chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO: 3;

[0221] Light chain variable structural domain, wherein the light chain variable structural domain comprises:

[0222] The light chain CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO: 5;

[0223] A light chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO: 6; and

[0224] A light chain CDR3 sequence containing the amino acid sequence shown in SEQ ID NO: 7.

[0225] In another embodiment of the treatment method, 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.

[0226] In some embodiments, the forms of asthma suitable for treatment with the formulations of the present invention include mild asthma, moderate asthma, severe asthma, non-eosinophilic asthma, hypoeosinophilic asthma, and hypereosinophilic asthma. In some embodiments, the formulations of the present invention can be used to treat mild asthma. In some embodiments, the formulations of the present invention can be used to treat moderate asthma. In some embodiments, the formulations of the present invention can be used to treat severe asthma. In some embodiments, the formulations of the present invention can be used to treat non-eosinophilic asthma. In some embodiments, the formulations of the present invention can be used to treat hypoeosinophilic asthma. In some embodiments, the formulations of the present invention can be used to treat hypereosinophilic asthma.

[0227] As used herein, the terms “mild asthma” and “moderate asthma” refer to asthma with a Global Initiative for Asthma (GINA) grade of 3 or lower, or appropriately a GINA grade of 2 or 3. GINA grades are based on the following criteria to measure the severity of asthma (see “Pocket Guide for Asthma Management and Prevention,” Global Initiative for Asthma; 2019).

[0228] As used herein, the term “severe asthma” refers to asthma requiring intensive treatment (e.g., GINA steps 4 and 5) to maintain good control, or asthma that is not well controlled 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-to-severe asthma. Moderate-to-severe asthma suitable for treatment with the formulations described herein may be asthma uncontrolled with moderate to high doses of ICS:LABA, characterized by one or more episodes and frequent symptoms. In some embodiments, severe asthma is further defined as severe asthma with type 2 inflammation, characterized by elevated blood eosinophil count (i.e., blood eosinophil count ≥150 cells / μL) and / or elevated FeNO (i.e., FeNO ≥20 ppb).

[0229] The term "FENO" refers to exhaled nitric oxide, a biomarker of bronchial or airway inflammation. FENO is produced by airway epithelial cells in response to inflammatory cytokines such as TSLP, IL-4, and IL-13. FENO levels in healthy adults range from 2 to 30 parts per billion (ppb). Exemplary assays for measuring FENO include administration of NIOX by the subject. The airway inflammation monitor inhaled the total lung volume, followed by exhalation at 50 ml / s for 10 seconds (assisted by visual and auditory cues).

[0230] As used in this article, the term "hypereoeosinophilic asthma" refers to asthma patients with a blood eosinophil count ≥250 cells / μL.

[0231] In particular, the formulation offers the possibility of treating less severe asthma patients who can typically be controlled in a primary care setting. For example, patients with a Global Initiative for Asthma (GINA) grade 3 or lower, or appropriately a GINA grade 2 or 3. GINA grades are based on criteria that measure asthma severity (see “Pocket Guide for Asthma Management and Prevention,” Global Initiative for Asthma; 2019).

[0232] Daytime asthma symptoms occur more than twice a week;

[0233] Nighttime awakenings due to asthma;

[0234] Using an asthma reliever (reliever) more than twice a week; and

[0235] Activity is limited due to asthma. A score of zero 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 "uncontrolled".

[0236] In some implementations, the formulation provides the possibility of treating patients with moderate-to-severe asthma who can be controlled in a primary care setting or who are difficult to treat with specialized care. For example, the formulation may be used to treat patients with moderate-to-severe asthma at a Global Asthma Initiative (GINA) level of 4-5. Appropriately, the formulation provides the possibility of treating uncontrolled moderate-to-severe asthma. Appropriately, the formulation provides the possibility of treating uncontrolled moderate-to-severe asthma with one or more episodes and frequent symptoms at moderate to high doses of ICS:LABA.

[0237] Other exemplary implementations

[0238] Embodiment 1 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 an anti-thymocyte stromal lymphopoietin (TSLP) antibody, the antigen-binding fragment comprising: a. a heavy chain variable domain, the heavy chain variable domain comprising: 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, and a heavy chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO: 3, wherein any one of heavy chain CDR1, 2, or 3 optionally comprises a single amino acid substitution; and b. a heavy chain variable domain, the heavy chain variable domain comprising: a light chain CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO: 5, a light chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO: 6, and a light chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO: 7, wherein the light chain CDR1 sequence comprises: a light chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 sequence comprising: a light chain CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO: 7, wherein the light chain CDR1 sequence comprises: a light chain CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 sequence comprising: a light chain CDR1 sequence comprising: a light chain CDR2 sequence comprising: ...2 sequence comprising: a light chain CDR3 sequence comprising: a light chain CDR1 sequence comprising: a light Any of 1, 2, or 3 may optionally contain a single amino acid substitution, wherein the leucine and the trileucine are present in a leucine:trileucine concentration ratio of about 0.1:1 to about 30:1.

[0239] Implementation scheme 2 is a dry powder formulation as described in implementation scheme 1, wherein the dry powder formulation has a compressed bulk density of about 0.4-1.0 g / cm3.

[0240] Implementation scheme 3 is a dry powder formulation as described in any of the preceding implementation schemes, wherein the dry powder formulation further comprises a glass stabilizer.

[0241] Implementation scheme 4 is a dry powder formulation as described in implementation scheme 3, wherein the glass stabilizer is an amorphous sugar or a buffer.

[0242] Embodiment 5 is a dry powder formulation as described in Embodiment 3, wherein the glass stabilizer comprises amorphous sugars and buffers.

[0243] Implementation Scheme 6 is a dry powder formulation as described in Implementation Scheme 4 or Implementation Scheme 5, wherein the amorphous sugar is selected from the group consisting of: trehalose, sucrose, raffinose, inulin, dextran, mannitol, and cyclodextrin.

[0244] Implementation Scheme 7 is a dry powder formulation as described in any one of Implementation Schemes 4-6, wherein the buffer is selected from the group consisting of: citrate buffer, phosphate buffer, histidine buffer, glycine buffer, acetate buffer and tartrate buffer.

[0245] Embodiment 8 is a dry powder formulation as described in any one of Embodiments 4-7, wherein the amorphous sugar is present in an amount of about 30% to about 70% by weight.

[0246] Implementation scheme 9 is a dry powder formulation as described in any one of implementation schemes 4-8, wherein the amorphous sugar is trehalose.

[0247] Embodiment 10 is a dry powder formulation as described in Embodiment 9, wherein the trehalose is present at about 30%-65% by weight.

[0248] Embodiment 11 is a dry powder formulation as described in any one of Embodiments 4-10, wherein the buffer is present at about 1% to about 10% by weight.

[0249] Embodiment 12 is a dry powder formulation as described in any one of Embodiments 1-11, wherein the concentration ratio of leucine to trileucine is about 1:1 to about 12:1.

[0250] Embodiment 13 is a dry powder formulation as described in any one of Embodiments 1-12, wherein the concentration ratio of leucine to trileucine is about 1:1 to about 7:1.

[0251] Embodiment 14 is a dry powder formulation as described in any one of Embodiments 1-13, wherein the concentration ratio of leucine to trileucine is about 5.25:1.

[0252] Embodiment 15 is a dry powder formulation as described in any one of Embodiments 1-14, wherein the dry powder formulation contains about 1% to about 7% by weight of trileucine.

[0253] Embodiment 16 is a dry powder formulation as described in any one of Embodiments 1-15, wherein the dry powder formulation comprises about 8% to about 11% leucine and about 2% to about 4% trileucine by weight.

[0254] Embodiment 17 is a dry powder formulation as described in any one of Embodiments 1-16, wherein the dry powder formulation comprises about 10.5% leucine and about 2% trileucine by weight.

[0255] Embodiment 18 is a dry powder formulation as described in any one of Embodiments 1-17, the dry powder formulation further comprising a surfactant, wherein the surfactant is optionally selected from polysorbate-20 (PS-20), polysorbate-40 (PS-40), polysorbate-60 (PS-60), polysorbate-80 (PS-80) and poloxamer-188.

[0256] Embodiment 19 is a dry powder formulation as described in Embodiment 18, wherein the surfactant is PS-80, wherein optionally PS-80 is present at a concentration ranging from about 0.27% to about 2.7% by weight.

[0257] Embodiment 20 is a dry powder formulation as described in Embodiment 18, wherein the surfactant is poloxamer-188, wherein poloxamer-188 is optionally present at a concentration ranging from about 1% to about 10% by weight.

[0258] Embodiment 21 is a dry powder formulation as described in any one of Embodiments 1-20, wherein the plurality of microparticles have an equivalent optical volume average diameter (oVMD) of about 1 μm to about 5 μm.

[0259] Embodiment 22 is a dry powder formulation as described in any one of Embodiments 1-21, wherein the plurality of particles have a median mass aerodynamic diameter (MMAD) of about 2 μm to about 4 μm when provided in aerosol form.

[0260] Embodiment 23 is a dry powder formulation as described in any one of Embodiments 2-22, wherein the compressed bulk density is about 0.5 g / cm3 to about 0.8 g / cm3.

[0261] Embodiment 24 is a dry powder formulation as described in any one of Embodiments 2-23, wherein the dry powder formulation comprises about 39% trehalose, about 10.5% leucine, about 2% trileucine and about 8.5% citrate buffer.

[0262] Embodiment 25 is a dry powder formulation as described in any one of Embodiments 1-24, wherein the plurality of microparticles have a specific surface area of ​​less than about 10 m² / g.

[0263] Embodiment 26 is a dry powder formulation as described in Embodiment 25, wherein the plurality of microparticles have a specific surface area of ​​about 4 mg² / g to about 7 m² / g.

[0264] Embodiment 27 is a dry powder formulation as described in any of the preceding embodiments, wherein the heavy chain variable domain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the heavy chain variable domain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, the heavy chain variable domain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, the light chain variable domain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 5, the light chain variable domain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable domain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 7.

[0265] Embodiment 28 is a dry powder formulation as described in 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.

[0266] Implementation scheme 29 is a dry powder formulation as described in any of the preceding implementation schemes, wherein the antigen-binding fragment is selected from Fab, Fab′, F(ab′)2, scFv, microantibody or biantibody.

[0267] Embodiment 30 is a dry powder formulation as described in Embodiment 29, wherein the antigen-binding fragment is Fab.

[0268] Implementation scheme 31 is a dry powder formulation as described in implementation scheme 30, wherein the Fab is human or anthropomorphized.

[0269] Embodiment 32 is a dry powder formulation as described in any of the preceding embodiments, wherein the anti-TSLP antibody from which the antigen-binding fragment is derived is IgG1.

[0270] Implementation scheme 33 is a method for treating a patient's asthma, the method comprising administering a dry powder formulation according to any one of implementation schemes 1-29 via inhalation.

[0271] Implementation scheme 34 is the method as described in implementation scheme 33, wherein the asthma is mild asthma.

[0272] Implementation scheme 35 is the method as described in implementation scheme 33, wherein the asthma is moderate asthma.

[0273] Implementation scheme 36 is the method as described in implementation scheme 33, wherein the asthma is severe asthma.

[0274] Implementation scheme 37 is the method as described in implementation scheme 33, wherein the asthma is eosinophilic or non-eosinophilic asthma.

[0275] Implementation scheme 38 is the method as described in implementation scheme 33, wherein the asthma is hypoeosinophilic asthma.

[0276] Implementation scheme 39 is the method as described in any one of implementation schemes 33-38, wherein the asthma is characterized by fewer than three of the following: daytime asthma symptoms more than twice a week; nighttime awakenings due to asthma; use of asthma relievers more than twice a week; and limited activity due to asthma.

[0277] Embodiment 40 is a dry powder formulation as described in any one of Embodiments 1-32, wherein the dry powder formulation is used in a treatment method, wherein the formulation is administered by inhalation.

[0278] Implementation scheme 41 is a dry powder preparation used according to implementation scheme 37, wherein the dry powder preparation is used in a method for treating asthma.

[0279] Implementation scheme 42 is a dry powder formulation used according to implementation scheme 38, wherein the asthma is mild asthma.

[0280] Implementation scheme 43 is a dry powder formulation used according to implementation scheme 38, wherein the asthma is moderate asthma.

[0281] Implementation scheme 44 is a dry powder formulation used according to implementation scheme 38, wherein the asthma is severe asthma.

[0282] Implementation scheme 45 is a dry powder formulation used according to implementation scheme 38, wherein the asthma is eosinophilic asthma or non-eosinophilic asthma.

[0283] Implementation scheme 46 is a dry powder formulation used according to implementation scheme 38, wherein the asthma is hypoeosinophilic asthma.

[0284] Implementation scheme 47 is a dry powder formulation used according to implementation scheme 38, wherein the asthma is characterized by fewer than three of the following: daytime asthma symptoms more than twice a week; nighttime awakenings due to asthma; use of asthma relievers more than twice a week; and limited activity due to asthma.

[0285] Example

[0286] Example 1 - Generation of Anti-TSLP FAB

[0287] Using standard molecular biology and cloning techniques, a series of antibody-binding fragments (Fabs) derived from the anti-TSLP monoclonal antibody “A5” disclosed in WO2009 / 035577 (which is incorporated herein by reference in its entirety) were generated. In short, the CDR sequence of A5 was cloned into an IgG1 Fab scaffold, resulting in the Fab fragment referred to herein as Fab1 or Fab1. The VH and VL sequences of Fab1 are disclosed as SEQ ID NO: 4 and 8, respectively.

[0288] Variant Fab was also derived from Fab1, which contains mutations in the CDR region. 2-9 Table 3 shows the combinations of VH and VL CDR for each of Fab 1-9.

[0289] Table 3 Anti-TSLP Fab 1-9 CDR sequence

[0290] VH CDR 1, 2 and 3 VL CDR 1, 2 and 3 <![CDATA[Fab1]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 5, 6 and 7 <![CDATA[Fab2]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 11, 6 and 7 <![CDATA[Fab3]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 14, 6 and 7 <![CDATA[Fab4]]> SEQ ID NO: 1, 15 and 3 SEQ ID NO: 5, 6 and 7 <![CDATA[Fab5]]> SEQ ID NO: 1, 17 and 3 SEQ ID NO: 5, 6 and 7 <![CDATA[Fab6]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 19, 6 and 7 <![CDATA[Fab7]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 19, 6 and 7 <![CDATA[Fab8]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 5, 6 and 23 <![CDATA[Fab9]]> SEQ ID NO: 1, 2 and 3 SEQ ID NO: 5, 6 and 25

[0291] The purity, stability, and aggregation tendency of Fab1 were analyzed. Briefly, 50 mg / mL Fab1 was prepared in 30 mM sodium citrate and 105 mM trehalose (pH 6.0). Samples were placed in stability chambers at 40°C and 5°C for different time periods. At different time points, samples were tested using relevant analytical techniques, such as high-performance size exclusion chromatography (HP-SEC). An Agilent HPLC system from Agilent Technologies (Santa Clara, CA, USA) with a temperature-controlled autosampler, DAD or VWD, and Agilent ChemStation software / OpenLAB ECM CDS was used. Guard columns from Tosoh Bioscience (Griesheim, Germany): a TSKgel column (7.9 mm ID, catalog number 08543) and a TSK-Gel G3000SWxl column (5 μm, (7.8 x 300 mm, catalog number 08541). The mobile phase used was 0.1 M anhydrous disodium hydrogen phosphate and 0.1 M sodium sulfate (pH 6.8). The results of stability and aggregation analyses are shown in Table 4.

[0292] Table 4. Stability and Aggregation of Fab1

[0293]

[0294]

[0295] The stability of Fab1 was also tested by differential scanning calorimetry (DSC). Tests were performed using a MicroCal Capillary VP DSC from Malvern Panalytical (Malvern, UK), and data analysis was performed using Origin 7.0 software (Northampton, MA, USA). Fab1 samples were diluted to 5 mg / mL with formulation buffer (30 mM sodium citrate, 105 mM trehalose, pH 6.0). For each independent run, 500 μL of the diluted Fab1 sample and reference (formulation buffer) were injected into the DSC sample and reference cells via an autosampler. The solution was heated from 25 °C to 100 °C at a scan rate of 95 °C / h. Buffer scans (filled in sample and reference cells) were also obtained as blanks for baseline correction of the samples.

[0296] The charge distribution of Fab1 was also determined using an iCE3 analyzer via imaging isoelectric focusing (IEF). The iCE3 capillary IEF analyzer, PrinCE MicroInjector autosampler, and MicroInjection coated transfer capillary column were purchased and supplied by Protein Simple. Samples were analyzed using an FC column (part number 101701, Protein Simple) with a fluorocarbon-coated capillary column and a built-in electrolyte reservoir. The autosampler was maintained at 4°C throughout the analysis. The pI range of Fab1 was determined to be 8.35 to 8.80.

[0297] Example 2 - FAB1 binds to human and cynomolgus monkey TSLP with PM affinity.

[0298] The affinity of Fab1 for binding to TSLP was determined using BIAcore.

[0299] The specificity and affinity of Fab1 for human and cynomolgus monkey TSLP expressing recombinant mammalian cells were determined using a Biacore 8K SPR instrument (GE Healthcare, Little Chalfont, Bucks, UK).

[0300] The S-Series C1 biosensor chip, amine conjugation kit, hepes-based buffer, and regeneration buffer were all obtained from GE Healthcare and used according to the manufacturer's instructions. Streptavidin surfaces were prepared using lyophilized streptavidin (reconstituted with D-PBS). Briefly, streptavidin was diluted to 4 μg mL⁻¹ in 10 mM sodium acetate (pH 4.5) and covalently immobilized to the surfaces of the three flow cells of the S-Series C1 biosensor chip using a standard amine conjugation method. A final streptavidin surface with 170 response units (RU) was achieved. The amine conjugation kit was also used to prepare control blank surfaces (without immobilized streptavidin) to serve as reference surfaces within each flow cell. N-terminal labeled biotinylated TSLP (human and cynomolgus monkey) was then titrated onto each streptavidin surface to achieve Fab1 binding at saturation (Rmax) of <100 RU. Low analyte binding levels ensured minimal mass transfer-induced artifacts, especially when combined with a relatively fast assay flow rate of 50 μL min⁻¹ used during the kinetic measurement steps. Monomerized Fab₁ diluents (multi-cycle kinetics) (2-fold dilution in HBS-EP+ buffer, ranging from 1.25 to 20 nM) were injected at an assay flow rate of 50 μL min⁻¹, with association for 2 min and dissociation for 10 min. Multiple buffer-only injections were performed under identical conditions throughout the experiment to allow for dual-reference processing of the final sensor atlas.

[0301] The chip surface was completely regenerated by two 30-second pulses of flowing 10 mM glycine (pH 1.7). Binding affinity and kinetics were determined using a 1:1 Langmuir model.

[0302] The results shown in Table 5 demonstrate that Fab1 binds to fixed human and cynomolgus monkey TSLP with similar affinity (within 2 times; 46 pM and 88 pM, respectively).

[0303] Table 5 shows the affinity of Fab1 for human and cynomolgus monkey TSLP determined using BIAcore.

[0304] Analytes <![CDATA[k a (M -1 s -1 )]]> <![CDATA[k d (s -1 )]]> <![CDATA[K D (pM)]]> Human TSLP 2.39E6 1.11E-4 46.3 Crab-eating macaques TSLP 1.75E6 1.55E-4 88.4

[0305] Binding affinity was determined by kinetic exclusion assay (KinExA).

[0306] The KinExA 3200 instrument (Sapidyne Instruments, Boise, Idaho, USA) was also used to determine the solution binding affinity (KbA1) of Fab1 to human and cynomolgus monkey TSLP. D The obtained data was processed using KinExA Pro software version 4.1.11. The KinExA method has been reviewed (Darling and Brault, 2004).

[0307] Fab1 was premixed with different concentrations of each human and cynomolgus monkey TSLP until equilibrium was reached (at least 12 concentrations of each human and cynomolgus monkey TSLP were prepared using a 2-fold serial dilution method). The amount of free Fab1 was then measured using a KinExA instrument by capturing free Fab1 with human TSLP-coated beads, washing away unbound material, and detecting bound Fab1 fluorescence with a commercial species-specific antibody (Alexa Fluor 647-labeled mouse anti-human heavy and light chain specific antibody (Jackson Immunoresearch 209-605-088)). The Ki ratio of Fab1 to human TSLP was determined by an overall 1:1 fit. D Three datasets were extracted, which were derived from TSLP titration of Fab1 solutions at fixed concentrations of 1000 pM (solid rhombus), 500 pM (inverted solid triangle), or 40 pM (hollow square). Figure 1 Ki of Fab1 to cynomolgus monkey TSLP was obtained by overall 1:1 fitting. D Two datasets were extracted, derived from cynomolgus monkey TSLP titrations to 1000 pM (solid rhombus) or 40 pM (hollow square) fixed Fab1 concentration solutions. Figure 2 ).

[0308] The amount of free Fab1 detected at each human and cynomolgus monkey TSLP concentration was plotted against the titration concentration of TSLP (respectively...). Figure 1 and 2 The KinExA software was used to calculate the equilibrium dissociation constant (KD). The results shown in Table 6 demonstrate that the affinity of Fab1 for human TSLP in free solution is 1.7 times that for cynomolgus monkey TSLP.

[0309] Table 6 shows the soluble phase affinity of Fab1 for human and cynomolgus monkey TSLP, determined using KinExA.

[0310] ligands <![CDATA[Affinity (K D ) pM]]> Human TSLP 8.0 (95% confidence interval: 6.27–10.01 pM) Crab-eating macaques TSLP 13.6 (95% confidence interval 9.07-19.22 pM)

[0311] Example 3 - FAB1 and terzoliumab bind to TSLP with similar binding characteristics

[0312] The binding signature of Fab1 to human TSLP was directly compared with that of terzolumab. Terzolumab is a human immunoglobulin G2 (IgG2) monoclonal antibody (mAb) that binds to TSLP, thereby preventing its interaction with the TSLP receptor complex. A proof-of-concept study in patients with mild atopic asthma demonstrated that terzolumab suppressed early and late asthma responses after inhaled allergen provocation and inhibited biomarkers of Th2 inflammation. Terzolumab is currently being investigated clinically as a specialty treatment for severe asthma.

[0313] Using homogeneous time-resolved fluorescence based on homogeneous fluorescence resonance energy transfer (FRET) Cisbio International's TSLP:mAb binding assay was used to determine the in vitro binding potency of Fab1. Streptavidin cavitary compounds were used to detect biotinylated TSLP. Briefly, unlabeled Fab1 samples were titrated into the HTRF assay to compete with DyLight-labeled terzerulumab for binding to biotinylated His-Avi human TSLP. Competitive assays were also performed using unlabeled terzerulumab and DyLight-labeled terzerulumab (as positive controls).

[0314] The results showed that Fab1 competed with terzegluzumab for binding to human TSLP and bound to human TSLP with similar potency as terzegluzumab (IC50: Fab1 -0.38 nM; terzegluzumab -0.23 nM). Figure 3 Also using Fab. 2-9 HTRF assays were performed, which showed that each of these Fabs also competed with terzeglund for binding to human TSLP and bound to human TSLP with similar potency to terzeglund (Table 7).

[0315] Table 7 shows the Fab values ​​determined by HTRF measurements. 2-9 IC 50

[0316] <![CDATA[IC 50 nM]]> <![CDATA[Fab2]]> 0.29 <![CDATA[Fab3]]> 0.24 <![CDATA[Fab4]]> 0.36 <![CDATA[Fab5]]> 0.42 <![CDATA[Fab6]]> 0.32 <![CDATA[Fab7]]> 0.24 <![CDATA[Fab8]]> 0.29 <![CDATA[Fab9]]> 0.29

[0317] Example 4 - Fab1 Neutralizing TSLP Activity in Peripheral Blood Mononuclear Cell (PBMC) Assay

[0318] Next, we determined whether the binding of Fab1 to TSLP had a functional blocking activity in primary cell assays by measuring TSLP-induced CCL17 release from PBMCs after Fab1 treatment.

[0319] Blood was obtained from healthy donors according to the donor procedure established by MedImmune, Cambridge, UK. Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll gradient according to a standard procedure. Briefly, 20 ml of blood diluted with PBS (10 ml blood: 30 ml PBS) was plated onto 15 ml of Ficoll. The tubes were rotated at 400 g for 40 min at room temperature without braking. The PBMC layer was collected and the cells were washed twice with 50 ml PBS. The PBMCs were counted using a cell counter and trypan blue to remove dead cells, and then resuspended in medium (RPMI containing 10% fetal bovine serum and 1% penicillin / streptomycin) and plated into 96-well plates. Cells were stimulated with TSLP (0.5 ng / ml) for 48 h in the presence of the TSLP-binding antibody fragment Fab1. Terzaluzumab, an antibody binding to TSLP, was also used for assays (as a positive control). After 48 h, the supernatant was removed according to the manufacturer's protocol, and CCL17 production was measured using an R&D duoset ELISA. Six donors were used in three separate experiments.

[0320] The results show that Fab1 inhibits the generation of CCL17 from PBMCs, where IC 50 1.39 nM ( Figure 4 In addition to using Fab1, Fab2, and Fab3 (containing the variable heavy chain and variable light chain sequences outlined in Table 3), the determinations were repeated, and similar results were obtained. Figure 5 ).

[0321] Example 5 - Determination of maximum tolerated dose and pharmacokinetics after inhalation of FAB1 in cynomolgus monkeys

[0322] The aim of this study was to determine the maximum tolerated dose (MTD) or maximum feasible dose (MFD) and pharmacokinetics (PK) of Fab1 nebulized after inhalation exposure via a mask in cynomolgus monkeys.

[0323] Female cynomolgus macaques received single 8-minute and 20-minute Fab1 inhalations (Groups 1 and 2, three animals per group). Based on 25% lung deposition, the doses delivered to the lungs in Groups 1 and 2 were 1 and 2 mg / kg, respectively. Group 3 was a repeated dose-escalation regimen. One female and one male cynomolgus macaque were treated as follows: 8-minute inhalation daily for the first two days, followed by 20-minute inhalation daily for two days, and then 60-minute inhalation daily for three days. Serial blood samples were collected to obtain Fab1 serum PK and urea concentrations. Bronchoalveolar lavage (BAL) samples were collected to obtain Fab1 PK and urea concentrations. Epithelial fluid (ELF) was calculated from BAL samples using urea concentration as a dilution marker. Mixed immunoaffinity LC-MS / MS was used to determine Fab1 concentrations in the serum and BAL sample matrices. The lower limits of quantification were 4 ng / mL in serum and 10 ng / mL in BAL. Non-compartmental (NCA) analysis of individual plasma PK data was performed using Phoenix WinNonlin (version 7.0, Certara, LP, St. Louis, MO).

[0324] Following inhalation of Fab1, serum PK, BAL, and ELF concentrations increased with dose, and Fab1 concentration exhibited high variability. Figures 6A to 6C The mean serum terminal half-life of Fab1 ranged from 9.75 to 13.6 hours. The median T1 time was 2 to 4 hours after inhalation. max When serum C reaches max The concentration of ELF after inhalation was much higher than that in serum (>2000 times higher), indicating that the distribution of Fab1 in serum was low after the inhaled dose.

[0325] Example 6: Evaluation of the physical characteristics of a spray-dried formulation containing leucine and trileucine.

[0326] The following method was used to evaluate the effect of the trileucine to leucine concentration ratio on particle properties.

[0327] At a total raw material solids concentration of 10%, using the same process parameters, a total of 24 powders with different wt% concentrations of trileucine, leucine, and trehalose (TLT) were spray-dried on a pilot-scale spray dryer. Since the raw materials were prepared at a total solids concentration of 10% (100 mg / mL), all wt% values ​​in this study are also consistent with the concentration values ​​(mg / mL). The concentration ranges for each particulate excipient are shown in Table 8.

[0328] Table 8: Particulate Component Composition Range

[0329] Components Minimum value Maximum value Trileucine 0.71 mg / mL 5.72 mg / mL Leucine 0.62 mg / mL 19.94 mg / mL Trehalose 65.84 mg / mL 90.16 mg / mL Trisodium citrate 8.5 mg / mL 8.5 mg / mL

[0330] Each feedstock was prepared by dissolving the excipients in water (Table 9). Once all excipients were completely dissolved, the feedstocks were spray-dried using the following process parameters: outlet temperature, 70°C; feedstock rate, 12 ml / min; atomizer gas flow, 13 kg / h; and drying gas flow, 80 kg / h. Parameters were selected to obtain the target particle and aerosol characteristics for the dry powder formulations used for inhalation. Each of the 24 formulations was manufactured in batches of 18 g to provide sufficient powder for characterization and product performance evaluation. Batch randomization was performed, and production was completed within two days.

[0331] Table 9: Raw material concentrations for formulations 1-24

[0332]

[0333] The following physical powder characteristics of all formulations were tested.

[0334] Table 10: Particle parameters analyzed

[0335]

[0336]

[0337] 1 The compressive force is 300,000 N / m 2 Alternatively, if a 12.7mm sample chamber is used, the value is 38N.

[0338] use A Model 1360 density analyzer (Micromeritics, Norcross, GA) was used to measure the compressive bulk density (CBD) of the powder. Powder samples were prepared in a low-humidity environment (<5% RH) and then transferred to the nitrogen-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 via a plunger at a rate of 300 consolidation steps per second. The linear distance traveled by the plunger in each consolidation step was converted into the volumetric displacement of the powder sample. The average of the measurements from each consolidation step was then converted into a calculated bulk density value in g / cm³. 3 express.

[0339] The results showed that the contents of leucine and trileucine had a significant impact on particle properties. Trileucine was identified as the major factor with the greatest influence, while leucine was identified as a minor factor with a significant influence. The results are summarized in Table 11.

[0340] Table 11: Results of particle characterization

[0341]

[0342]

[0343] 1 The compressive force is 300,000 N / m 2 Alternatively, if a 12.7mm sample chamber is used, the value is 38N.

[0344] Example 7 - Aerosol performance characteristics of leucine / trileucine formulation

[0345] The following examples evaluated the aerosol performance of formulations containing leucine and trileucine in a dry powder inhaler device. The aerosol performance outputs listed in Table 7 were tested for 20 of the 24 formulations listed in Table 4. All product performance characterizations were performed using a Monodose RS01 device with capsule #3. Next-Generation Impactor (NGI) analysis was conducted at a flow rate of 60 L / min.

[0346] According to USP <601> Cascade impaction tests were conducted to measure the aerosol properties of the spray-dried formulation during delivery from a dry powder inhaler device. The cascade impactor equipment used was a new generation impactor (NGI; USP41, No. 1). <601> (Chapter). For the aerosol measurements performed in these embodiments, a No. 3 HPMC capsule containing a spray-dried powder formulation was dispersed from a dry powder inhaler device and delivered to the NGI under vacuum at 60 L / min according to the USP methodology. Samples from each stage of the NGI were recovered, and protein content was determined by UV absorption 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 aerodynamic particle size measurement of the powder fraction ejected from the device being < 5 μm; and b) mass median aerodynamic diameter (MMAD).

[0347] Table 12: Aerosol Characterization

[0348]

[0349] The results of the aerosol analysis are summarized in Table 13.

[0350] Table 13: Results of aerosol characterization

[0351]

[0352] Example 8 - Production of an inhalable leucine / trileucine formulation containing an anti-TSLP antibody-binding fragment (Fab)

[0353] Another formulation comprising different Fabs was tested. An anti-TSLP Fab, derived from a human IgG1 monoclonal antibody that specifically binds to TSLP (thymic stromal lymphopoietin) (see sequences shown in SEQ ID NO: 1-8 provided herein), was used. Different formulations containing the mass concentrations outlined in Table 14 were produced.

[0354] Table 14: Composition of spray-dried formulations containing anti-TSLP Fab

[0355]

[0356] First, anti-TSLPFab was received in a liquid buffer containing 105 mM trehalose and 30 mM citrate (pH 6.0). Leucine, trileucine, trehalose, and citrate were dissolved in separate aqueous solutions and then added to the anti-TSLPFab solution to produce a large volume of liquid feedstock solution for spray drying. Table 15 summarizes the feedstock compositions prepared to obtain the target powder formulation composition. The liquid feedstock solution was then spray-dried using the process parameters listed in Table 16. Parameters were selected to obtain the target particle and aerosol properties for inhalation of the dry powder formulation.

[0357] Table 15 Composition of liquid feedstocks used for spray drying

[0358]

[0359] Table 16: Key Spray Drying Process Parameters

[0360] Formulation #1 Formulation #2 Formulation #3 Outlet temperature (°C) 70 70 70 Raw material feed rate (mL / min) 20 17 3 Atomizer airflow (kg / h) 13 13 2.1 Dry airflow (kg / h) 155 155 59.5

[0361] Table 17 summarizes the results of powder and aerosol performance characterization of the spray-dried formulations. For aerosol performance measurements, all three formulations were tested using 20 mg of spray-dried powder filled in HPMC No. 3 capsules and dispersed from a dry powder inhaler device.

[0362] Table 17 Powder and aerosol properties of spray-dried formulations containing anti-TSLP Fab

[0363]

[0364]

[0365] Of particular note is the successful filling of 50 mg of formulation #3 into a single HPMC capsule #3, thanks to the high packing density of the powder. This high packing density (cBD) enables the delivery of a very high effective load from a single capsule (approximately 14 mg with FPM < 5 μm, FPF 82%, and MMAD 2.4 μm).

[0366] In addition, formulation #3 exhibited similar cBD (0.58 g / cm3) and SSA (4.6 m2 / g) to anti-IL-4 Fab formulation #2 (cBD = 0.59 g / cm3, SSA = 4.5 m2 / g), indicating that powder characteristics can be converted between drug formulations containing different active ingredients.

[0367] Example 9 - Powder and aerosol properties of spray-dried anti-TSLP formulations in three batch sizes

[0368] This embodiment utilizes a larger batch size to provide analysis of the powder and aerosol properties of the TSLP Fab leucine / trileucine formulation, enabling both non-GLP and GLP inhalation toxicology studies. Scaling up would require the use of alternative-scale spray dryer equipment and adjustments to spray drying process parameters to address the increased heat and mass flowing through the system, as well as the need for extended processing runs.

[0369] Three batches of spray-dried anti-TSLP Fab formulation were manufactured at increased batch sizes. These batches contained: anti-TSLP Fab 40% w / w, trehalose 39% w / w, leucine 10.5% w / w, trileucine 2% w / w, and citrate (pH 6.0) 8.5% w / w. The selected process parameters for each batch are shown in Table 18.

[0370] Table 18. Spray dryer process parameters for three batches of anti-TSLP Fab formulations with increased batch size.

[0371]

[0372] * Weight of the powder processed.

[0373] Aerosol performance testing was conducted on batch #1 using a powder filling mass of 50 mg from HPMC capsule #3, while batches #2 and #3 were tested using a filling mass of 20 mg. Although oVMD increased slightly with increasing batch size from 8.5 g to 1.2 kg, it was achieved within the range of 0.45 to 0.85 g / cm³. 3 The compressed powder bulk density (cBD) was maintained. The aerosol properties of the powder were also maintained regardless of batch size, with high-capsule delivery of anti-TSLP Fab from capsule-based inhaler devices. This demonstrates the scalability of the formulation, requiring minimal adjustments to the spray dryer process. Table 19 summarizes the full results of powder characterization and aerosol performance testing.

[0374] Table 19. Powder properties and aerosol performance of three anti-TSLP Fab batches with increased batch size.

[0375]

[0376] Example 10: Further characterization of the leucine / trileucine formulation containing a surfactant.

[0377] Further batches of trileucine / leucine formulations containing varying amounts of PS-80 were produced. Table 20 shows the formulation composition and process parameters used to produce each batch. Otherwise, the formulation was produced as described in Example 6.

[0378]

[0379] The aerosol properties of the formulations in Table 20 were analyzed using the methods disclosed in Example 7. The results of the analysis are shown in Table 21.

[0380] Table 21: Aerosol properties of formulations containing PS-80

[0381]

[0382] The aggregate content, oVMD, residual moisture content, Tg, cBD, and SSA were also measured using the methods described in the foregoing embodiments. The results of the powder property analysis are shown in Table 22.

[0383] Table 22: Powder properties of dry powder formulations containing FAB1 and different (w / w) amounts of PS-80

[0384]

[0385] *nm - Not measured

[0386] Analysis showed that, regardless of the % (w / w) amount of PS-80, the powder characteristics were largely equivalent to the control formulation.

[0387] Next, the subvisible particle (SVP) content of the formulations described in Table 22 was analyzed. The SVP count was measured using microfluidic imaging (MFI). MFI combines microfluidic microscopy and high-resolution imaging particle analysis to quantify the SVP count and classify these counts across the entire particle size range. Prior to testing, the powder sample was dissolved in water and gently vortexed to ensure uniform particle distribution, then loaded onto a Protein Simple MFI 5200 (CA, USA). Results were reported as counts per ml for different particle sizes (≤1 μm, ≤2 μm, ≤5 μm, ≤10 μm, and ≤25 μm). Figure 14A shows that the inclusion of 0.27% (w / w) PS-80 in the dry powder formulation reduced the absolute number of SVPs per ml after reconstitution. The reduction in SVP count decreased with increasing PS-80 concentration. A significant reduction in SVP was observed after the addition of 0.67% (w / w) PS-80, where the amount of SVP with a particle size greater than 5 μm was negligible. This trend was observed when the formulation was reconstituted to a concentration of 30 mg / ml FAB1 or 2.5 mg / ml FAB1. Figure 14 B).

[0388] As described above, the second formulation containing the excipient underwent formulation characterization and analysis using SVP. In this study, poloxamer 188 was used as the excipient, compared to PS-80.

[0389] Multiple % w / w values ​​of poloxamer 188 were examined. The formulation composition and process parameters used to generate each formulation batch are as described in Table 20 for formulations containing PS-80. The amount of trehalose was modified to compensate for the variable in poloxamer 188.

[0390] The aggregate content, oVMD, residual moisture content, Tg, cBD, and SSA were also measured using the methods described in the foregoing embodiments. The results of the powder property analysis are shown in Table 23.

[0391] Table 23: Powder properties of dry powder formulations containing FAB1 and different (w / w) amounts of poloxamer-188.

[0392]

[0393]

[0394] *nm - Not measured

[0395] The aerosol properties of the poloxamer-188 formulation were also analyzed using the method disclosed in Example 7. The results are shown in Table 24.

[0396] Table 24: Aerosol properties of formulations containing poloxamer-188 (P188)

[0397]

[0398] The method described above was used to analyze the SVP content of the P188 formulation. Figure 15A The study showed that the inclusion of 0.67% (w / w) P188 in the dry powder formulation reduced the absolute amount of SVP per ml after reconstitution. When the formulation was reconstituted to a concentration of 30 mg / ml FAB1 (…),… Figure 15A ) or 2.5 mg / ml FAB1 ( Figure 15B This trend was observed when ( ).

[0399] Example 11 Characterization of a leucine / trileucine formulation containing 1.1% (w / w) PS-80.

[0400] In this embodiment, the powder properties of a dry powder formulation comprising 1% or 40% (w / w) Fab1 and 1.1% (w / w / ) PS-80 were analyzed. The complete formulation composition is shown in Table 25. The formulation was manufactured as described in Example 6.

[0401] Table 25: Amount of excipients by weight in dry powder formulations containing 1.1% (w / w) PS-80 and 40% (w / w) or 1% (w / w) Fab1

[0402] <![CDATA[Fab1 40%(w / w)]]> <![CDATA[Fab1 1%(w / w)]]> Trileucine 2 2 Leucine 10.5 10.5 Trehalose 37.9 76.9 citrate buffer 8.5 8.5

[0403] The stability of the formulation was analyzed after storage at 40°C and 75% relative humidity (40 / 75) or 25°C and 60% relative humidity (25 / 60) for one or three months. Particle size distribution, moisture content, and surface wrinkling were tested. Figure 16A and Figure 16B The moisture content and particle size distribution of the formulation containing 40% (w / w) Fab1 remained stable over time. Figure 16C shows that the particle morphology remained consistent over time. Figure 17A and Figure 17B The moisture content and particle size distribution of the formulation containing 1% (w / w) Fab1 remained stable over time. Figure 17C The particle morphology remains consistent over time.

[0404] The formation of SVPs after reconstruction and storage at 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 The results show that the amount of SVP formed remains unchanged under each condition after 40% (w / w) of the Fab1 formulation is reconstituted to a Fab1 concentration of 30 mg / ml. Figure 18B The results show that the amount of SVP formed remains unchanged under each condition after recombining 1% (w / w) Fab1 formulation to a Fab1 concentration of 0.75 mg / ml.

[0405] Aerosol characteristics were also tested after storage. The results are shown in Tables 26 and 27.

[0406] Table 26: Aerosol properties of formulations containing 40% (w / w) Fab1 and 1.1% (w / w) PS-80 immediately following manufacturing and after storage at 40 / 75 for 1 or 3 months or at 25 / 60 for 3 months.

[0407]

[0408] Table 27: Aerosol properties of formulations containing 1% (w / w) Fab1 and 1.1% (w / w) PS-80 immediately following manufacturing and after storage at 40 / 75 for 1 or 3 months or at 25 / 60 for 3 months.

[0409]

[0410] After storing each formulation under each condition, the percentage of delivered dose (DD) was also characterized. The results are shown in Tables 26 and 27.

[0411] The potency of Fab1 in each formulation described in Table 25 was also tested after storage at 40 / 75 for 1 or 3 months or at 25 / 60 for 3 months.

[0412] Power was determined using homogeneous time-resolved fluorescence (HTRF). HTRF combines fluorescence resonance energy transfer (FRET) with time-resolved measurement (TR). When two fluorophores (donor and acceptor) are brought close together, excitation of the donor induces energy transfer to the acceptor, resulting in a FRET signal. In this assay, the streptavidin-europene cavitary compound bound to biotinylated human TSLP is the donor, while the d2-labeled anti-TSLP mAb is the acceptor. FAB1 binds to human TSLP and prevents the labeled mAb from binding. This, in turn, increases the distance between the donor and acceptor fluorophores, leading to a decrease in the FRET signal.

[0413] After assessing the parallelism between the reference standard and the assay control or between the reference standard and the test sample, a constrained four-parameter logarithmic (4PL) curve fitting was performed, and the relative potency of the FAB1 assay control and the test sample was calculated 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%.

[0414] The potency level of Fab1 is between 85% and 110% of the potency of Fab1 immediately after remodeling of the equivalent formulation (i.e., t=0).

[0415] References:

[0416] Darling RJ, Brault PA. Assay and Drug Development Technologies. 2004; 2: 647-657

[0417] Gauvreau GM, O'Byrne PM, Boulet LP, et al. N Engl J Med 2014;370:2102-10

[0418] Tepper, JS, et al Int J Toxicol 2016;35:376-92

[0419] Rennard, SI, et al J Appl Physiol 1986;60:532-538

[0420] Those skilled in the art will readily understand that other suitable modifications and adjustments can be made to the methods and applications described herein without departing from the scope of any implementation. The following examples are included herein for illustrative purposes only and are not intended to be limiting.

[0421] It should be understood that although certain embodiments have been described and illustrated herein, the claims are not limited to the specific form or arrangement of the described and shown portions. Illustrative embodiments have been disclosed in this specification, and although specific terminology has been used, it is used only in a general and descriptive sense and not for limiting purposes. In view of the foregoing teachings, modifications and changes may be made to these embodiments. Therefore, it should be understood that these embodiments can be practiced in ways other than those specifically described.

[0422] Although various embodiments have been described above, it should be understood that they are presented only as illustrations and examples of the invention and not as limitations. Those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of the invention. Therefore, the breadth and scope of the invention should not be limited by any of the embodiments described above, but should be defined solely by the appended claims and their equivalents. It should also be understood that each feature of each embodiment discussed herein and each feature of each reference cited herein may be used in conjunction with features of any other embodiment. All patents and publications discussed herein are incorporated herein by reference in their entirety. sequence list <110> Immunomedical Co., Ltd. <120> Dry powder formulation of antibodies conjugated to thymic stromal lymphopoietin (TSLP) and its method of use <130> LC22310013P <160> 31 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> HCDR1 FAB1 <400> 1 Thr Tyr Gly Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> HCDR2 FAB1 <400> 2 Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 13 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> HCDR3 FAB1 <400> 3 Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile 1 5 10 <210> 4 <211> 122 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Heavy chain VH FAB1 <400> 4 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Asn 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 5 <211> 11 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> LCDR1 FAB1 <400> 5 Gly Gly Asn Asn Leu Gly Ser Lys Ser Val His 1 5 10 <210> 6 <211> 7 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> LCDR2 FAB1 <400> 6 Asp Asp Ser Asp Arg Pro Ser 1 5 <210> 7 <211> 11 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> LCDR3 FAB1 <400> 7 Gln Val Trp Asp Ser Ser Ser Asp His Val Val 1 5 10 <210> 8 <211> 108 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Light chain VL <220> <221> MISC_FEATURE <223> Light chain VL FAB1 <400> 8 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 9 <211> 366 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Variable heavy chain FAB1 <400> 9 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 <210> 10 <211> 324 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> FAB1 variable light chain <400> 10 tcatatgttc ttacacaacc accgtcggtt tcggttgctc caggacaaac agctcgaatt 60 acatgcggag gaaacaacct cggatcgaag tcggttcact ggtatcaaca aaagccagga 120 caagctccag ttctcgtggt gtacgatgat tcagatcgac catcatggat cccagagcga 180 ttctcaggat caaactcggg aaatactgcc acgctcacaa tttcacgcgg agaagcggga 240 gatgaagctg attactattg ccaagtgtgg gactcgtcgt cagatcatgt tgttttcgga 300 ggtggaacaa agctcacagt gctc 324 <210> 11 <211> 11 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> LCDR1 FAB2 <400> 11 Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His 1 5 10 <210> 12 <211> 108 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Light chain FAB2 <220> <221> MISC_FEATURE <223> Light chain VL FAB2 <400> 12 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 13 <211> 11 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> LCDR1 FAB3 <400> 13 Gly Gly Asn Asn Val Gly Ser Lys Ser Val His 1 5 10 <210> 14 <211> 108 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> light chain FAB3 <220> <221> MISC_FEATURE <223> light chain VL FAB3 <400> 14 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Val Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 15 <211> 17 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> HCDR2 FAB4 <400> 15 Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Glu Ser Val Lys 1 5 10 15 Gly [[ID=三十五]]<210> 16 <211> 122 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Heavy chain FAB4 <220> <221> MISC_FEATURE <223> Heavy chain VH FAB4 <400> 16 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 It should be noted that there is an error in the original text where "三十五" is used instead of the correct number "35" in the translation of line 35. The corrected translation is provided above.Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Asn 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 17 <211> 17 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> HCDR2 FAB5 <400> 17 Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val Lys 1 5 10 15 Ala <210> 18 <211> 122 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Heavy chain FAB5 <220> <221> MISC_FEATURE <223> Heavy chain VH FAB5 <400> 18 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val 50 55 60 Lys Ala Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Asn 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 19 <211> 11 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> LCDR1 FAB6 <400> 19 Gly Gly Gln Asn Leu Gly Ser Lys Ser Val His 1 5 10 <210> 20 <211> 108 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Light chain FAB6 <220> <221> MISC_FEATURE <223> Light chain VL FAB6 <400> 20 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Gln Asn Leu Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 21 <211> 11 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> LCDR1 FAB7 <400> 21 Gly Gly Asn Gln Leu Gly Ser Lys Ser Val His 1 5 10 <210> 22 <211> 108 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Light chain FAB7 <220> <221> MISC_FEATURE <223> Light chain VL FAB7 <400> 22 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Gln Leu Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 23 <211> 11 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> LCDR3 FAB8 <400> 23 Gln Val Trp Asp Thr Ser Ser Asp His Val Val 1 5 10 <210> 24 <211> 108 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Light chain FAB8 <220> <221> MISC_FEATURE <223> Light chain VL FAB8 <400> 24 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 25 <211> 11 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> LCDR3 FAB9 <400> 25 Gln Val Trp Asp Ser Thr Ser Asp His Val Val 1 5 10 <210> 26 <211> 108 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Light chain FAB9 <220> <221> MISC_FEATURE <223> Light chain VL FAB9 <400> 26 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Thr Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 27 <211> 159 <212> PRT <213> Homo sapiens <400> 27 Met Phe Pro Phe Ala Leu Leu Tyr Val Leu Ser Val Ser Phe Arg Lys 1 5 10 15 Ile Phe Ile Leu Gln Leu Val Gly Leu Val Leu Thr Tyr Asp Phe Thr 20 25 30 Asn Cys Asp Phe Glu Lys Ile Lys Ala Ala Tyr Leu Ser Thr Ile Ser 35 40 45 Lys Asp Leu Ile Thr Tyr Met Ser Gly Thr Lys Ser Thr Glu Phe Asn 50 55 60 Asn Thr Val Ser Cys Ser Asn Arg Pro His Cys Leu Thr Glu Ile Gln 65 70 75 80 Ser Leu Thr Phe Asn Pro Thr Ala Gly Cys Ala Ser Leu Ala Lys Glu 85 90 95 Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile Trp Cys Pro Gly 100 105 110 Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met Lys Lys Arg Arg 115 120 125 Lys Arg Lys Val Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu 130 135 140 Gln Gly Leu Trp Arg Arg Phe Asn Arg Pro Leu Leu Lys Gln Gln 145 150 155 <210> 28 <211> 227 <212> PRT <213> Homo Sapiens <400> 28 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Asn 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys 225 <210> 29 <211> 214 <212> PRT <213> Homo Sapiens <400> 29 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 30 <211> 681 <212> DNA <213> Homo sapiens <400> 30 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 tcctcaggac tctactccct cagcagcgtg gtgacagtgc cctccagcag cttgggcacc 600 cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagagagtt 660 cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagagagtt 660 gagcccaaat cttgtgacaa a 681 gagcccaaat cttgtgacaa a 681 <210> 31<210> 31 <211> 642<211> 642 <212> DNA<212> DNA <213> 智人(Homo sapiens)<213> Homo sapiens <400> 31<400> 31 tcatatgttc ttacacaacc accgtcggtt tcggttgctc caggacaaac agctcgaatt 60 tcatatgttc ttacacaacc accgtcggtt tcggttgctc caggacaaac agctcgaatt 60 acatgcggag gaaacaacct cggatcgaag tcggttcact ggtatcaaca aaagccagga 120 acatgcggag gaaacaacct cggatcgaag tcggttcact ggtatcaaca aaagccagga 120 caagctccag ttctcgtggt gtacgatgat tcagatcgac catcatggat cccagagcga 180 caagctccag ttctcgtggt gtacgatgat tcagatcgac catcatggat cccagagcga 180 ttctcaggat caaactcggg aaatactgcc acgctcacaa tttcacgcgg agaagcggga 2[ ttctcaggat caaactcggg aaatactgcc acgctcacaa tttcacgcgg agaagcggga 240 gatgaagctg attactattg ccaagtgtgg gactcgtcgt cagatcatgt tgttttcgga 300 gatgaagctg attactattg ccaagtgtgg gactcgtcgt cagatcatgt tgttttcgga 300 ggtggaacaa agctcacagt gctcggtcag cccaaggctg ccccctcggt cactctgttc 360 ggtggaacaa agctcacagt gctcggtcag cccaaggctg ccccctcggt cactctgttc 360 ccgccctcct ctgaggagct tcaagccaac aaggccacac tggtgtgtct cataagtgac 420 ccgccctcct ctgaggagct tcaagccaac aaggccacac tggtgtgtct cataagtgac 420 ttctacccgg gagccgtgac agtggcctgg aaggcagata gcagccccgt caaggcggga 480 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

Claims

1. An antigen-binding fragment of an anti-thymic stromal lymphopoietin (TSLP) antibody, the antigen-binding fragment comprising a heavy chain and a light chain, wherein the antigen-binding fragment is a Fab, and wherein the sequence of the heavy chain is set forth in SEQ ID NO: 28 and the sequence of the light chain is set forth in SEQ ID NO:

29.

2. A dry powder formulation comprising a plurality of microparticles, the microparticles comprising: a. 8% to 11% by weight leucine; b. 2% to 4% by weight tri-leucine; and c. a Fab of an anti-thymic stromal lymphopoietin (TSLP) antibody according to claim 1.

4. The dry powder formulation of claim 2, further comprising a glass stabilizer.

5. The dry powder formulation of claim 4, wherein the glass stabilizer is an amorphous sugar or a buffer.

6. The dry powder formulation of claim 4, wherein the glass stabilizer comprises an amorphous sugar and a buffer.

3. The dry powder formulation of claim 2, wherein the dry powder formulation has a compressive bulk density of 0.4-1.0 g / cm3. 3 3. The dry powder formulation of claim 2, wherein the dry powder formulation has a compressive bulk density of 0.4-1.0 g / cm3.

7. The dry powder formulation of claim 5, wherein the dry powder formulation comprises an amorphous sugar, and wherein the amorphous sugar is selected from the group consisting of trehalose, sucrose, raffinose, inulin, dextran, mannitol, and cyclodextrin.

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

9. The dry powder formulation of claim 5, wherein the amorphous sugar is trehalose.

10. The dry powder formulation of any one of claims 2 to 9, comprising 10.5% by weight leucine and 2% by weight tri-leucine.

11. The dry powder formulation of any one of claims 2 to 9, further comprising a surfactant, wherein the surfactant is selected from the group consisting of polysorbate-20 (PS-20), polysorbate-40 (PS-40), polysorbate-60 (PS-60), polysorbate-80 (PS-80), and poloxamer-188.

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

13. The dry powder formulation of claim 12, wherein PS-80 is present at a concentration ranging from 0.67% by weight to 1.33% by weight.

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

15. The dry powder formulation of claim 10, further comprising a surfactant, wherein the surfactant is selected from the group consisting of polysorbate-20 (PS-20), polysorbate-40 (PS-40), polysorbate-60 (PS-60), polysorbate-80 (PS-80), and poloxamer-188. ​ ​ ​ 16. The dry powder formulation of claim 15, wherein the surfactant is PS-80, wherein PS-80 is present at a concentration ranging from 0.27% by weight to 2.7% by weight.

17. The dry powder formulation of claim 16, wherein PS-80 is present at a concentration ranging from 0.67% by weight to 1.33% by weight.

18. The dry powder formulation of claim 17, wherein the PS-80 is present at a concentration of 1.1% by weight.

19. The dry powder formulation of any one of claims 2 to 9, wherein the formulation comprises a plurality of microparticles, the microparticles comprising: a. 10.5% by weight leucine; b. 2% by weight tri-leucine; c. the Fab of an anti-thymic stromal lymphopoietin (TSLP) antibody according to claim 1 ; d. a buffer; and e. trehalose.

20. The dry powder formulation of claim 19, wherein the formulation further comprises a surfactant, wherein the surfactant is selected from the group consisting of polysorbate-20 (PS-20), polysorbate-40 (PS-40), polysorbate-60 (PS-60), polysorbate-80 (PS-80), and poloxamer-188.

21. The dry powder formulation of claim 20, wherein the surfactant is PS-80, wherein PS-80 is present at a concentration ranging from 0.27% by weight to 2.7% by weight.

22. The dry powder formulation of claim 21, wherein PS-80 is present at a concentration ranging from 0.67% by weight to 1.33% by weight.

23. The dry powder formulation of claim 22, wherein the PS-80 is present at a concentration of 1.1% by weight.

24. Use of the dry powder formulation according to any one of claims 2 to 23 in the manufacture of a medicament, wherein the medicament is for the treatment of asthma.

25. The use of claim 24, wherein the asthma is mild asthma, moderate asthma, severe asthma, eosinophilic asthma, or non-eosinophilic asthma.

26. The use of claim 24, wherein the asthma is low-eosinophilic asthma.

27. The use of any one of claims 24 to 26, wherein the medicament is administered by inhalation.

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